Compositions and methods for producing them

COMPOSITIONS AND METHODS FOR PRODUCING A COMPOSITION

[0001] The present patent application claims the priority of U.S. Serial No. 60/752,267, filed on December 20,2005, U.S. Serial No. 60/849,543, filed on October 5,2006, and U.S. Serial No. 60/752,150, filed on December 20, 2005, all of which are hereby incorporated by reference in their entireties, this application also incorporates by reference in its entirety the patent application entitled “Stable Protein Formulations” with Attorney Docket Number 10739 PCT filed on December 19, 2006.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

[0004] Cytotoxic T lymphocyte antigen 4 (CTLA4), a member of the immunoglobulin superfamily, is a molecule expressed by activated T cells. CTLA4 is similar to the T-cell costimulatory molecule CD28, and both molecules bind to B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells (APCs). However, CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal.

[0005] CTLA4-Ig molecules are fusion proteins of the ligand-binding domain of cytotoxic T lymphocyte antigen 4 (CTLA4) and an immunoglobulin (Ig) heavy chain constant region. This soluble molecule exerts its physiological effects by binding to B7 antigens (CD80 and CD86) on the surface of various antigen-presenting cells (APC), thus blocking the functional interaction of B7-1 and B7-2 with CD28 on the surface of T-cells. This blockade results in the suppression of T-cell activation, and hence, the suppression of the immune response. CTLA4-Ig molecules can therefore provide a method for inhibiting tissue and/or solid organ transplant rejections, as well as a therapeutic use for diseases or disorders that relate to disregulated immune responses in general, including autoimmunity. For example, CTLA4-Ig molecules can suppress the production of anti-dsDNA antibodies and decrease nephritis in lupus prone mice; can reduce proteinuria and prolong survival in mice with advanced nephriti; and can improve clinical outcomes for psoriasis and rheumatoid arthritis.

[0006] To improve the therapeutic usefulness of CTLA4-Ig molecules, it is important to determine molecular alterations that can be made to enhance the efficacy of the molecule as an inhibitor of T cell stimulation, for example, by increasing the avidity and potency of the molecule for B7 antigens. An increase in the avidity and potency of CTLA4-Ig molecules may allow for administration of a decreased amount of CTLA4-Ig molecules to a patient to achieve a desired therapeutic effect (i.e., administration of a lower dose). An increase in the avidity and potency of CTLA4-Ig molecules may also decrease the number of doses or the frequency of doses that are administered to a patient to achieve a desired therapeutic effect.

SUMMARY OF THE INVENTION

[0007] The present invention relates to improved compositions and methods for producing CTLA4-Ig compositions. The invention is directed to CTLA4-Ig molecules, improved compositions comprising CTUA4-Ig molecules, and improved methods for producing (including mass-producing) CTLA4-Ig molecules and other recombinant proteins.

[0008] The invention includes any permutations and/or combinations of any of the elements and characteristics described herein, whether described singly or in certain combinations or permutations.

[0009] Cells: The invention provides for a clonal Chinese Hamster Ovary cell population capable of producing CTLA4-Ig. The invention provides for a clonal Chinese Hamster Ovary cell population capable of producing CTLA4-Ig, each cell comprising 30 or more copies of a CTLA4-Ig expression cassette. The invention also provides for a clonal Chinese Hamster Ovary cell population capable of producing CTLA4-Ig, each cell comprising 30 or more copies of a CTLA4-Ig expression cassette, wherein the 30 or more copies are integrated at a single site in the genome of each cell. The invention provides for a clonal Chinese Hamster Ovary cell population capable of producing CTLA4-Ig, wherein a CTLA4-Ig expression cassette is stable over about 105 passages. In one embodiment, the CTLA4-Ig is encoded by an expression cassette comprising a nucleic acid sequence described by Koduri R., et al. (Gene, 2001, 280:87-95) and in U.S. Patent Nos. 6,800,457 and 6,521,419, which are hereby incorporated by reference in their entireties. In another embodiment, the CTLA4Ig is encoded by an expression cassette integrated into a cell genome from the cell population at a specific locus described by Koduri R., et al. (Gene. 2001, 280:87-95) and in U.S. Patent

Nos. 6,800,457 and 6,521,419, which are hereby incorporated by reference in their entireties.

In one embodiment, the population comprises a sub-population of cells comprising 33 or more copies of the CTLA4-Ig expression cassette, wherein the 33 or more copies are integrated at a single site in the genome of each cell of the subpopulation.

[0010] The invention provides for a clonal Chinese Hamster Ovary cell population capable of producing CTLA4-Ig, wherein at least 75% of the population of cells has 30 or more copies of a CTLA4-Ig expression cassette, wherein the 30 or more copies are integrated at a single site in the genome of each cell of the 75% of the population. The invention provides for a clonal Chinese Hamster Ovary cell population capable of producing CTLA4Ig, wherein at least 85% of the population of cells has 30 or more copies of a CTLA4-Ig expression cassette, wherein the 30 or more copies are integrated at a single site in the genome of each cell of the 85% of the population. Hie invention provides for a clonal Chinese Hamster Ovary cell population capable of producing CTLA4-Ig, wherein at least 95%> of the population of cells has 30 or more copies of a CTLA4-Ig expression cassette, wherein the 30 or more copies are integrated at a single site in the genome of each cell of the 95% of the population. In one embodiment, the cell population is capable of producing greater than 0.5 or more grams of CTLA4-Ig protein per liter of liquid culture, and wherein the CTLA4-Ig exhibits acceptable carbohydrate characteristics, where the molar ratio of sialic acid to CTLA4-Ig is from about 6 to about 14 at a culture scale of 1,000 L or more. In another embodiment, the cell population has been adapted to serum-free, chemically defined medium. In another embodiment, CTLA4-Ig produced from culture of the cell population has an extinction coefficient of 1.00 ± 0.05 AU mL cm-1 mg-1. In another embodiment, the cell population, when grown in culture, is capable of producing CTLA4-Ig polypeptides, wherein: (a) about 90% of the CTLA4-Ig polypeptides comprise an amino acid sequence of SEQ ID NO:2 beginning with the methionine at residue 27; (b) about 10% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 beginning with the alanine at residue number 26; (c) about 4% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 ending with the lysine at residue number 383, (d) about 96% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 ending with the glycine at residue number 382; and optionally, (e) about less than 1 % of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 beginning with the methionine at residue number 25.

[0011] The invention provides for a progeny cell of the clonal cell, wherein the progeny cell produces CTLA4-Ig. In one embodiment, the progeny cell is obtained from culturing the clonal parental cell over at least 5 generations. In another embodiment, the progeny cell is obtained from culturing a cell over at least 10 generations, over .at least 20 generations, over at least 40 generations, over at least 50 generations, over at least 75 generations, or over at least 100 generations. The invention provides for a cell line produced from the clonal cell. In one embodiment, the cell line is clonal. The invention provides for a cell line capable of producing: (a) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO: 10 (methionine at amino acid position 27 and glycine at amino acid position 382; Figures 1A and IB); (b) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO: 7 (methionine at amino acid position 27 and lysine at amino acid position 383; Figures 1A and IB); (c) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO: 9 (alanine at amino acid position 26 and glycine at amino acid position 382; Figures 1A and IB); (d) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO: 6 (alanine at amino acid position 26 and lysine at amino acid position 383; Figures 1A and IB);

(e) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:8 (methionine at amino acid position 25 and glycine at amino acid position 382; Figures 1A and IB); or (f) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:5 (methionine at amino acid position 25 and lysine at amino acid position 383; Figures 1A and IB). In another embodiment, the cell line is capable of producing CTLA4-Ig fusion proteins, wherein: (a) about 90% of the CTLA4-Ig polypeptides comprise an amino acid sequence of SEQ ID NO:2 beginning with the methionine at residue 27; (b) about 10% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 beginning with the alanine at residue number 26; (c) about 4% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 ending with the lysine at residue number 383, (d) about 96% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 ending with the glycine at residue number 382; and optionally, (e) about less than 1 % of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 beginning with the methionine at residue number 25.

[0012] In one embodiment, the CTLA4-Ig fusion proteins, which are produced from culturing the cell line, have an extinction coefficient of 1.00 ± 0.05 AU mL cm-1 mg-1. The invention provides for a cell population derived from the clonal cell line. In an embodiment, the cell population consists of at least one additional genetic change as compared to the .

original clonal cell line and wherein the derived cell population is capable of producing CTLA4-Ig. In another embodiment, the cell population consists of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, or at least 20 additional genetic changes as compared to the parental cell, and wherein the derived cell population is capable of producing CTLA4-Ig. In one embodiment, the genetic change comprises at least one non-conservative mutation in the cellular genome or in the recombinant expression cassette encoding CTLA4-Ig. In another embodiment, the genetic change comprises at least one additional recombinant nucleic acid within the cell. In a further embodiment, the change comprises a mutation of the cellular genome. In another embodiment, the change comprises the addition of a nucleic acid to either the cell genome or as a trans nucleic acid, which encodes an anti-apoptotic polypeptide. In another embodiment, the anti-apoptotic polypeptide relates to glycosylation. In another embodiment, genetic change comprises at least one mutation of the cellular genome or of the recombinant expression cassette encoding CTLA4-Ig.

[0013] Compositions: The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about 6 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about 8 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about 11 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-lg dimer or molecule of from about 12 to about

18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about 13 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about 14 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about 15 to about

17. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer or molecule of about 16. The invention provides for a population of CTLA4-Ig molecules, wherein greater than 95% of the molecules are CTLA4-Ig dimers. In one embodiment, greater than 98% of the molecules are CTLA4-Ig dimers. In another embodiment, greater than 99% of the molecules are CTLA4-Ig dimers. In another embodiment, greater than 99.5% of the molecules are CTLA4-Ig dimers. In another embodiment, from about 95% to about 99.5% of the molecules are CTLA4-Ig dimers and about 0.5% to about 5% of the molecules are CTLA4-Ig tetramers or high molecular weight species. In another embodiment, about 98.6% of the molecules are CTLA4-Ig dimers and about 1.2% of the molecules are CTLA4-Ig tetramers or high molecular weight species and about less than 0.7 % of the molecules are CTLA4-Ig monomers. The invention provides for a population consisting of CTLA4-Ig dimers. The invention provides for a population of CTLA4-Ig molecules, wherein the population is substantially free of CTLA4-Ig monomer. The invention provides for a population of CTLA4-Ig molecules, wherein the population is substantially free of CTLA4-Ig tetramer. The invention provides for a population of CTLA4-Ig monomer molecules substantially free of CTLA4-Ig dimer and tetramer. In one embodiment, each monomer of each CTLA4-Ig dimer has at least 3 sialic acid groups. In another embodiment, each monomer of each CTLA4-Ig dimer has from at least 3 sialic acid groups to at least 8 sialic acid groups. The invention provides for a purified population of CTLA4-Ig tetramer molecules, the population being substantially free of CTLA4-Ig dimer, and optionally wherein the population comprises an amount that is greater than about 100 grams. The invention provides for a purified population of CTLA4-Ig tetramer molecules, the population being substantially free of CTLA4-Ig monomer, and optionally wherein the population comprises an amount that is greater than about 100 grams. In one embodiment, each tetramer molecule comprises two pairs of CTLA4-Ig polypeptides, wherein each polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOS: 5-10, and wherein each member of the pair of polypeptides is covalently linked to the other member, and wherein the two pairs of polypeptides are non-covalently associated with one another. In another embodiment, each tetramer molecule is capable of binding to a CD80 or CD86. In a further embodiment, each tetramer molecule has at least a 2-fold greater avidity for CD80 or CD86 as compared to a CTLA4-Ig dimer molecule. In another embodiment, each tetramer molecule has at least a 2fold greater inhibition of T cell proliferation or activation as compared to a CTLA4-Ig dimer molecule. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the composition comprises dominant isoforms visualizable on an isoelectric focusing gel of CTLA4-Ig which have an isoelectric point, pl, less than or equal to 5.1 as determined by isoelectric focusing. In one embodiment, the invention provides for a composition comprising CTLA4-Ig molecules, wherein the composition comprises dominant isoforms visualizable on an isoelectric focusing gel of CTLA4-Ig which have an isoelectric point, pl, less than or equal to 5.8 as determined by isoelectric focusing. In one embodiment, the pl increases after neuraminidase treatment. In one embodiment, the composition comprises dominant isoforms visualizable on an isoelectric focusing gel of CTLA4-Ig which have an isoelectric point, pl, less than or equal to 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, or 4.5 as determined by isoelectric focusing. In another embodiment, at least 40% of the CTLA4-Ig molecules exhibit an isoelectric point less than or'equal to about 5.1 as determined by isoelectric focusing. In another embodiment, at least 70% of the CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 5.1 as determined by isoelectric focusing. In another embodiment, at least 90% of the CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 2.5 as determined by isoelectric focusing. The invention provides for a population of CTLA4-Ig molecules having a pl of from about 2.0 ± 0.2 to about 5.0 ± 0.2. The invention provides for a population of CTLA4-Ig molecules having a pl of from about 4.0 ± 0.2 to about 5.0 ± 0.2. The invention provides for a population of CTLA4-lg molecules having a pl from about 4.3 ± 0.2 to about 5.0 ± 0.2. The invention provides for a population of CTLA4-Ig molecules having a pl of about 3.3 ± 0.2 to about 4.7 ± 0.2. The invention provides for a method for preparing a composition, the composition comprising a CTLA4-Ig molecule with a pl of from about 2.0 ± 0.2 to about 5.0 ± 0.2, the method comprising: (a) subjecting a mixture of CTLA4-Ig molecules to isoelectric focusing gel electrophoresis, wherein a single band on the gel represents a population of CTLA4-Ig molecules with a particular pl, and (b) isolating the population of CTLA4-Ig molecules having a pl of from about 2.0 ± 0.2 to about 5.0 ± 0.2 so as to prepare the composition.

[0014] The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule of from about 15 to about 35. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4Ig molecules are characterized by an average molar ratio of GalNAc per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule of from about 1.7 to about 3.6. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of galcatose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule of from about 8 to about 17. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of fucose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule of from about 3.5 to about 8.3. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of mannose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule of from about 7.2 to about 22. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of sialic acid per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule of from about 6 to about 12.

[0015] The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 15 to about 35; and (b) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 1.7 to about 3.6; and (c) an average molar ratio of sialic acid per mole of CTLA4-lg dimer or CTLA4-Ig molecule from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 1.7 to about 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 8 to about 17; and (d) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 1.7 to about 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 8 to about 17; (d) an average molar ratio of fucose per mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 3.5 to about 8.3; and (e) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer or molecule from about 1.7 to about 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer or molecule from about 8 to about 17; (d) an average molar ratio of fucose per mole CTLA4-Ig dimer or molecule from about 3.5 to about 8.3; (e) an average molar ratio of mannose per mole CTLA4-Ig dimer or molecule from about 7.2 to about 22; and (f) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules, wherein composition exhibits an NGNA chromatogram peak of about 9.589+/-0.3 and an NANA chromatogram peak of about 10.543+/-0.3. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA-Ig molecules exhibit a carbohydrate profile as shown in Figure 68. The invention provides for a composition comprising CTLA4Ig molecules, wherein the CTLA4-Ig molecules exhibit a carbohydrate profile of Domains I — V (e.g., I-IV), wherein Domain I comprises peaks which represent a-sialylated oligosaccharides, Domain II comprises peaks which represent mono-sialylated oligosaccharides, Domain III comprises peaks which represent di-sialylated oligosaccharides, and Domain IV comprises peaks which represent tri-sialylated oligosaccharides. Domain V comprises peaks that represent tetra-sialyated oligosaccharides. In one embodiment, the difference in retention times of N-linked oligosaccharides between a first peak in Domain I and a main peak in Domain II is from about 22 to about 28 minutes. The invention provides for a composition comprising CTLA4-Ig dimer molecules, wherein at least 0.5 % of the CTLA4-Ig dimer molecules are cysteinylated. In one embodiment, at least 1.0% of the CTLA4-Ig dimer molecules are cysteinylated. The invention provides for a population of CTLA4-Ig molecules, wherein the population exhibits a mass spectrometry profile as shown in Figures 8 and 10. The invention provides for a population of CTLA4-Ig molecules, wherein the population exhibits a capillary electrophoresis profile as shown in Figures 20 and

21. The invention provides for a composition of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of from about 6 to about 18. The invention provides for a CTLA4-Ig composition obtained by any of the methods described herein. The invention provides for a population of CTLA4-Ig molecules, wherein the molecules are glycosylated at an aparagine amino acid residue at position 102 of SEQ ID NO:2, an aparagine amino acid residue at position 134 of SEQ ID NO:2, an aparagine amino acid residue at position 233 of SEQ ID NO:2, a serine amino acid residue at position 155 of SEQ ID NO:2, or a serine amino acid residue at position 165 of SEQ ID NO:2.

[0016] The invention provides for a population of CTLA4-Ig molecules, wherein the population of molecules is characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer or CTLA4-Ig molecule from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer or molecule from about 1.7 to about 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer or molecule from about 8 to about 17; (d) an average molar ratio of fucose per mole CTLA4-Ig dimer or molecule from about 3.5 to about 8.3; (e) an average molar ratio of mannose per mole CTLA4-Ig dimer or molecule from about 7.2 to about 22; (f) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer or molecule from about 6 to about 12; (g) a pl as determined from visualization on an isoelectric focusing gel in a range from about 2.4 ± 0.2 to about 5.0 ± 0.2; (h) MCP-1 of less than or equal to 5 ppm; (i) less than 3.0 % tetramer (e.g., 2.5% high molecular weight species or tetramer, 2.0% high molecular weigh species or tetramer; (j) less than 0.5% monomer; (k) CTLA4-Ig polypeptides of the population having an amino acid at least 95% identical to any of SEQ ID NOS: 2-8; (1) wherein CTLA4-Ig molecules within the population is capable of binding to CD80 and CD86.

[0017] Compositions: The invention provides for a composition comprising an effective amount of the CTLA4-Ig molecules of the invention and a pharmaceutically acceptable carrier. The invention provides for a composition comprising excipients as described in U.S. Application No. 60/752,150, filed December 20, 2005. In one embodiment, the composition includes CTLA4-Ig. In one embodiment, the composition further comprises a pharmaceutically acceptable diluent, adjuvant or carrier. In another embodiment, the composition further comprises maltose, sodium phosphate monobasic monohydrate, sodium chloride, sodium hydroxide, and sterile water. In another embodiment, the composition comprises sucrose, poloxamer, sodium phosphate monobasic monohydrate, sodium phosphate dibasic anhydrous, sodium chloride, sodium hydroxide, and sterile water.

[0018] Formulations and Kits: The invention provides for a lyophilized CTLA4-Ig mixture comprising at least 95% CTLA4-Ig dimer, and not more than 5% CTLA4-Ig tetramer. In one embodiment, the mixture comprises at least 98% CTLA4-Ig dimer and no more than 2% CTLA4-Ig high molecular weight species or tetramer. In another embodiment, the mixture comprises at least 99% CTLA4-Ig dimer and no more than 1 % CTLA4-Ig high molecular weight species or tetramer. In another embodiment, the mixture comprises at least 8.0 moles of sialic acid per mole of CTLA4-Ig dimer or molecule. In another embodiment, the mixture comprises from about 15.7 to about 31 moles of GlcNAc per mole of CTLA4-Ig dimer or molecule. In another embodiment, the mixture comprises from about 1.6 to about

3.2 moles of GalNAc per mole of CTLA4-Ig dimer or molecule. In another embodiment, the mixture comprises from about 9.3 to about 15.5 moles of galactose per mole of CTLA4-Ig dimer or molecule. In one embodiment, the mixture comprises from about 3.6 to about 7.9 moles of fucose per mole of CTLA4-Ig dimer or molecule. In one embodiment, the mixture comprises from about 9.7 moles of mannose per mole of CTLA4-Ig dimer or molecule. The invention also provides for a pharmaceutical kit comprising: (a) a container containing a lyophilized CTLA4-Ig mixture of the invention; and (b) instructions for reconstituting the lyophilized CTLA4-Ig mixture into solution for injection.

[0019] Illustrative Methods of Treatment: The invention provides for a method for inhibiting T cell proliferation (or activation), the method comprising contacting a T cell with an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a method for inhibiting an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a method for inducing immune tolerance to an antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a method for treating inflammation in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a method for treating rheumatoid arthritis comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of ־ the invention. The invention provides for a method for treating psoriasis in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a method for treating lupus in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a method for treating or preventing an allergy in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a method for treating or preventing graft vs host disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a method for treating or preventing rejection of a transplanted organ in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a. method for treating multiple sclerosis in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides for a method for treating Crohn’s Disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides a method for treating type I diabetes in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides a method for treating inflammatory bowel disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides a method for treating oophoritis in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides a method for treating glomerulonephritis in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides a method for treating allergic encephalomyelitis in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention. The invention provides a method for treating myasthenia gravis in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention.

[0020] The invention provides for the use of a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about 6 to about 1S in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of an immune disorder. The invention provides for the use of a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about 6 to about 18 in the manufacture of an anti-rheumatoid arthritis agent in a package together with instructions for its use in the treatment of rheumatoid arthritis.

[0021] Illustrative Combination therapies: The invention provides for a method for inhibiting T cell proliferation (or activation), the method comprising contacting a T cell with an effective amount of a CTLA4-Ig composition of the invention in combination with methotrexate. The invention provides a method for inhibiting an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a CTLA4-Ig composition of the invention in combination with methotrexate. The invention provides a method for inducing immune tolerance to an antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of a

CTLA4-Ig composition of the invention in combination with methotrexate.

[0022] Methods for Producing CTLA4-Ig : The invention provides a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein, wherein the expanding is from a seed culture to a liquid culture, wherein the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the liquid culture. The liquid culture can be at least 1,000 L, at least 5,000 L, at least 10,000 L, at least 15,000 L, at least 20,000 L, at least 25,000 L, at least 30,000 L, at least 40,000 L. In one embodiment, the expanding of step (a) comprises: (i) culturing the cells in a serum-free, chemically defined medium with at least four passages so as to obtain a cell density of at least about 1.0 x 10 viable cells per mL, wherein each seed stage starts at about 2 x 10⁵ per ml and goes to 1-2 mil cells per ml; (ii) maintaining the cells in culture for a time sufficient to produce from the culture at least about 0.5 g / L. In one embodiment, the protein is a glycoprotein. In one embodiment, the protein is a CTLA4-Ig protein. In one embodiment, the mammalian cells are progeny of a CHO clonal cell line capable of producing CTLA4-Ig fusion protein, wherein the CHO cells have stably integrated in their genome at least 3 0 copies of a CTLA4-Ig expression cassette. In one embodiment, the time sufficient is a time by which the cells’ viability does not fall below 30%. In another embodiment, the time sufficient is a time by which the cells’ viability does not fall below 40%. In another embodiment, the time sufficient is a time by which the cells’ viability does not fall below 50%. In another embodiment, the time sufficient is a time by which the cells’ viability does not fall below 60%. In another embodiment, the time sufficient is a time by which the cells’ viability does not fall below 70%, or 80% or 90% or 95%.

[0023] In a further embodiment, the at least four passages comprises: (i) growing the cells in a culture volume of at least 50 mL until a cell density of from about 1 million to about 2.5 mill cels per ml is reached, (ii) growing the cells in a culture volume of at least 10 L until a cell density of about 1 million to about 2.5 million cels per ml is reached; (iii) growing the cells in a culture volume of at least 100 L until a cell density of about 1 million to about 2.5 million cels per ml is reached; and (iv) growing the cells in a culture volume of 200 L until a cell density of about 1 million to about 2.5 million cels per ml is reached. .In one embodiment, galactose is added to the serum-free, chemically defined medium. In one embodiment, the maintaining comprises (i) lowering the temperature of the culture from 37±2°C to 34±2°C; and (ii) lowering the temperature of the culture from 34±2°C to 32±2°C. In another embodiment, the temperature is kept within the range of 32±2°C for at least 5 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 6 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 7 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 8 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 9 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 10 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 11 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 12 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 13 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 14 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 15 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 16 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 17 days. In another embodiment, the temperature is kept within the range of 32±2°C for at least 18 days. In another embodiment, the temperature is kept within the range of 32±2°C for up to 18 days. In another embodiment, the temperature is kept within the range of 32±2°C until the cell density of the culture is from about 30 x 10⁵ to about 79 x 10⁵ cells per mL of liquid culture.

[0024] The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the liquid culture, wherein the isolating occurs only when the liquid culture contains greater than or equal to about 6.0 moles of NANA per mole of CTLA4-Ig protein or dimer. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the liquid culture, wherein the isolating occurs only when the liquid culture has a cell density of from about 33 x 10^s to about 79 x 10⁵ cells per mL. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the liquid culture, wherein the isolating occurs when cell viability in the liquid culture has not fallen below about 20%, or about 30%, or about 38%. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000 L so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from. the liquid culture, wherein the isolating occurs only when endotoxin is less than or equal to about 76.8 EU per mL of liquid culture. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000 L so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the at least 10,000 L liquid culture, wherein the isolating occurs only when bioburden is less than 1 colony forming unit per mL of liquid culture. The liquid culture of the invention can be of a volume of at least 5,000 L, at least 10,000 L, at least 15,000 L, at least 20,000 L, at least 25,000 L, at least 30,000 L, at least 40,000 L, at least 50,000 L, at least 60,000 L.

[0025] The invention provides a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the liquid culture, wherein the isolating occurs only if at least two of the following conditions are met: (i) the liquid culture contains greater than or equal to about 6:0 moles of NANA per mole of protein, (ii) the liquid culture has a cell density of from about 33 x 10⁵ to about 79 x 10⁵ cells per mL,(iii) cell viability in the liquid culture has not fallen below about 20%, or about 38%, or (iv) amount of CTLA4-Ig in the culture is greater than 0.5 g / L. In one embodiment, the isolating comprises: (i) obtaining a cell culture supematent; (ii) subjecting the supernatant to anion exchange chromotagraphy to obtain an eluted protein product; (iii) subjecting the eluted protein product of step (ii) to hydrophobic interaction chromatography so as to obtain an enriched protein product; (iv) subjecting the enriched protein product to affinity chromatography to obtain an eluted and enriched protein product; and (v) subjecting the eluted and enriched protein product of (iv) to anion exchange chromatography. In another embodiment, the enriched protein product obtained in step (iii) is characterized in that a percentage of any high molecular weight contaminant is less than 25% by weight. In another. embodiment, the anion exchange chromatography of step (ii) is earned .out using a wash buffer comprising about 75 mM HEPES, and about 360 mM NaCI, and having a pH of about 8.0. In another embodiment, the anion exchange chromatography of step (ii) is carried out using an elution buffer comprising about 25 mM HEPES, and about 325 mM NaCI, and having a pH of about 7.0. In another embodiment, the hydrophobic interaction chromatography of step (iii) is carried out using a single wash buffer comprising about 25 mM HEPES, and about 850 mM NaCI, and'having a pH of about 7.0. In another embodiment, the affinity chromatography of step (iv) is carried out using a wash buffer comprising about 25 mM Tris, and about 250 mM NaCI, and having a pH of about 8.0. In another embodiment, the affinity chromatography of step (iv) is carried out using an elution buffer comprising about 100 mM Glycine and having a pH of about 3.5. In another embodiment, the anion exchange chromatography of step (v) is carried out using a wash buffer comprising about 25 mM HEPES, and from about 120 mM NaCI to about 130 mM NaCI, and having a pH of about 8.0. In another embodiment, the anion exchange chromatography of step (v) is carried out using an elution buffer comprising about 25 mM HEPES, and about 200 mM NaCI, and having a pH of about 8.0. In another embodiment, the anion exchange chromatography of step (ii) is carried out using a column having an anion exchange resin having a primary, secondary, tertiary, or quartenary amine functional group. In another embodiment, the resin has a quartenary amine functional group. In another embodiment, the hydrophobic interaction chromatography of step (iii) is carried out using a hydrophobic interaction resin having a phenyl, an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group. In another embodiment, the functional group is a phenyl functional group. In another embodiment, the affinity chromatography of step (iv) is carried out using a column containing Protein A.

[0026] The invention provides for a method for preparing CTLA4-Ig, the method comprising purifying CTLA4-Ig from a liquid cell culture so that the purified CTLA4-Ig (a) has about 38 ng of MCP-1 per mg of CTLA4-Ig dimer, and (b.) comprises less than 2.5% of CTLA4-Ig high molecular weight species (e:g;, tetramer) by weight. The invention provides for a method for producing CTLA4-Ig, the method comprising: (a) expanding progeny cells or CHO cells that are capable of producing CTLA4-Ig, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L, wherein the CTLA4-Ig concentration is at least

0.5 grams / L of liquid culture; and (b) isolating CTLA4-Ig from the at least 10,000 L liquid culture, wherein the chromotagraphy is on a column with hydrophobic interaction resin with at least a phenyl functional group, wherein the isolating comprises a step of hydrophobic interaction chromatography carried out using a single wash buffer comprising about 25 mM

HEPES, and about 850 mM NaCl, and having a pH of about 7.0.

[0027] CTLA4-Ig molecules include beta polypeptide molecules. CTLA4^A29YL104E-Ig is a beta polypeptide molecule. The present invention relates to methods for producing (including mass-producing) beta polypeptide compositions or beta polypeptide molecule compositions, and improved compositions. The invention is directed to beta polypeptide molecules, improved compositions comprising beta polypeptide molecules, and improved methods for producing (including mass-producing) beta polypeptide molecules and other recombinant glycoproteins.

[0028] Methods for producing beta polypeptides and other glycoproteins: The invention provides for a method for producing a recombinant glycoprotein, the method comprising: (a) expanding mammalian cells that secrete a recombinant glycoprotein, wherein the expanding is from a seed culture to a liquid culture of at least about 10,000 L, wherein the recombinant protein concentration is at least about 0.5 g/L of liquid culture, wherein the expanding comprises: (i) culturing the cells in a serum-free, chemically defined medium with at least four passages so as to obtain a cell density of at least about 1.0 χ 10⁵ viable cells per mL, wherein each seed stage starts at about 2 χ 10⁵ per ml and goes to about 1-2 million cells per ml, wherein the culturing comprises: (1) culturing the cells in a serum-free, chemicallydefined inoculum medium for from about 15 days to about 25 days; then (2) culturing the cells in a serum-free, chemically-defined basal medium until a cell density of about at least 4 million cells per mL is reached; and (ii) maintaining the cells in culture for a time sufficient to produce the recombinant protein from the culture at least about 0.5 g/L; and (b) isolating the recombinant protein from the at least about 10,000 L liquid culture.

[0029] The invention provides for a method for producing a recombinant glycoprotein, the method comprising: (a) expanding mammalian cells that secrete a recombinant glycoprotein, wherein the expanding is from a seed culture to a liquid culture of at least about 10,000 L, wherein the recombinant protein concentration is at least about 0.5 g/L of liquid culture, wherein the expanding comprises: (i) culturing the cells in a serum-free, chemically defined medium with at least four passages so as to obtain a cell density of at least about 1.0 x 10⁵ viable cells per mL, wherein each seed stage starts at about 2 x 10⁵ per ml and goes to about 1-2 million cells per ml; and (ii) maintaining the cells in culture for a time sufficient to produce the.recombinant protein from the-culture at least about 0.5 g/L, wherein the . . maintaining comprises: (1) lowering the temperature of the culture from 37±2°C to 34±2°C; and (2) adding a polyanionic compound to the culture; and (b) isolating the recombinant protein from the at least about 10,000 L liquid culture.

(0030] The invention provides for a method for producing a recombinant glycoprotein, the method comprising: (a) expanding mammalian cells that secrete a recombinant glycoprotein, wherein the expanding is from a seed culture to a liquid culture of at least about 10,000 L, wherein the recombinant protein concentration is at least about 0.5 g/L of liquid culture; and (b) isolating the recombinant protein from the at least about 10,000 L liquid culture, wherein the isolating comprises: (i) obtaining a soluble fraction of the culture of step (a); (ii) subjecting the soluble fraction to affinity chromotagraphy to obtain an eluted protein product; (iii) subjecting the eluted protein product of step (ii) to anion exchange chromatography so as to obtain an eluted and enriched protein product; and (iv) subjecting the enriched protein product to hydrophobic interaction chromatography to obtain an enriched protein product.

[0031] In one embodiment of the invention, the protein comprises a CTLA4-Ig. In another embodiment, the protein comprises a beta polypeptide or beta polypeptide molecules. In another embodiment, the protein comprises beta polypeptides having SEQ ID NO: 11,. 12, 13, 14, 15, or 16.

[0032] In one embodiment of the invention, the at least four passages comprises: (i) growing the cells in a culture volume of at least 50 mL until a cell density of from about 1 million to about 2.5 million cells per ml is reached; (ii) growing the cells in a culture volume of at least 10 L until a cell density of about 1 million to about 2.5 million cells per ml is reached; (iii) growing the cells in a culture volume of at least 100 L until a cell density of about 1 million to about 2.5 million cells per ml is reached; and (iv) growing the cells in a culture volume of 200 L until a cell density of about 1 million to about 2.5 million cells per ml is reached.

[0033] In one embodiment of the invention, the isolating comprises: (i) obtaining a soluble fraction of the culture of step (a); (ii) subjecting the soluble fraction to affinity chromotagraphy to obtain an eluted protein product; (iii) subjecting the eluted protein product of step (ii) to anion exchange chromatography so as to obtain an eluted and enriched protein product; and (iv) subjecting the enriched protein product to hydrophobic interaction chromatography to obtain an enriched protein product.

[0034] In one embodiment, the enriched protein product obtained in step (iv) is characterized in that a percentage of any high molecular weight multimer is less than 25% by weight. In another embodiment, the anion exchange chromatography of step (iii) is carried out using a wash buffer comprising about 50 mM HEPES, and about 135 mM NaCl, and having a pH of about 7. In another embodiment, the anion exchange chromatography of step (iii) is carried out using an elution buffer comprising about 50 mM HEPES, and about 200 mM NaCl, and having a pH of about 7. In another embodiment, the hydrophobic interaction chromatography of step (iv) is carried out using a wash buffer comprising about 50 mM HEPES, and about 1.2 M (NH₄)2SO4, and having a pH of about 7. In another embodiment, the affinity chromatography of step (ii) is carried out using a wash buffer comprising about 25 mM NaH2PO4, and about 150 mM NaCl, and having a pH of about 7.5. In another embodiment, the affinity chromatography of step (ii) is carried out using an elution buffer comprising about 250 mM Glycine and having a pH of about 3. In another embodiment, the anion exchange chromatography of step (iii) is carried out using a column having an anion exchange resin having a primary, secondary, tertiary, or quartenary amine functional group. In another embodiment, the resin has a quarternary amine functional group. In another embodiment, the hydrophobic interaction chromatography of step (iii) is carried out using a hydrophobic interaction resin having a phenyl, an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group. In another embodiment, the functional group is a phenyl functional group. In another embodiment, the affinity chromatography of step (ii) is carried out using a column containing Protein A.

[0035] In another embodiment, the expanding comprises: (i) culturing the cells in a serum-free, chemically defined medium with at least four passages so as to obtain a cell density of at least about 1.0 x 10⁵ viable cells per mL, wherein each seed stage starts at about 2 x 10^s per ml and goes to about 1 -2 million cells per ml; and (ii) maintaining the cells in culture for a time sufficient to produce the recombinant protein from the culture at least about 0.5 g/L. In another embodiment, the culturing comprises: (i) culturing the cells in a serumfree, chemically-defined inoculum medium for from about 15 days to about 25 days; then (ii) culturing the cells in a serum-free, chemically-defined basal medium until a cell density of about at least 4 million cells per mL is reached.

[0036] In another embodiment, the maintaining comprises (i) lowering the temperature of the culture from 37±2°C to 34±2°C; and (ii) adding a polyanionic compound to the culture. In one embodiment, the polyanionic compound is dextran sulfate and wherein the dextran sulfate is added to the culture at a final concentration-of about 50 mg/ml. In another embodiment, the temperature is’kept within the range of 34±2°C for at least •5 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 6 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 7 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 8 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 9 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 10 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 11 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 12 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 13 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 14 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 15 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 16 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 17 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 18 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 19 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 20 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 21 days. In another embodiment, the temperature is kept within the range of34±2°C for at least 22 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 23 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 24 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 25 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 26 days. In another embodiment, the temperature is kept within the range of 34±2°C for at least 27 days. In another embodiment, the temperature is kept within the range of

34±2°C for at least 28 days. In another embodiment, the temperature is kept within the range of 34±2°C for up to 28 days. In another embodiment, the temperature is kept within the range of 34±2°C until the cell density of the culture is from about 30 x 10⁵ to about 79 x 10^s cells per mL of liquid culture.

[0037] In one embodiment, the time sufficient is a time by which the cells’ viability does not fall below 30%. In another embodiment, the time sufficient is a time by which the cells’ viability does not fall below 40%. In another embodiment, the time sufficient is a time by which the cells’ viability does not fall below 50%. In another embodiment, the time sufficient is a time by which the cells’ viability does not fall below 60%. In another embodiment, the time sufficient is a time by which the cells’ viability does not fall below 70%, or 80% or 90% or 95%.

[0038] In one embodiment, galactose is added to the serum-free, chemically defined medium. In another embodiment, isolating occurs when the liquid culture contains greater than or equal to about 6 moles of sialic acid per mole of protein. In another embodiment, isolating occurs when the liquid culture contains from about 5.2 to about 7.6 moles of sialic acid per mole of protein. In another embodiment, the isolating occurs when the liquid culture has a cell density of from about 33 x 10⁵ to about 79 x 10⁵ cells per mL. In another . embodiment, the isolating occurs when cell viability in the liquid culture has not fallen below about 37%. In another embodiment, the isolating occurs when endotoxin is less than or equal to about 4.8 EU per mL of liquid culture. In another embodiment, the isolating occurs when bioburden is less than about 1 colony forming unit per mL (cfu/ml) of liquid culture. In another embodiment, the isolating occurs if at least two of the following conditions are met: (i) the liquid culture contains greater than or equal to about 6 moles of sialic acid per mole of protein, (ii) the liquid culture has a cell density of from about 33 x 10⁵ to about 79 x 10⁵ cells per mL, (iii) cell viability in the liquid culture has not fallen below about 37%, or (iv) the amount of glycoprotein in the culture is from about 0.46 g/L to about 0.71 g/L.

[0039] . In one embodiment, the mammalian cells are progeny of a Chinese Hamster Ovary clonal cell line that produces any combination of beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises SEQ ID NO: 11,12, 13,14,15, or 16, wherein the Chinese Hamster Ovary cells each have stably integrated in their genome at least 30 copies of an expression cassette comprising SEQ ID NO:3. In one embodiment, the liquid culture comprises a cell of or a progeny cell of a cell a production cell line of the invention.

[0040] The invention provides for a beta polypeptide comprising SEQ ID NO: 11, 12, 13, 14, 15, or 16 obtained by a method provided by the invention. The invention provides for a composition comprising beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises SEQ ID NO: 11, 12, 13, 14, 15, or 16 obtained by a method provided by the invention. The invention provides for a beta polypeptide obtained by a method provided by the invention.

[0041] Cells: The invention provides for a clonal Chinese Hamster Ovary cell comprising a nucleic acid encoding a beta polypeptide or a beta polypeptide molecule. The invention provides for a clonal Chinese Hamster Ovary cell population that produces beta polypeptides or beta polypeptide molecules. In one. embodiment, the beta polypeptide comprises SEQ ID NO: 11, 12,13, 14, 15, or 16. The invention provides for a clonal Chinese Hamster Ovary cell comprising a nucleic acid comprising an expression cassette encoding the amino acid sequence of SEQ ID NO: 11,12,13, 14,15, or 16. In one embodiment, the expression cassette comprises SEQ ID NO:3. The invention provides for a clonal Chinese Hamster Ovary cell population that produces a beta polypeptide or beta polypeptide molecule, wherein the beta polypeptide is expressed from a nucleotide sequence derived from a plasmid having ATCC Accession No. PTA-2104 deposited under the provisions of the Budapest Treaty on June 19, 2000 with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA, 20110.

[0042] The invention provides for a clonal Chinese Hamster Ovary cell population that produces a beta polypeptide or beta polypeptide molecules, each cell comprising 30 or more copies of a beta polypeptide expression cassette. The invention provides for a clonal Chinese Hamster Ovary cell population that produces a beta polypeptide or beta polypeptide molecules, each cell comprising 30 or more copies of a beta polypeptide expression cassette, wherein the 30 or more copies are integrated at a single site in the genome of each cell. The invention provides for a clonal Chinese Hamster Ovary cell population that produces a beta polypeptide or beta polypeptide molecules, wherein a beta polypeptide expression cassette is stable over about 105 passages. In one embodiment, the beta polypeptide is encoded by an expression cassette integrated into a cell genome.

[0043] The invention provides for a clonal Chinese Hamster Ovary cell population that produces a beta polypeptide, wherein at least 75% of the population of cells has 30 or more copies of a beta polypeptide expression cassette per cell, wherein the 30 or more copies are integrated at a single site in the genome of each cell of the 75% of the population. The invention provides for a clonal Chinese Hamster Ovary cell population that produces a beta polypeptide, wherein at least 85% of the population of cells has 30 or more copies of a beta polypeptide expression cassette per cell, wherein the 30 or more copies are integrated at a single site in the genome of each cell of the 85% of the population. The invention provides for a clonal Chinese Hamster Ovary cell population that produces a beta polypeptide, wherein at least 95% of the population of cells has 30 or more copies of a beta polypeptide expression cassette per cell, wherein the 30 or more copies are integrated at a single site in the genome of each cell of the 95% of the population. In one embodiment, the expression cassette is derived from a plasmid deposited as ATCC Accession No. PTA-2104. In another embodiment, the expression cassette comprises a nucleic acid having the sequence of SEQ ID NOG. In one embodiment, the cell population produces at least about 0.5 grams of the beta polypeptide per liter of liquid culture, and wherein the beta polypeptide has a molar ratio of sialic acid to beta polypeptide dimer or beta polypeptide molecule of from about 5.5 to about

8.5 at a culture scale of 1,000 L or more. In another embodiment, the cell population produces at least 5, at least 10 or at least 20 grams of the beta polypeptide per liter of liquid culture. In another embodiment, the beta polypeptide has a molar ratio of sialic acid to beta polypeptide dimer or beta polypeptide molecule of from about 5 to about 10 at a culture scale of 1,000 L or more. In another embodiment, the cell population has been adapted to a serumfree, chemically defined medium. In another embodiment, a beta polypeptide produced from culture of the cell population has an extinction coefficient of 1.0 ± 0.05 AU mL cm'¹ mg'¹. In another embodiment, the cell population, when grown in culture, produces beta polypeptides, wherein: (a) about 90% or about 80% of the beta polypeptides comprise an amino acid sequence of SEQ ID NO:4 beginning with the methionine at residue 27; (b) about 10% or about 20% of the beta polypeptides comprise the amino acid sequence of SEQ ID NO:4 beginning with the alanine at residue number 26; (c) from about 4% to about 8% of the beta polypeptides comprise the amino acid sequence of SEQ ID NO:4 ending with the lysine at residue number 383; (d) from about 92% to about 96% of the beta polypeptides comprise the amino acid sequence of SEQ ID NO:4 ending with the glycine at residue number 382; and optionally, (e) about less than 1 % of the beta polypeptides comprise the amino acid sequence of SEQ ID NO:4 beginning with the methionine at residue number 25.

[0044] The invention provides for a progeny cell of a cell population of the invention, wherein the progeny cell produces a beta polypeptide. In one embodiment, the progeny cell is obtained from culturing a cell over at least 5, at least 10, at least 20, at least 40, at least 50, at least 75 generations. In another embodiment, the progeny cell is obtained from culturing a cell for 27 generations.

[0045] The invention provides for a cell line produced from any cell provided by the invention. In one embodiment, the cell line is clonal. In one embodiment, the cell line produces: (a) a beta polypeptide having an amino acid sequence of SEQ ID NO:16 (methionine at amino acid position 27 and glycine at amino acid position 382 of SEQ ID NO:4); (b) a beta polypeptide having an amino acid sequence of SEQ ID NO: 13 (methionine at amino acid position 27 and lysine at amino acid position 383 of SEQ ID NO:4); (c) a beta polypeptide having an amino acid sequence of SEQ ID NO: 15 (alanine at amino acid position 26 and glycine at amino acid position 382 of SEQ ID NO:4); (d) a beta polypeptide having an amino acid sequence of SEQ ID NO: 12 (alanine at amino acid position 26 and lysine at amino acid position 383 of SEQ ID NO:4); (e) a beta polypeptide having an amino acid sequence of SEQ ID NO: 11 (methionine at amino acid position 25 and lysine at amino acid position 383 of SEQ ID NO:4); (f) a beta polypeptide having an amino acid sequence of SEQ ID NO: 14 (methionine at amino acid position 25 and glycine at amino acid position 382 of SEQ ID NO:4); or (g) any combination thereof. In one embodiment, the cell line produces beta polypeptides or beta polypeptide molecules, wherein: (a) about 90% or about 80% of the beta polypeptides comprise an amino acid sequence of SEQ ID NO:4 beginning with the methionine at residue 27; (b) about 10% or about 20% of the beta polypeptides comprise the amino acid sequence of SEQ ID NO:4 beginning with the alanine at residue number 26; (c) from about 4% to about 8% of the beta polypeptides comprise the amino acid sequence of SEQ ID NO:4 ending with the lysine at residue number 383; (d) from about 92% to about 96% of the beta polypeptides comprise the amino acid sequence of SEQ ID NO:4 ending with the glycine at residue number 382; and optionally, (e) about less than 1% of the beta polypeptides comprise the amino acid sequence of SEQ ID NO:2 beginning with the methionine at residue number 25. In one embodiment, wherein the beta polypeptides, which are produced from culturing the cell line, have an extinction coefficient of 1.0 ± 0.05 AU mL cm-1 mg-1.

[0046] The invention provides for a cell population derived from a cell of the invention. In one embodiment, the cells of the population contain at least one additional genetic change as compared to the cell of the invention from which the population was derived, and wherein the cells produce a beta polypeptide. In another embodiment, the cells of the population contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 20 additional genetic change as compared to the cell of the invention from which the population was derived, and wherein the cells produce a beta polypeptide. In one embodiment, the genetic change comprises* at least one non-conservative mutation in the cellular genome or in the expression cassette encoding the beta polypeptide. In another embodiment, the genetic change comprises at least one additional recombinant nucleic acid within the cell. In another embodiment, the genetic change comprises a mutation of the cellular genome. In another embodiment, the genetic change comprises addition of a nucleic acid to the cell genome or as a trans nucleic acid, and wherein the nucleic acid encodes an anti-apoptotic polypeptide. In.another embodiment, the anti-apoptotic polypeptide relates to glycosylation. In another embodiment, the genetic change comprises at least one mutation of the cellular genome or of the expression cassette encoding a beta polypeptide. In another embodiment, the cell population, when grown in culture, produces: (a) a beta polypeptide having an amino acid sequence of SEQ ID NO: 16 (methionine at amino acid position 27 and glycine at amino acid position 382 of SEQ ID NO:4); (b) a beta polypeptide having an amino acid sequence of SEQ ID NO:7 (methionine at amino acid position 27 and lysine at amino acid position 383 of SEQ ID NO:4); (c) a beta polypeptide having an amino acid sequence of* SEQ ID NO: 15 (alanine at amino acid position 26 and glycine at amino acid position 382 of SEQ ID NO:4); (d) a beta polypeptide having an amino acid sequence of SEQ ID NO: 12 (alanine at amino acid position 26 and lysine at amino acid position 383 of SEQ ID NO:4); (e) a beta polypeptide having an amino acid sequence of SEQ ID NO:11 (methionine at amino acid position 25 and lysine at amino acid position 383 of SEQ ID NO:4); (f) a beta polypeptide having an amino acid sequence of SEQ ID NO: 14 (methionine at amino acid position 25 and glycine at amino acid position 382 of SEQ ID NO:4); or (g) any combination thereof.

[0047] Compositions: The invention provides for an isolated population of beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO: 11,12,13,14, 15, or 16, having an average molar ratio of sialic acid groups to beta polypeptide dimer or beta polypeptide molecule of from about 5 to about 10. In one embodiment, the average molar ratio of sialic acid groups to beta polypeptide dimer or beta polypeptide molecule of from about 5.5 to about 8.5. In one embodiment, average molar ratio of sialic acid groups to beta polypeptide dimer or beta polypeptide molecule of from about 5.2 to about 7.6. The invention provides for an isolated population of beta polypeptides, wherein each polypeptide comprises the sequence of SEQ. ID NO: 11,12, 13, 14, 15, or 16, having an average molar ratio of sialic acid groups to beta polypeptide dimer or beta polypeptide molecule of about 6. The invention provides for an isolated population of beta polypeptides, wherein each polypeptide comprises, the sequence of SEQ . ID NO11, 12, 13, 14, 15, or 16, and wherein greater than 95% of the polypeptides are formed into dimers. In one greater than 98%, greater than 99%, or greater than 99.5% of the polypeptides are formed into dimers. In another embodiment, from about 95% to about 99.5% of the polypeptides are formed into dimers and about 0.5% to about 5% of the polypeptides are formed into tetramers or high molecular weight species. In another embodiment, about 98.6% of the polypeptides are formed. into dimers and about 1.2% of the polypeptides are formed into tetramers or high molecular weight species and about less than 0.7 % of the polypeptides are monomers. In another embodiment, about.95% of the polypeptides are formed into dimers and about.4% of the polypeptides are formed into tetramers or high molecular weight species and about 1% of the polypeptides are isolated population of beta polypeptide dimers, wherein each polypeptide monomer comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16. In one embodiment, the population is substantially free of beta polypeptide monomer. In another embodiment, the population is substantially free of beta polypeptide tetramer. The invention provides for an isolated population of beta polypeptide monomers substantially free of beta polypeptide dimer and tetramer. In one embodiment, each monomer of each beta polypeptide dimer comprises the sequence of SEQ ID NO: 11,12, 13, 14,15, or Idand has at least 2.5 sialic acid groups.

[0048] The invention provides for an isolated population of beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, having a potency of from about 70% to about 130% in a B7 binding assay, compared to a CTLA4-Ig standard, wherein the assay comprises measuring surface plasmon resonance. The invention provides for an isolated population of beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12,13,14,15, or 16, having a potency of from about 5.0% to about 150% in a human cell IL-2 inhibition assay, compared to a standard. The invention provides for a purified population of beta polypeptide tetramers or high molecular weight species, wherein each polypeptide monomer comprises the sequence of SEQ ID NO: 11,12, 13, 14, 15, or 16, the population being substantially free of beta polypeptide dimers, and optionally wherein the population comprises an amount that is greater than about 100 grams.

The invention provides for a purified population of beta polypeptide tetramers or high . molecular weight species, wherein each polypeptide monomer comprises the sequence of SEQ ID NO: 11,12,13, 14, 15, or 16, the population being substantially free of beta polypeptide monomer, and optionally wherein the population comprises an amount that is greater than about 100 grams. In one embodiment, each tetramer molecule comprises two pairs of beta polypeptides, wherein each polypeptide monomer comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein each member of the pair of polypeptides is covalently linked to the other member, and wherein the .two pairs of polypeptides are noncovalently associated with one another. In one embodiment, each tetramer molecule is capable of binding to a CD80 or CD86. In one embodiment, each tetramer molecule has at least a 2-fold greater avidity for CD80 or CD86 as compared to a beta polypeptide dimer, wherein each polypeptide monomer of the dimer, comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16. In another embodiment, each tetramer molecule has at least a 2-fold greater avidity for CD80 or CD86 as compared to a CTLA4-Ig tetramer molecule comprising the sequence of SEQ ID NO:2. In another embodiment, each tetramer molecule has at least a 2-fold greater inhibition of T cell proliferation or activation as compared to a beta polypeptide dimer, wherein each polypeptide monomer of the dimer comprises the sequence of SEQ ID NO: 11,12,13, 14, 15, or 16. In another embodiment, each tetramer molecule has at least a 2-fold greater inhibition of T cell proliferation or activation as compared to a CTLA4-Ig tetramer molecule comprising the sequence of SEQ ID NO:2.

[0049] The invention provides for an isolated composition comprising beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the composition comprises dominant isoforms visualizable on an isoelectric focusing gel which have an isoelectric point, pl, less than or equal to 5.5 as determined by isoelectric focusing. In one embodiment, the pl increases after neuraminidase treatment. In one embodiment, at least 40% of the beta polypeptides or beta polypeptide molecules exhibit an isoelectric point less than or equal to about 5.3 as . determined by isoelectric focusing. In one embodiment, at least 70% of the beta polypeptides or beta polypeptide molecules exhibit an isoelectric point less than or equal to about 5.3 as determined by isoelectric focusing. In one embodiment, at least 90% of the beta polypeptides or beta polypeptide molecules exhibit an isoelectric point less than or equal to about 5.3 as determined by isoelectric focusing. The invention provides for an isolated population of beta polypeptides or beta polypeptide molecules having a pl of from about 2.0 ± 0.2 to about 5.2 +

0.2. The invention provides for an isolated population of beta polypeptides or beta polypeptide molecules having a pl from about 4.5 ± 0.2 to about 5.2 ± 0.2. The invention ־ provides for an isolated population of beta polypeptides or beta polypeptide molecules having a pl of about 4.7 ± 0.2 to about 5.1 + 0.2. The invention provides for a method for preparing a composition, the composition comprising beta polypeptides or beta polypeptide molecules with a pl of from about 2.0 ± 0.2 to about 5.2 ± 0.2, wherein each polypeptide comprises the sequence of SEQ ID NO: 11,12, 13, 14, 15, or 16, the method comprising: (a) subjecting a mixture of beta polypeptides to isoelectric focusing gel electrophoresis, wherein a single band on the gel represents a population of beta polypeptides of beta polypeptide molecules witha particular pl, and (b) isolating the population of beta polypeptides of beta polypeptide molecules having a pl of from about 2.0 ± 0.2 to about 5.2 ± 0.2 so as to prepare the composition.

[0050] The invention provides for an isolated composition comprising beta polypeptides. or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13,14, 15, or 16, and wherein the polypeptides are characterized by an average molar ratio of GlcNAc per mole of beta polypeptide dimer or beta polypeptide molecule of from about 24 to about 28. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12,

13, 14, 15, or 16, and wherein the polypeptides are characterized by an average molar ratio of GalNAc per mole of beta polypeptide dimer or beta polypeptide molecule of from about 2.7 to about 3.6. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13,

14, 15, or 16, and wherein the polypeptides are characterized by an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule of from about 11 to about 13. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides are characterized by an average molar ratio of fucose per mole of beta polypeptide dimer or beta polypeptide molecule of from about 6.4 to about 7.0. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, '14,15, or 16, and wherein the polypeptides are characterized by an average molar ratio of mannose per mole of beta polypeptide dimer or beta polypeptide molecule of from about 14 to about 16. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14,-15, or 16, and wherein the molecules are characterized by an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule of from about 5-.5 to about 8.5. . The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11,12,13, 14, 15, or 16, and wherein the molecules are characterized by an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule of from about 5 to about 10. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14,15, or 16, and wherein the polypeptides are characterized by: (a) an average molar ratio of GlcNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 24 to about 28; and (b) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11,12, 13, 14, 15, or 16, and wherein the molecules are characterized by: (a) an average molar ratio of GlcNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 24 to about 28; (b) an average molar ratio of GalNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 2.7 to about 3.6; and (c) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the molecules are characterized by: (a) an average molar ratio of GlcNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 24 to about 28; (b) an average molar ratio of GalNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 2.7 to about 3.6; (c) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 11 to about 13; and (d) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides are characterized by: (a) an average molar ratio of GlcNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 24 to about 28; (b) an average molar ratio of GalNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 2.7 to about 3.6; (c) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 11 to about 13; (d) an average molarratio of fucose per mole of beta polypeptide dimer or beta polypeptide molecule from about 6.4 to about 7.0; and (e) an average molar ’ ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from.

about 5.5 to about 8.5. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13,

14, 15, or 16, and wherein the polypeptides are characterized by: (a) an average molar ratio of GlcNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 24 to about 28; (b) an average molar ratio of GalNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 2.7 to about 3.6; (c) an average molar ratio.of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 11 to about 13; (d) an average molar ratio of fucose per mole of beta polypeptide dimer or beta polypeptide molecule from about 6.4 to about 7.0; (e) an average molar ratio of mannose per mole of beta polypeptide dimer or beta polypeptide molecule from about 14 to about 16; and (f) an average molar ratio of sialic acid per mole of beta polypeptide dimer or .beta polypeptide molecule from about 5.5 to about 8.5. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11,12, 13,14,15, or 16, and wherein the polypeptides are characterized by: (a) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 8 to about 17; (b) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5; and (c) a carbohydrate profile substantially the same as Figure 8. The invention provides for an isolated composition comprising beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides are characterized by: (a) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 8 to about 17; (b) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5; (c) a carbohydrate profile substantially the same as Figure 8; and (d) a beta polypeptide tetramer content less than about 5%. The invention provides for an isolated composition comprising beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14,

15, or 16, and wherein the polypeptides are characterized by: (a) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 11 to about 13; and (b) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11,12,13,14,15,-or 16, and wherein the polypeptides are characterized by: (a) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 11 to about 13; (b) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5; and (c) a beta polypeptide tetramer content less than about 5%. The invention provides for an isolated composition comprising beta polypeptides, wherein each. polypeptide comprises the sequence of SEQ ID NO: 11, 12,13,14,15, or 16, and wherein polypeptides are characterized by: (a) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5; and (b) a carbohydrate profile substantially the same as Figure 8. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides are characterized by: (a) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 11 to about 13; and (b) a.carbohydrate profile substantially . the same as Figure 8. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein polypeptides are characterized by: (a) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5; and (b) a beta polypeptide tetramer or high molecular weight species content less than about 5%. The invention provides for an isolated composition comprising beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14,15, or 16, and wherein the polypeptides are characterized by: (a) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 11 to about 13; and (b) a beta polypeptide tetramer or high molecular weight species content less than about 5%.

[0051] The invention provides for an isolated composition comprising beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides exhibit a carbohydrate profile substantially the same as Figure 8. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides exhibit a carbohydrate profile of Domains I - IV, wherein Domain I comprises peaks which represent a-sialylated oligosaccharides, Domain II comprises peaks which represent, mono-sialylated oligosaccharides, Domain III comprises peaks which represent di-sialylated oligosaccharides, and Domain IV comprises peaks which represent tri-sialylated oligosaccharides. In one embodiment, the difference in retention times of N-linked oligosaccharides between a first peak in Domain I and a main peak in Domain II is from about 11 to about 13 minutes. In one embodiment, the sum of Domains III and IV comprises from about 25% to about 36% of the total carbohydrate profile.

[0052] The invention provides for an isolated composition comprising beta polypeptide dimers or beta polypeptide molecules, wherein each polypeptide monomer comprises the sequence of SEQ ID NO: 11, 12, 13,14, 15, or 16, and wherein at least about 0.5 % of the molecules are cysteinylated. The invention provides for an isolated population of beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the population exhibits a mass spectrometry profile as shown in Figure 11. The invention provides for an isolated population of beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12,13, 14, 15, or 16, having an average molar ratio of sialic acid groups to beta polypeptide dimer or beta polypeptide molecule of from about 5.5 to about 8.5, wherein the beta polypeptide dimer or beta polypeptide molecules is produced from cells of a production cell line. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11,12, 13, 14, 15, or 16, wherein the polypeptides are glycosylated at an asparagine amino acid residue at position 102 of SEQ ID NO:4, an asparagine amino acid residue at position 134 of SEQ ID NO:4, an asparagine amino acid residue at position 233 of SEQ ID NO:4. The invention provides for an isolated composition comprising beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein the molecules are characterized by: (a) an average molar ratio of GlcNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 24 to about 28; (b) an average molar ratio of GalNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 2.7 to about 3.6; (c) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 11 to about 13; (d) an average molar ratio of fucose per mole of beta polypeptide dimer or beta polypeptide molecule from about 6.4 to about 7.0; (e) an average molar ratio of mannose per mole of beta polypeptide dimer or beta polypeptide molecule from about 14 to about 16; (f) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5; (g) a pl as determined from visualization on an isoelectric focusing gel in a range from about 2.4 ± 0.2 to about 5.2 + 0.2; (h) MCP-1 ־of less than or equal to 5 ppm; (i) less than 5% tetramer or high molecular weight species; (j) less than beta polypeptide 1% monomer; and (k) beta polypeptides or beta polypeptide molecules of the population having an amino acid at least 95% identical to any ofSEQ ID N0S:4, 11,12, 13,14, 15, or 16, wherein the beta polypeptides within the population are capable of binding to CD80 and CD86. The invention provides for an isolated population of beta polypeptides, wherein each polypeptide comprises the sequence of SEQ ID NO: 11,12,13,1415 _ג, or 16, and wherein the population of molecules is characterized by: (a) an average molar ratio of GlcNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 24 to about 28; (b) an average molar ratio of GalNAc per mole of beta polypeptide dimer or beta polypeptide molecule from about 2.7 to about 3.6; (c) an average molar ratio of galactose per mole of beta polypeptide dimer or beta polypeptide molecule from about 11 to about 13; (d) an average molar ratio of fucose per mole of beta polypeptide dimer or beta polypeptide molecule from about 6.4 to about 7.0; (e) an average molar ratio of mannose per mole of beta polypeptide dimer or beta polypeptide molecule from about 14 to about 16; (f) an average molar ratio of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule from about 5.5 to about 8.5; (g) a pl as determined from visualization on an isoelectric focusing gel in a range from about 2.4 ± 0.2 to about 5.2 ± 0.2; (h) MCP-1 of less than or equal to 5 ppm; (i) less than 5% beta polypeptide tetramer or high molecular weight; (j) less than 1% monomer; and (k) beta polypeptides of the population having an amino acid at least 95% identical to any of SEQ ID NOS:4,11, 12, 13, 14,15, or 16, wherein beta polypeptide molecules within the population are capable of binding to CD80 and CD86; or pharmaceutical equivalents thereof.

[0053] The invention provides for a composition comprising an effective amount of the beta polypeptide of the invention and a pharmaceutically acceptable carrier. The invention provides for a composition comprising excipients as described in U.S. Application No. 60/752,150; filed December 20, 2005. In one embodiment, the composition includes beta polypeptide molecules. In one embodiment, the composition further comprises a pharmaceutically acceptable diluent, adjuvant or carrier. In one embodiment, the composition further comprises sucrose, sodium phosphate monobasic monohydrate, sodium chloride, sodium hydroxide, hydrochloric acid, and sterile water. In another embodiment, the composition comprises sucrose, poloxamer, sodium phosphate monobasic monohydrate, sodium phosphate dibasic anhydrous, sodium chloride, sodium hydroxide, and sterile water.

In one embodiment, the composition is lyophilized. The invention provides for a lyophilized composition comprising an effective amount of the beta polypeptides of the invention, sucrose, sodium phosphate monobasic monohydrate, sodium chloride, sodium hydroxide, and hydrochloric acid. . .

[0054] Formulations and kits: The invention provides for lyophilized beta polypeptide mixture, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, comprising at least 95% beta polypeptide dimer, and not more than 5% beta polypeptide tetramer (high molecular weight species). In one embodiment, the mixture comprises at least 98% beta polypeptide dimer and no more than 2% beta polypeptide tetramer (high molecular weight species). In one embodiment, the mixture comprises at least 99% beta polypeptide dimer and no more than 1% beta polypeptide tetramer (high molecular weight species). In one embodiment, the mixture comprises at least 5 moles of sialic acid per mole of beta polypeptide dimer or beta polypeptide molecule. In one embodiment, the mixture comprises from about 24 to about 28 moles of GlcNAc per mole of beta polypeptide dimer (high molecular weight species). In one embodiment, the mixture comprises from about 2.7 to about 3.6 moles of GalNAc per mole of beta polypeptide dimer or beta polypeptide molecule. In one embodiment, the mixture comprises from about 11 to about 13 moles of galactose per mole of beta polypeptide dimer or beta polypeptide molecule. In one embodiment, the mixture comprises from about 6.4 to about 7.0 moles of fucose per mole of beta polypeptide dimer or beta polypeptide molecule. Tn one embodiment, the mixture comprises from about 14 to about 16 moles of mannose per mole of beta polypeptide dimer or beta polypeptide molecule. The invention also provides for a pharmaceutical kit comprising: (a) a container containing a lyophilized beta polypeptide mixture of the invention and (b) instructions for reconstituting the lyophilized beta polypeptide mixture into solution for injection.

[0055] Illustrative methods of treatment: A method for inhibiting T cell proliferation, activation or both, the method comprising contacting a T cell with an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for inhibiting an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating an immune disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. .The invention provides for a method for inducing immune tolerance to an antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The.method provides for a method for treating inflammation in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The method provides for a method for treating rheumatoid arthritis comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating psoriasis in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating lupus in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating or preventing an allergy in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating or preventing graft versus host disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating or preventing rejection of a transplanted organ in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. ’ The invention provides for a method for treating or preventing rejection of transplanted tissue in a subject, the method comprising administering to a subject in need thereof an effective amount of the composition a beta polypeptide composition of the invention. The invention provides for a method for treating or preventing rejection of a transplanted cell in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. In one embodiment, the transplanted cell is a bone marrow cell. In another embodiment, the transplanted cell is an islet cell. In another embodiment, the transplanted cell is an insulinproducing pancreatic islet cell. The invention provides for a method for treating multiple sclerosis in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating Crohn’s Disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating type I diabetes in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating inflammatory bowel disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating oophoritis in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating glomerulonephritis in a subject, the method comprising administering to.a subject in need ־ thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating allergic encephalomyelitis in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention. The invention provides for a method for treating myasthenia gravis in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention.

[0056] The invention provides for the use of a population of beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14,15, or 16, and wherein the population has an average molar ratio of sialic acid groups to beta polypeptide dimer or beta polypeptide molecule of from about 5 to about 10 in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of an immune disorder. The invention provides for the use of a population of beta polypeptides or beta polypeptide molecules, wherein each polypeptide comprises the sequence of SEQ ID NO11, 12,13,14, 15, or 16, and wherein the population has an average molar ratio of sialic acid groups to beta polypeptide dimer or beta polypeptide molecule of from about 5 to about 10 in the manufacture of an anti-rheumatoid arthritis agent in a package together with . . instructions for its use in the treatment of rheumatoid arthritis. In one embodiment, the population has an average molar ratio of sialic acid groups to beta polypeptide dimer or beta polypeptide molecule of from about 5.5 to about 8.5.

[0057] Illustrative combination therapies: The invention provides for a method for inhibiting T cell proliferation, activation or both, the method co'mprising contacting a T cell with an effective amount of a beta polypeptide composition of the invention in combination with methotrexate. The invention provides for a method for inhibiting an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention in combination with methotrexate.

The invention provides for a method for inducing immune tolerance to an antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of a beta polypeptide composition of the invention in combination with methotrexate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIGS. 1A-1B presents the nucleotide sequence (SEQ ID NO:1) of a portion of an expression cassette for a CTLA4-Ig molecule. Also shown is the amino acid sequence (SEQ ID NO:2) encoded by the nucleic acid. CTLA4-Ig molecules that can be produced from this expression cassette include molecules having the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 26-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2. The expression cassette comprises the following regions: (a) an Oncostatin M signal sequence (nucleotides 11-88 of SEQ ID NO:1; amino acids 1-26 of SEQ ID NO:2); (b) an extracellular domain of human CTLA4 (nucleotides 89-463 of SEQ ID NO:1; amirio acids 27-151 of SEQ ID NO:2); (c) a modified portion ofthe human IgGl constant region (nucleotides 464-1159 of SEQ ID NO:1; amino acids 152-383 of SEQ ID NO:2), including a modified hinge region (nucleotides 464-508 of SEQ ID NO:1; amino acids 152-166 of SEQ ID NO:2), a modified human IgGl Ch2 domain (nucleotides 509-838 of SEQ ID NO:1; amino acids 167-276 of SEQ ID NO:2), and a human IgGl Ch3 domain (nucleotides 839-1159 of SEQ ID NO: 1; amino acids 277-383 of SEQ ID NO:2).

[0059] FIG. 2 presents the nucleic acid (top row) and amino acid (bottom row) sequences corresponding to CTLA4^A29YLI04E-Ig. The amino acid sequence contains an amino acid change from the sequence shown in Fig. 1, wherein the changes are at position 29 (A to Y) and at position 104 (L to E) compared to that of SEQ ID NO: 2, wherein numbering of amino acid residues begins at Methionine (M) marked by “+1.” The nucleotide sequence of CTLA4^A29YL104E-Ig is shown in this figure starting from the A at position 79 (i.e., the position marked by the “+1” below the M) through the A at nucleotide position 1149 (SEQ ID NO:3). In particular, the nucleotide sequence encoding CTLA4^A29YL104E-Ig is from the nucleotide at position 79 to the nucleotide at position 1149, designated SEQ ID NO:3. The full nucleotide sequence shown in Fig. 2 is designated SEQ ID NO:23 and includes the nucleic acid sequence encoding the Oncostatin M signal peptide.

[0060] FIG. 3 presents the amino acid sequence (SEQ ID NO:4) of CTLA4^A29YLI04E-Ig molecule including an Oncostatin M prosequence (see bold italics). Polypeptides that can be produced that are CTLA4^A29YLI04E-Ig molecules include molecules having the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 26-382 of SEQ IDNO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4.

[0061] FIG. 4 is a model of a CTLA4^A29YL104E-Ig shown with the N-linked glycosylation sites (N76, N108, and N207), the C120-C120 disulfide bond, and the two amino acid substitutions made in the CTLA-4 domain (LI 04E and A29Y).,

[0062] FIG. 5 represents the theoretical cDNA-derived amino acid sequence of a CTLA4^A29YL104E-Ig (SEQ ID NO:4). Two amino acid substitutions were made in the CTLA4 extracellular domain (L104E and A29Y) to generate CTLA4^A29YLI04E-Ig. The sequence identifies the signal peptide (pro-sequence) of oncostatin M along with the N-linked glycosylation sites.

[0063] FIG. 6 is a graph depicting binding of CTLA4^A29YL104E-Ig samples to goat antihuman IgG Fc antibody. Binding of CTLA4^A29YL104E-Ig samples was detected by measuring the response obtained on this surface, compared to an unmodified sensorchip surface. The various lots represent three different CTLA4^A29YL104E-Ig samples. ...

[0064] FIG. 7 is a graph that shows the apparent molecular weights which correspond to multimer, tetramer, and dimer fractions of a CTLA4-Ig HIC cleaning peak as determined by an overlay of two-column SEC. with dynamic light scattering detection (DSL) and retention time on SEC.

[0065] FIG. 8A (top) and 8B (bottom) show representative IEF gels of fractions of glycosylated CTLA4-Ig molecules (comprising SEQ ID NO:2 monomers) isolated and purified from HIC cleaning peak. The loading order for the top gel is: lane 1, pl markers (Amersham); lane 2, CLTA4-Ig dimer standard; lane 3, Protein A eluate; lane 4, Multimer; lane 5, tetramer; lane 6, dimer. The loading order for the bottom gel is: lane 1, pl marker (Amersham); lane 2, lane 2, CLTA4-Ig dimer standard; lane 3, tetramer; lane 4, dissociated tetramer. The panels show that the tetramer is less sialylated than the dimer.

[0066] FIG. 9 shows the predominant carbohydrate structures and relative amounts of carbohydrates observed on-a CTLA4-Ig dimer comprising monomers of SEQ ID NO:2. The amino acid residue numbering in the figure is not consistent with SEQ ID NO:2. For the amino acid residue numbering in the figure to be consistent with SEQ ID NO:2, the

. 102 ״ 76 numeration needs to increase by 26, z.e., N isN .

[0067] FIG. 10 INTENTIONALLY LEFT BLANK

[0068] FIG. 11 shows a representative IEF gel (pH 4.0 to 6.5) of a CTLA4-Ig dimer comprising SEQ ID NO:2 monomers. Lanes ’1 and 5 show a calibration standard, lane 2, 3, 4 each show 20 μg/μl of CLTA4-Ig dimer.

[0069] FIG. 12 shows a representative IEF gel (pH 4.0 to 6.5) of a CTLA4^A29YLI04E-Ig dimer comprising SEQ ID NO:4 monomers. Lanes 1 .and 8 show a calibration standard, lane 2-7 each show 10 μg/μl of CTLA4^A29YLI04E-Ig dimer.

[0070] FIG. 13 shows the N-linked carbohydrate profile of a CTLA4-Ig molecule population comprising monomers of SEQ ID NO:2. The carbohydrates were collected from glycopeptides and separated using the LC/MS PGC N-linked Oligosaccharide technique. The chromatograms provide the population profile for each N-link attachment site. A) The Asn⁷⁶ (Asn¹⁰² of SEQ ID NO:2) carbohydrates from the T5 peptide and B) the Asn¹⁰⁸(Asn¹³⁴ of SEQ ID NO:2) carbohydrates from the T7 peptide both show distributions among mono- and multi-sialylated species. C) The Asn²⁰⁷ (Asn ²³³ of SEQ ID NO:2) carbohydrates from the T14 peptide consist of predominantly asialylated species. D) The distribution of N-linked carbohydrates for CTLA4-Ig molecules is shown. E) A selected raw spectrum from the T5 peptide shows a major peak corresponding to the bi-antennary monosialylated structure depicted. F) A selected raw spectrum from the T14 shows a major peak corresponding to the bi-antennary asialo structure. G) A selected raw spectrum consists of a minor species which coelutes with the peak at 64.23 minutes, which corresponds to the tri-antennary di-sialylated structure. H) A selected raw spectrum reveals the major species in the peak at 64.23 minutes, which corresponds to the bi-antennary di-sialylated structure.

[0071] FIG. 14A-B shows a UV and TIC trace of an N-linked oligosaccharide profile of a CTLA4-Ig SEQ ID NO:2 monomer from PGC chromatography under acidic elution conditions (0.05% TFA). The trace of FIG. 14A shows negative ion total count (TIC) for

PGC chromatograpm under acidic elution conditions (0.05% TFA). The trace of FIG. 14B shows UV trace at 206nm for PGC chromatogram under acidic elutions (0.05% TFA).

[0072] FIG. 15A-B shows a UV and TIC trace of an N-linked oligosaccharide profile of a CTLA4-Ig SEQ ID NO:2 monomer from PGC chromatography under basic elution conditions (0.4% NH4OH). The trace of FIG. 15A shows negative ion total count (TIC) for PGC chromatograpm under basic elution conditions (0.4%NH4OH). The trace of FIG. 15B shows UV trace at 206nm for PGC chromatogram under basic elutions (0.4% NH4OH).

[0073] FIG. 16 represents the comparative TV-linked oligosaccharide carbohydrate profiles for CTLA4^A29YU04E-Ig molecules comprising SEQ ID NO:4. Four oligosaccharide domains are observed: Domain I contains non-sialylated species, while Domains II, III, and IV contain mono-sialylated, di-sialylated and tri-sialylated species, respectively. Isolating the oligosaccharides chromatographically and analyzing them by mass spectroscopy determined the domains.

[0074] FIG. 17 shows an HPAEC-PAD profile of N-linked oligosaccharides of CTLA4Ig molecules comprising SEQ ID NO:2 monomers. Domains are shown in order of increasing sialic acid content for oligosaccharides. Domains I, II, III an IV contain oligosaccharide structures having 0,1,2, and 3 sialic acids respectively. Peak labels represent oligosaccharide structures assigned by HPAEC-PAD profiling of peaks collected from PGC profiling. The structural identification of carbohydrate structure is consistent with previous determinations.

[0075] FIG. 18A-B shows a PGC profile of CTLA4-Ig molecules comprising monomers of SEQ ID NO:2. The profile is obtained from direct injection.of carbohydrate digest mixture. prepared as described in Example 3. Direct injection results in detection of structure P4144 eluting at 130 minutes. The tetra-sialylated structure P4144 is not observed in profiles of oligosaccharides which are isolated prior to injection. . . .

[0076] FIG. 19 presents a LC/MS deconvoluted positive electrospray spectrum for the T9 fragment of a SEQ ID NO:2 monomer. The spectrum illustrates three major O-linked structures. The spectrum illustrates the base peptide with sugar ladder consistent with the Olinked structure (GalNAc)!(Gal)1(NeuAc)1. ^,The bold portion of the spectrum has been enhanced 10-fold with respect to the non-bold portion of the spectrum and illustrates two additional O-linked structures with (GalNAc)1(Gal)1(NeuAc)₂ and (GalNAc)1(GlcNAc)1(Gal)₂(NeuAc)₂.

[0077] FIG. 20 shows the attachment points and relative populations of O-linked carbohydrate structures of a CTLA4-Ig single chain having a SEQ ID NO:2 monomer sequence. The relative amounts at each site show data generated by two or more orthogonal techniques and are subject to variability. The location of the covalent cysteinylation is also depicted.

[0078] . FIG. 21 depicts amap of the intermediate .plasmid piLN-huCTLA4-Ig. This. plasmid has comprises a sequence that can encode a human CTLA4-Ig molecule (huCTLA4Ig) (i.e., SEQ ID NO: 1) flanked by the restriction enzyme sites Hindlll and Xbal.

[0079] FIG. 22 depicts a map of the plasmid pDl 6LEA29Y. This plasmid comprises a sequence that can encode a human CTLA4^A29YL104E-Ig molecule (i.e., SEQ ID NO:4).

[0080] FIG. 23 is a photograph of a Southern blot of DNA extracted from CHO cells expressing the CTLA4-Ig expression cassette derived fromlD5-100Al (for example, clone 17). ׳The lanes for the gel from left to right are: lane M, DNA molecular weight marker; lane N, EcoRI/Xbal digested untransfected CHO DNA (5 mg); lane 1, EcoRI/Xbal digested untransfected CHO DNA (2.5 pg) + 1 ng pcSDhuCTLA4-Ig; lane 2, EcoRI/Xbal digested untransfected CHO DNA (2.5 pg) + 0.5 ng pcSDhuCTLA4-Ig; lane 3, EcoRI/Xbal digested untransfected CHO DNA (2.5 pg) + 0.25 ng pcSDhuCTLA4-Ig; lane 4, EcoRI/Xbal digested untransfected CHO DNA (2.5 pg) + 0.125 ng pcSDhuCTLA4-Ig; lane 5, EcoRI/Xbal digested untransfected CHO DNA (2.5 pg) + 0.0625 ng pcSDhuCTLA4-Ig; lane 6, EcoRI/Xbal digested untransfected CHO DNA (2.5 pg) + 0.03125 ng pcSDhuCTLA4-lg; lane 7, EcoRI/Xbal digested DNA: MCB (5.0 pg); lane 8, EcoRI/Xbal digested DNA: EPCB Lot Number C20030618A-01 (5.0 pg); lane 9, EcoRI/Xbal digested DNA: EPCB Lot Number C20030712A-01 (5.0 pg); lane 10, EcoRI/Xbal digested DNA: EPCB Lot Number C20030801A-01 (5.0 pg); lane 11, EcoRI/Xbal digested DNA: MCB (2.5 pg); lane 12, EcoRI/Xbal digested DNA: EPCB Lot Number C20030618A-01 (2.5 pg); lane 13, EcoRI/Xbal digested DNA: EPCB Lot Number C20030712A-01 (2.5 pg); lane 14, EcoRI/Xbal digested DNA: EPCB Lot Number C20030801A-01 (2.5 pg); lane 15, EcoRI/Xbal digested DNA: MCB (1.25 pg); lane 16, EcoRI/Xbal digested DNA: EPCB Lot Number C20030618A-01 (1.25 pg); lane 17, EcoRI/Xbal digested DNA: EPCB Lot Number C20030712A-01 (1.25 pg); lane!8, EcoRI/Xbal digested DNA: EPCB Lot Number C20030801A-01 (1.25 pg).

[0081] FIG. 24 depicts a flow diagram of a production-scale culturing process. This process allows for the mass-production of recombinant proteins in a 25,000-L production bioreactor.

[0082] FIG. 25 shows a representative chromatogram of NGNA and NANA system suitability standard. The peak at ~9.7 min is NGNA, and the peak at -10.7 min is NANA.

[0083] FIG. 26 shows a representative chromatogram of hydrolyzed CTLA4-Ig molecules comprising SEQ ID NO:2 monomers. The peak at -8.4 min is the solvent peak. The peak at -9.6 min is NGNA. The peak at ~10.5min is NANA. The peak at -11.3 min is degraded NANA, resulting from the hydrolysis conditions. The area counts of NANA and degraded NANA are combined for calculations of the NANA molar ratio.

[0084] FIGS. 27A, 27B, and 27C show the MALDI spectra of CTLA4-Ig cysteinylated peptide. The MALDI spectra were obtained for CTLA4-Ig trypsin/chymotrypsin fragment containing Cys¹⁴⁶ of SEQ ID NO:2. FIG. 27A shows the single chain peptide spectrum ־ illustrating cysteinylation modification. FIG. 27B shows the spectrum of the single-chain ־ peptide following reduction and demonstrates that the modification occurs at Cys¹⁴⁶. FIG. 27C shows alkylation of the reduced single-chain peptide, which demonstrates that the cysteinylation occurs at Cys¹⁴⁶.

[0085] FIG. 28 presents a cloning scheme useful for generating the vector pcSD. pcDNA3 was digested with the restriction enzyme Nael in order to isolate a 3.821 Kb fragment that contains the CMV promoter, an ampicillin resistance gene, and an origin of replication for E. coli. pSV2-dhfr was digested with the restriction enzymes PvuII and BamHI in order to isolate a 1.93 Kb fragment, which contains the SV40 promoter and the dhfr gene, and was subsequently blunt-ended. To generate pcSD, both fragments were ligated. The map of plasmid pcSD is shown at the bottom of the figure.

[0086] FIG. 29 presents a cloning scheme useful for generating the expression vector pcSDhuCTLA4-Ig. pcSD was digested with the restriction enzymes EcoRVand XbaL piLN-huCTLA4-Ig was digested with the restriction enzyme Hindlll, blunt-ended, and then digested with the restriction enzyme Xbal in order to isolate the 1.2 Kb huCTLA4-Ig fragment. To generate pcSDhuCTLA4-Ig, the CTLA4-Ig fragment was ligated to the digested pcSD vector. The map of plasmid pcSDhuCTLA4-Ig is shown at the bottom of the figure. This plasmid was linearized and transfected into CHO cells that do not have a functional dhfr gene. As the plasmid contains a functional dhfr gene, stable.transfectants can be selected on the basis of cell survival. The pcSDhuCTLA4-Ig has the expression cassette comprising the CMV promoter, a sequence that can encode a human CTLA4-Ig molecule (huCTLA4-Ig) (i.e., SEQ ID NO:1) and a poly(A) tail sequence from BGH.

[0087] FIG. 30 shows an electropherogram of system suitability amino monosacchrarides depicted as relative fluorescence units (RFU) versus time (min).

[0088] FIG. 31 shows an electropherogram of system suitability neutral monosacchrarides depicted as relative fluorescence units (RFU) versus time (min).

[0089] FIG. 32 represents a tryptic peptide map of CTLA4^A29YL104E-Ig with peptides labeled. Table 23 corresponds with the labeled peptides.

[0090] FIG. 33 shows a Northern Hybridization Analysis of the CTLA4^A29YL104E-Ig. Panel A depicts an Ethidium bromide-stained agarose gel wherein Lane M is RNA marker; Lane 1 is total CHO RNA; Lane 2 is total MCB RNA; and Lane 3 is total EPCB RNA. Panel B is the corresponding autoradiogram wherein Lane M is RNA marker; Lane 1 is total CHO RNA; Lane 2 is total MCB RNA; and Lane 3 is total EPCB RNA.

[0091] FIG. 34A-C depict size exclusion chromatograms, which distinguish CTLA4^A29YL104E-Ig dimers from high and low molecular weight species.

[0092] FIG. 35 shows an SDS-PAGE (Reduced and Non-Reduced) analysis of CTLA4^A29YL104E-Ig stained with Coomassie Blue. Lane 1 is loaded with molecular weight markers; Lanes 2, 7, and 12 are blank; Lanes 3-6 are CTLA4^A29YL104E-Ig samples (reduced); Lanes 8-11 are CTLA4^A29YU04E-Ig samples (non-reduced).

[0093] FIG. 36 shows an SDS-PAGE (Reduced and Non-Reduced) analysis of CTLA4^A29YLI04E-Ig subjected to silver-staining. Lane 1 is loaded with molecular weight markers; Lanes 2, 7, and 12 are blank; Lanes 3-6 are CTLA4^A29YL104E-Ig samples (reduced); Lanes 8-11 are CTLA4^A29YLI04E-Ig samples (non-reduced).

[0094] FIG. 37 depicts a peptide map of non-reduced CTLA4^A29YLI04E-Ig using a combination of trypsin and chymotrypsin digestion.

[0095] FIG. 38 .depicts a peptide map of non-reduced CTLA4^A29YL104E־Ig using a .

combination of trypsin and elastase digestion.

[0096] FIG. 39 is a diagram that depicts patients with anti-CTLA4-Ig or anti-CTLA-4 responses. Antibody response to the whole CTLA4-Ig molecule (CTLA-4 and Ig portion) and the CTLA-4 portion only were determined using Assays A and B, as outlined in the Example 32.

[0097] FIG. 40 is a schematic demonstrating the distribution of clearance and the volume of central compartment by immunogenicity status.

[0098] FIG. 41 is a graph demonstrating profiles of mean (SD) CTLA4-Ig serum concentrations over time in monkeys adminstered 10 mg/kg of drug substance produced by a process of the invention.

[0099] FIG. 42 is a graph of a Size Exclusion Chromatography (SEC) chromatogram of

Protein A (MAbSelect) purified from control and disaggregated CTLA4-Ig material.

[00100] FIG. 43 is a graph of an N-glycan analysis comparing the Disaggregation Processed Material (ii) to Control (i).

[00101] FIG. 44 is a graph depicting the mean CTLA4-Ig serum concentrations ^g/ml] versus time (over 71 days).

[00102] FIG. 45 shows an electropherogram of neutral monosacchrarides depicted as relative fluorescence units (RFU) versus time (min).

[00103] FIG. 46 shows an electropherogram of amino monosacchrarides depicted as relative fluorescence units (RFU) versus time (min).

[00104] FIG. 47 represents the comparative ?/-linked oligosaccharide carbohydrate profiles for CTLA4^A29YLI04E-Ig molecules comprising SEQ ID NO:4. Four oligosaccharide domains are observed, wherein Domain I contains non-sialylated species, while Domains II, III, and IV contain mono-sialylated, di-sialylated and tri-sialylated species, respectively.

[00105] FIG. 48 is a graph of a capillary electrophoretic separation of CTLA4-Ig that was mixed 1:1 with CTLA4^A29YLI04E-Ig. The main peak migration times are approximately 0.8 minutes apart.

[00106] FIG. 49 is a chromatogram of hydrolyzed CTLA4^A29YL104E-Ig material , wherein a NANA Peak is observed at 3.4 minutes.

[00107] FIG. 50 depicts several of the various N-linked carbohydrate structures found in mammalian proteins. All chains share a common core structure containing two GlcNAc and three mannose residues.

[00108] FIG. 51 is graph depicting CTLA4-lg exposure (AUC) as a function of sialylation of the glycoprotein (NANA ratio).

[00109] FIG. 52 is graph depicting CTLA4-Ig exposure (AUC) as a function CTLA4-Ig’s carbohydrate profile. A large number of peaks were generated by anion exchange HPLC which were resolved into four or five domains. Domains 1 and 2 are largely asialylated and mono-sialylated structures, while domains 3 and 4 are largely di- and tri-sialylated structures.

[00110] Fig. 53 represents a tryptic peptide map of CTLA4^A29YL104E-Ig with peptides labeled. Table 56 corresponds with the labeled peptides.

[00111] FIG. 54 represents the comparative TV-linked oligosaccharide carbohydrate profiles for CTLA4-Ig molecules comprising SEQ ID NO:2. Four oligosaccharide domains are observed, wherein Domain I contains non-sialylated species, while Domains II, III, and IV contain mono-sialylated, di-sialylated and tri-sialylated species, respectively.

[00112] FIG. 55A-D is a graph that represents the oligosaccharide profiles of CTLA4-Ig and Peptides T5, T7, and T14 by HPAEC-PAD.

[00113] FIG. 56 is a graph depicting the labeled oligosaccharide profile of CTLA4-Ig obtained from PGC (Hypercarb) Column.

[00114] Fig. 57 depicts a graph of pharmacokinetic data showing monkey AUC on the Y axis and percent of N-linked glycosylation as shown in Domains I and 11 from a carbohydrate profile on the X axis. See methods of determining the N-linked carbohydrate profile in, for example, Examples 3, 44, 22 and 37. As the percentage of Domains I and II increases (and the percentage of Domains III, IV and V decreases), clearance increases. ־ Note that the negative control, the CTLA4-Ig with low sialic acid is cleared very rapidly. Note that the

CTLA4-Ig variant, LEA (CTLA4-Ig^A29YL104E-Ig) is included in this graph.

[00115] Fig. 58 A and 58 B depict a trace of a N-linked carbohydrate chromatogram of the N-linked carbohydrates released from CTA4-Ig (as obtained from methods such as those described in Examples 3, 44, 22 and 37). The trace in Fig. 58A is of from an analysis of CTLA4-Ig produced in a culture method without additional galactose added to the culture. The trace in Fig. 58 B did have galactose added to the culture. The percentages of N-linked carbohydrates in each Domain is shown in the inset table.

[00116] Fig. 59 depicts a trace of a N-linked carbohydrate chromatogram of the N-linked carbohydrates released from CTA4-Ig (as obtained from methods such as those described in Examples 3, 44, 22 and 37), This is from an analysis of CTLA4-Ig produced in a culture method with galactose added to the culture at day 8. This trace of from an analysis of CTLA4-Ig produced in a culture method without additional galactose added to the culture. The percentages of N-linked carbohydrates in each Domain is shown in the inset table.

[00117] Fig. 60 depicts a trace of a N-linked carbohydrate chromatogram of the N-linked carbohydrates released from CTA4-Ig (as obtained from methods such as those described in Examples 3, 44, 22 and 37). This is from an analysis of CTLA4-Ig produced in a culture method with galactose added to the culture at day 14. This trace of from an analysis of CTLA4-Ig produced in a culture method without additional galactose added to the culture. The percentages of N-linked carbohydrates in each Domain is shown in the inset table. ־

[00118] Fig. 61A and 61B depicts a trace of a N-linked carbohydrate chromatogram of the N-linked carbohydrates released from CTA4-Ig (as obtained from methods such as those described in Examples 3, 44, 22 and 37). Fig. 61A is from an analysis of CTLA4-Ig produced in a culture method without galactose added, and Fig. 61B is from an analysis where galactose was added to the culture at day 14. This trace of from an analysis of CTLA4-Ig produced in a culture method without additional galactose added to the culture. The percentages of N-linked carbohydrates in each Domain is shown in the inset table.

[00119] Fig. 62 depicts a trace of a N-linked carbohydrate chromatogram of the N-linked carbohydrates released from CTA4-Ig (as obtained from methods such as those described in Examples 3, 44, 22 and 37). ^,This trace was obtained from CTLA4-Ig material that was recovered from the wash step of the QFF column, producing a cut of CTLA4-Ig material with low sialic acid. The relative amount of Domain I and II is increased and Domains III and Iv are decreased, compared to the traces shown in Figures 61, 60 and 59. The percentages of Nlinked carbohydrates in each Domain is shown in the inset table.

[00120] FIG. 63 shows the tryptic peptide map of CTLA4-Ig indicating that T8 elutes at . the end of the solvent front, and T9 elutes at the shoulder of T27.

[00121] FIG. 64 is a graph that represents the full mass spectrum corresponding to glycopeptide T8 from CTLA4-Ig.

[00122] FIG. 65 is a graph that represents the full mass spectrum corresponding to glycopeptide T9 from CTLA4-Ig.

[00123] FIG. 66 is a graph that represents the MALDI-TOF data for the T9 peptide fragment from CTLA4-Ig.

[00124] FIG. 67A-B depicts ion chromatograms and mass spectra of oxidized and native tryptic peptides from CTLA4-Ig.

[00125] FIG. 68 depicts a typical N-Linked Oligosaccharide Profile (Domains I, II, III, IV and V, and Peaks 1A and IB within 5% of Lot averages). Peaks 1A, IB and 1C represent the asialo N-linked oligosaccharide structures of GO, G1 and G2. The data for the profile is in the table directly below. See Example 44.

<td></td><td> Peak Name</td><td> RT</td><td> Area</td><td> % Area</td>

<td> 1</td><td> Domain I</td><td> 19.413</td><td> 47807873</td><td> 31.3</td>

<td> 2</td><td> Domain II</td><td> 29.076</td><td> 50746179</td><td> 33.2</td>

<td> 3</td><td> Domain III</td><td> 42.819</td><td> 36640805</td><td> 24.0</td>

<td> 4</td><td> Domain V</td><td> 67.546</td><td> 3421324</td><td> 2.2</td>

<td> 5</td><td> Domain IV</td><td> 55.899</td><td> 14331509</td><td> 9.4</td>

<td> 6</td><td> Peak 1A</td><td> 19.413</td><td> 11115168</td><td> 7.3</td>

<td> 7</td><td> Peak IB</td><td> 20.290</td><td> 16331761</td><td> 10.7</td>

<td> 8</td><td> Peak 1C</td><td> 21.032</td><td> 13507144</td><td> 8.8</td>

<td> 9</td><td> Peak 2</td><td> 21.925</td><td> 4285.962</td><td> 2.8</td>

<td> 10</td><td></td><td> 22.685</td><td> 2567838</td><td> 1.7</td>

<td> 11</td><td></td><td> 29.076</td><td> 2808537</td><td> 1.8</td>

<td> 12</td><td></td><td> 30.763</td><td> 27989176</td><td> 18.3</td>

<td> 13</td><td></td><td> 31.577</td><td> 19948466</td><td> 13.0</td>

<td> 14</td><td></td><td> 42.819</td><td> 4555254</td><td> 3.0</td>

<td> 15</td><td> Peak 3</td><td> 43.823</td><td> 22213064</td><td> 14.5</td>

<td> 16</td><td></td><td> 46.626</td><td> 9872487</td><td> 6.5</td>

<td> 17</td><td></td><td> 55.899</td><td> 3898179</td><td> 2.5</td>

<td> 18</td><td> Peak 4</td><td> 57.368</td><td> 6789516</td><td> 4.4</td>

<td> 19</td><td></td><td> 60.333</td><td> 3643813</td><td> 2.4</td>

<td> 20</td><td></td><td> 67.546</td><td> 3421324</td><td> 2.2</td>

[00126]

[00127] FIG. 69 depicts an isoelectric focusing gel of CTLA4-Ig. The bands are characterized by:

<td> Lane</td><td> Description</td><td> Protein Load (micrograms)</td><td> Band No.</td><td> Cumulative % Band Intensity</td><td> Relative Band Percent (%)</td>

<td> 1</td><td> IEF Markers</td><td> NA</td><td> NA</td><td> NA</td><td> NA</td>

<td> 2</td><td> CTLA4-Ig material</td><td> 20</td><td> 16</td><td> 100</td><td> NA</td>

<td> 3</td><td> CTLA4-IgDrug Substance</td><td> 20</td><td> 16</td><td> 100</td><td> 100</td>

<td> 4</td><td> Staining Control</td><td> 1</td><td> NA</td><td> NA</td><td> NA</td>

[00128]

[00129] Fig. 70 depicts a representative isoelectric focusing gel quantitative analysis report of CTLA4-Ig. The quantitation of the gel was performed and the data is as follows:

<td colspan="3"> IEF Marker</td><td colspan="3"> Mi j aauu 1-2uu3-015</td><td colspan="3"> BQC060082 (DS ABC04014)</td><td colspan="3"> Staining Control</td>

<td> Lane 1</td><td> Lane 1</td><td> Lane I</td><td> Lane 2</td><td> Lane 2</td><td> Lane 2</td><td> Lane 3</td><td> Lane 3</td><td> Lane 3</td><td> Lane. 4</td><td> Lane 4</td><td> Lane 4</td>

<td> Band</td><td> Band %</td><td> Pl</td><td> Band</td><td> Band %</td><td> Pl</td><td> Band</td><td> Band %</td><td> Pl</td><td> Band</td><td> Band %</td><td> Pl</td>

<td> 1</td><td> 20.95</td><td> 5.85</td><td> 1</td><td> 0.51</td><td> 5.28</td><td> 1</td><td> 1.34</td><td> 5.26</td><td> 1</td><td> too</td><td> 5.68</td>

<td> 2</td><td> 22.39</td><td> 5.2</td><td> 2</td><td> 0.17</td><td> 5.25</td><td> 2</td><td> 2.34</td><td> 5.19</td><td></td><td></td><td></td>

<td> 3</td><td> 9.62</td><td> 4.55</td><td> 3</td><td> 0.71</td><td> 5.23</td><td> 3</td><td> 0.99</td><td> 5.17</td><td></td><td></td><td></td>

<td> 4</td><td> 47.04</td><td> 3.5</td><td> 4</td><td> 1.61</td><td> 5.2</td><td> 4</td><td> 3.2</td><td> 5.14</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 5</td><td> 1.15</td><td> 5.18</td><td> 5</td><td> 3.23</td><td> 5.11</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 6</td><td> 7.44</td><td> 5.15</td><td> 6</td><td> 3.41</td><td> 5.08</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> ר</td><td> 10.96 .</td><td> .5.09</td><td> 7</td><td> 10.29.</td><td> 5.03</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 8</td><td> 9.32</td><td> 5.02</td><td> 8</td><td> 5.95</td><td> 5.01</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 9</td><td> 18.22</td><td> 4.94</td><td> 9</td><td> 5.66</td><td> 4.97</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 10</td><td> 5.52</td><td> 4.91</td><td> 10</td><td> 13.4</td><td> . 4.91</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> II</td><td> 4.19</td><td> 4.88</td><td> 11</td><td> 17.11</td><td> 4.85</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 12</td><td> 22.58</td><td> 4.82</td><td> 12</td><td> 7.39</td><td> 4.8</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 13</td><td> 3.8</td><td> . 4.67</td><td> 13</td><td> .16.03</td><td> 4.72</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 14</td><td> 10.02</td><td> 4.63</td><td> 14</td><td> 3.88</td><td> 4.56</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 15</td><td> 3.44</td><td> 4.55</td><td> 15</td><td> ' 4.97</td><td> 4.52 '</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 16</td><td> 0.35</td><td> 4.48</td><td> 16</td><td> 0.8</td><td> 4.45</td><td></td><td></td><td></td>

<td></td><td></td><td colspan="2"> Bands (4.3-5.6)</td><td> 16</td><td colspan="2"> Bands (4.3-5.6)</td><td> 16</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td colspan="2"> % Bands (4.3-5.3)</td><td> .100</td><td colspan="2"> % Bands (4.3-5.3)</td><td> 100</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td colspan="3"> Sample Relative Percent (%)</td><td> 100</td><td></td><td></td><td></td><td></td><td></td><td></td>

Sample Relative Percent (%) = (Sample %Band Intensity/Ref %Band Intensity) x 100 NOTE: For the pl range of 4.3 to 5.3.

[00130] Fig. 71A depicts Typical 20 pL Injection of System Suitability Standard on TOSO HAAS 3000 SWXL Column Equipped with a Guard Column. Fig. 71B depicts a 20 pL Injection of CTLA4-Ig Reference Material on TOSO HAAS 3000 SWXL Column Equipped with a Guard Column.

[00131] Fig. 72 - Example of digitally acquired image of SDS-PAGE Analysis of

CTLA4-Ig by Coomassie Blue stained Polyacrylamide (4-20 ) Gel Electrophoresis

<td> Lane</td><td> Description</td><td> Protein Load (micro grams)</td><td> Non-Reduced (NR)/Reduced (R) Condition</td><td> Percent (%) Band Intensity</td>

<td> 1</td><td> CTLA4-Ig drug substance</td><td> 10</td><td> NR</td><td> 100</td>

<td> 2</td><td> CTLA4-Ig material</td><td> 10</td><td> NR</td><td> 99</td>

<td> 3</td><td> Blank</td><td> NA</td><td> NR</td><td> NA</td>

<td> 4</td><td> CTLA4-Ig drug substance</td><td> 10</td><td> R</td><td> 100</td>

<td> י 5</td><td> CTLA4-Ig material '.</td><td> 10</td><td> R</td><td> 100</td>

<td> 6</td><td> Molecular Weight Marker</td><td> NA</td><td> NA</td><td> NA</td>

<td> 7</td><td> • CTLA4-Ig drug product</td><td> 10</td><td> NR</td><td> 99</td>

<td> 8</td><td> CTLA4-Ig material</td><td> 10</td><td> NR</td><td> 100</td>

<td> 9</td><td> Blank</td><td> NA</td><td> NR</td><td> NA</td>

<td> 10</td><td> CTLA4-Ig drug product</td><td> 10</td><td> R</td><td> 99</td>

<td> 11</td><td> CTLA4-Ig material</td><td> 10</td><td> R</td><td> 99</td>

<td> 12</td><td> Trypsin Inhibitor Staining Control</td><td> NA</td><td> NA</td><td> NA</td>

[00132] Fig. 73 depicts an example of quantitative analysis report for Coomassie Blue stained SDS-PAGE.

[00133] Fig. 74 shows a table setting out the quantitative analysis of the stained SDSPAGE gel in Fig. 73.

[00134] Fig. 75 depicts Example of an enhanced image of SDS-PAGE Analysis of CTLA4-Ig Coomassie Blue Stained Gel for illustrating the migrating positions of the major and expected minor bands relative to the Molecular Weight Markers.

[00135] Fig. 76 is a depiction of a representative N-Linked Carbohydrate Profile of CTLA4-Ig reference / standard material. This is a representative carbohydrate profile of run on the Waters system. Retention times are system dependent.

[00136] Fig. 77 is a depiction of a representative Stachyose System Suitability Chromatogram.

[00137] Fig. 78 is a trace of a Representative Chromatogram of Hydrolyzed CTLA4-Ig Material.

[00138] Fig. 79 depicts a ccanned gel image of SDS- PAGE Analysis of

CTLA4^A29YL104E-Ig Coomassie Blue Stained Polyacrylamide (4---20%) Gel Electrophoresis

Coomassie Blue Staining.

[00139f

<td> Lane#</td><td> Description</td><td> Protein Load (pg)</td><td> Condition R/NR</td><td> % Purity (%)</td>

<td> 1</td><td> CTLA4^A29YL'^lu4b-Ig Drug Substance</td><td> 10</td><td> NR</td><td> 99</td>

<td> 2</td><td> CTLA4^A29YL104E-Ig Reference Material</td><td> 10</td><td> NR</td><td> 99</td>

<td> 3</td><td> Blank</td><td> NA</td><td> - NR</td><td> NA</td>

<td> 4</td><td> CTLA4^A29YL104E-Ig Drug Substance</td><td> 10</td><td> R</td><td> 100</td>

<td> 5</td><td> CTLA4^A29YL104E-Ig Reference Material.</td><td> 10</td><td> R.</td><td> 100</td>

<td> 6</td><td> Molecular Weight Marker</td><td> NA</td><td> R</td><td> NA</td>

<td> ר</td><td> CTLA4^A29YL'^04E-Ig Reference Material</td><td> 10</td><td> R</td><td> 99</td>

<td> . 8</td><td> CTLA4^A29YL104E-Ig Drug Product</td><td> 10</td><td> R</td><td> 99</td>

<td> 9 .</td><td> Blank</td><td> 0</td><td> NR</td><td> 0</td>

<td> 10</td><td> CTLA4^A29YL104E-Ig Reference Material</td><td> 10</td><td> NR</td><td> 100</td>

<td> 11</td><td> CTLA4^A29YL104E-Ig Drug Product</td><td> 10</td><td> NR</td><td> 100</td>

<td> 12</td><td> Staining control</td><td> ____________0.1___________</td><td> NR</td><td> 100</td>

[00140] Figure 80 depicts a flow diagram of the harvest steps, see Example 28.

[00141] Figure 81 depicts an electropherogram of system suitability of amino monosaccharides. See Example 16.

[00142] Figure 82 depicts a graph of pharmacokinetic data showing monkey AUC on the Y axis and percent of N-linked glycosylation as shown in Domains I and II from a carbohydrate profile on the X axis. See methods of determining the N-linked carbohydrate profile in, for example, Examples 3,44, 22 and 37. As the percentage of Domains I and II increases (and the percentage of Domains III, IV and V decreases), AUC increases. Note that the negative control, the CTLA4-Ig with low sialic acid is cleared very rapidly. Note that the mutant CTLA4-Ig molecules, CTLA4-Ig^A29YLI04E-Ig (designated LEA) is included in this graph.

[00143] Figure 83 depicts a graph of pharmacokinetic data showing AUC on the Y axis and percent of N-linked glycosylation as shown in Domains III and IV (as determined from an N-linked carbohydrate profile) on the X-axis. As the percentage of Domains III and

IV increase, the AUC increases. Note that the negative control, the CTLA4-Ig with low sialic acid is cleared very rapidly. See methods of determining the N-linked carbohydrate profile in, for example, Examples 3, 44, 22 and 37. Note that the mutant CTLA4-Ig molecules, .

CTLA4-Ig^A29YL104E-Ig (designated LEA) is included in this graph.

[00144] Figure 84 depicts another graph of pharmacokinetic data showing AUC on the

Y axis and percent of N-linked glycosylation as shown in Domains III and IV from a carbohydrate profile on the X-axis. As the percentage of Domains III and IV increase, the AUC increases. Note that the negative control, the CTLA4-Ig with low sialic acid is cleared very rapidly. See methods of determining the N-linked carbohydrate profile in, for example, Examples 3, 44, 22 and 37. Note that the mutant CTLA4-Ig molecules, CTLA4-Ig^A29YLI04EIg (designated LEA) is included in this graph.

[00145] Fig. 85 depicts a tryptic Map of CTLA4-Ig standard see Table at end of Example 65 for peak assignments. The small peak labeled Tl+A is the T1 tryptic peptide extended by an N-terminal alanine residue. The small peak labeled T31+K is the T31 tryptic peptide extended by a C-terminal lysine residue.

[00146] Fig. 86 depicts an overlay of 280 nm Data for Tryptic Map of CTLA4-Ig Standard Plus Same Spiked with 5 mole % of T6ox Indicator Peptide, Met(O) 85 (84-93). See Example 65.

[00147] Fig. 87 depicts an expanded View of 215 nm Data for Tryptic Map of CTLA4-Ig Standard Plus Same Spiked with 5 mole% of T26deaml Indicator Peptide, isoAsp294(281-302). See Example 65.

DETAILED DESCRIPTION OF THE INVENTION

[00148] CTLA4-Ig molecules can be used to treat a variety of disorders, including disorders relating to aberrant immunoproliferative and immunoreactive phemonena such as autoimmunity and allergy. The invention provides CTLA4-lg compositions that comprise, for example, populations of CTLA4-Ig molecules having particular glycosylation modifications, having particular carbohydrate profiles or characteristics, having particular multimeric structures, and/or having particular avidity strengths. Documents that are hereby incorporated by reference in their entirety that also describe CTLA4-Ig molecules, uses and methods thereof, include U.S. Patent No. 5,434,131; 5,851,795; 5,885,796; 5,885,579; and

7,094,874.

[00149] The invention also provides cell lines that are capable of producing large amounts of CTLA4-Ig molecules via the mass-production and culturing methods provided herein.

One particular cell line of the invention is a clonal cell line that can be used to mass-produce CTLA4-Ig molecules such that it has a particular glycosylation and carbohydrate profile. As compared to the heterogeneous and non-clonal cell population having ATCC Accession No. 68629 (see U.S. Patent No. 5,434,131, which is hereby incorporated by.reference in its entirety), the clonal cell lines of the invention can secrete a population of CTLA4-Ig molecules having a more consistent or more uniform glycosylation .or.carbohydrate profile. Further, as compared to the heterogeneous and non-clonal cell population having ATCC Accession No. 68629, the clonal cell lines of the invention can secrete a greater amount of CTLA4-Ig molecules, in part because the present clonal cell lines are selected to have a highcopy number of CTLA4-Ig expression cassettes integrated into a single site in the genome of the cell. ..

[00150] The invention provides for the discovery that the avidity and potency of CTLA4Ig (SEQ ID NO:2) can be increased by making two amino acid substitutions in the B7 binding region of the CTLA-4 binding domain: (i) alanine at position 29 of SEQ ID NO:2 is substituted by tyrosine (A29Y), and (ii) lysine at position 104 of SEQ ID NO:2 is substituted by glutamate (L104E). The invention provides a subgenus of CTLA4-Ig molecules, called “beta polypeptide molecules,” which comprise beta polypeptides which have B7 binding activity and may comprise the amino acid sequence in SEQ ID NO: 24 (CTLA4 extracellular domain with A29Y and L104E mutations), linked to an immunoglobulin constant region, or portion thereof.

[SEQ ID NO: 24]

MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI YVIDPEPCPDSD

[SEQ ID NO: 18]- A CTLA4 extracellular domain

MHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG

NELTFLDDSICTGTSSGNQVNLTIQGLRAMDT.GLYICKVELMYPPPYYLGIGNGTQ1

YVIDPEPCPDSD

[00151] Terms

[00152] As used herein, the term “clonal” refers to a cell. population that is expanded from a single cell. With respect to a clonal cell line or clonal cell population capable of expressing a CTLA4-Ig molecule, the clonal cell line or population is expanded from a single cell that was isolated from a population of cells that were transfected with an expression vector encoding the CTLA4-Ig molecule. The transfected population of cells can be a heterogeneous population. A clonal cell line or population can be considered to be homogeneous in the sense that all of the cells in the population came from a single transfectant.

[00153] As used herein, the term “B7-1” refers to CD80; the term “B7-2” refers CD86; and the term “B7” refers to either or both of B7-1 and B7-2 (CD80 and CD86). The term “B7-1-Ig” or “B7-lIg” refers to CD80-Ig; the term “B7-2-Ig”or “B7-2Ig” refers CD86-Ig.

[00154] As used herein, the terms “CTLA4-Ig” or “CTLA4-Ig molecule” or “CTLA4Ig molecule” or “CTLA4-Ig protein” or “CTLA4Ig.protein” are used interchangeably, and refer to a protein molecule that comprises at least a CTLA4-Ig polypeptide having a CTLA4 extracellular domain and an immunoglobulin constant region or portion thereof. In some embodiments, for example, a CTLA4-Ig polypeptide comprises at least the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the CTLA4 extracellular domain and the immunoglobulin constant region or portion thereof can be wild-type, or mutant or modified. A mutant CTLA4-Ig polypeptide is a CTLA4-Ig polypeptide comprising a mutant CTLA4 extracellular domain. A mutant CTLA4Ig molecule comprises at least a mutant CTLA4-Ig polypeptide. In some embodiments, the CTLA4 extracellular domain and the immunoglobulin constant region or portion thereof can be mammalian, including human or mouse. In some embodiments, a mutant CTLA4 extracellular domain can have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the CTLA4 extracellular domain shown in Figure 1 or SEQ ID NO:18. In some embodiments, a mutant immunoglobulin constant region or portion thereof can have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the immunoglobulin (g) constant region as shown in Figure 1. The polypeptide can further comprise additional protein domains. A CTLA4-Ig molecule can refer to a monomer of the CTLA4-Ig polypeptide, and also can refer to multimer forms of the polypeptide, such as dimers, tetramers, and hexamers, etc. (or other high molecular weight species). CTLA4-Ig molecules are also capable of binding to CD80 and/or CD86. CTLA4Ig molecules include mutant CTLA4Ig molecules, such as “beta polypeptides molecules,”

e.g., CTLA4^A29YL104E-Ig. For example, CTLA4-Ig comprises CTLA4-Ig molecules, and CTLA4^A29YLI04E-Ig comprises beta polypeptides molecules (an example of mutant CTLA4-Ig molecules).

[00155] As used herein, the term “CTLA4 extracellular domain” refers to a protein domain comprising all or a portion of the amino acid sequence shown in SEQ ID NO: 18, that binds to B7-1 (CD80) and/or B7-2 (CD86). In some embodiments, a CTLA4 extracellular domain can comprise a polypeptide having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 27-150 of SEQ ID NO:2, which are the same as amino acids shown SEQ ID NO: 18. The amino acid 151 ofSEQ ID NO:2 is a junction amino acid.

[00156] As used herein, the term “beta polypeptide” refers to a mutant CTLA4-Ig polypeptide that (1) comprises the amino acid sequence of SEQ ID NO: 18 wherein the amino acid at position 29 is mutated to tyrosine and the amino acid at position 104 is mutated to glutamate, optionally with various additional mutations, and an immunoglobulin constant region, or a portion thereof; and (2) is capable of binding to CD80 and/or CD86. In some embodiments, for example, a beta polypeptide comprises at least the amino acid sequence of the extracellular domain of CTLA4^^29YEI04E-Ig (as shown in SEQ ID NO:24). Non-limiting examples of beta polypeptides include belatacept and SEQ ID NOS: 4 and 11-16. In certain embodiments, the immunoglobulin constant region or portion thereof can be wild-type, or mutant or modified. In certain embodiments, the immunoglobulin constant region or portion thereof can be mammalian, including human or mouse. Additional non-limiting examples of beta polypeptides include a beta polypeptide comprising one or more amino acid mutations in the immunoglobulin constant region or portion thereof (for example, substitution of cysteine 120 ofSEQ ID NO:4), and a beta polypeptide comprising further mutations at one or more of amino acid position 25, 30, 93, 96, 103 or 105 of SEQ ID NO: 18. A beta polypeptide molecule comprises a beta polypeptide. A beta polypeptide molecule can refer to a monomer of the beta polypeptide and smultimer forms of the beta polypeptide, such as dimers,, tetramers and hexamers, etc. For example, belatacept comprises beta polypeptide molecules.

Beta polypeptide molecules are further described in U.S. Provisional Application No.

60/849,543 filed on October 5, 2006, which is hereby incorporated by reference in its .

entirety.

[00157] As used herein, the terms “glutamate” and “glutamic acid” are used interchangeably.

[00158] As used herein, the term “dimer” refers to a CTLA4-Ig protein or CTLA4-Ig molecule composed of two CTLA4-Ig polypeptides or monomers linked or joined together. The linkage between monomers of a dimer can be a non-covalent linkage or interaction, a covalent linkage or interaction, or both. An example of a CTLA4-Ig dimer is shown in Figure 4. A CTLA4-Ig protein or CTLA4-Ig molecule composed of two identical monomers is a homodimer. A CTLA4-Ig homodimer also encompasses a molecule comprising two monomers’that may differ slightly in sequence. A homodimer encompasses a dimer where the monomers joined together have substantially the same sequence. The monomers comprising a homodimer share considerable structural homology. For example, the differences in sequence may be due to N-termal processing modifications of the monomer.

[00159] As used herein, “conservative mutation” refers to a change in a nucleic acid sequence that substitutes one amino acid for another of the same class (e.g., substitution of one nonpolar amino acid for another, such as isoleucine, valine, leucine, or methionine; or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).

[00160] As used herein, “non-conservative mutation” refers to a change in a nucleic acid sequence that substitutes one amino acid for another of a different class (e.g., substitution of one basic amino acid, such as lysine, arginine or histidine, with an acidic amino acid, such as aspartic acid or glutamic acid). For example, an amino acid can be biochemically dissimilar from another amino acid based on size, charge, polarity, reactivity or other such ' characteristics of amino acids.

[00161] As used herein, “isolated” refers to a molecule that is taken out of its native environment and is in an environment different from that in which the molecule naturally occurs, or a substance (e.g., a protein) that is partially or completely recovered or separated from other components of its environment such that the substance (e.g., protein) is the predominant species (e.g., protein species) present in the resultant composition, mixture, or collection of components (for example, on a molar basis it is more abundant than any other individual species in the composition). For example, a preparation may consist of more than about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or or 95%, of isolated CTLA4-Ig. “Isolated” does not exclude mixtures of CTLA4-Ig molecules with other CTLA4-Ig molecules from the environment in which the molecule naturally occurs. “Isolated” does not exclude pharmaceutically acceptable excipients combined .with CTLA4-Ig, wherein the CTLA4-Ig has been recovered from its environment* such as a cell culture, a batch culture, or a bioreactor, etc. As used herein, “isolating” refers to carrying out a process or method to obtain an isolated CTLA4-Ig molecule.

[00162] As used herein, the term “soluble CTLA4” means a molecule that can circulate in vivo or CTLA4 which is not bound to a cell membrane. For example, the soluble CTLA4 can include CTLA4-lg which includes the extracellular region of CTLA4, linked to an Ig.

[00163], As used herein, the term “soluble fraction of a cell culture” refers to the liquid portion of a cell culture other than, or which is substantially free of, insoluble, particulate or solid components of the cell culture, such as cells, cell membranes and nuclei. The soluble fraction may be, for example, the resulting supernatant following centrifugation of the cell culture, or the resulting filtrate following filtration of the cell culture.

[00164] As used herein, the term “expression cassette” refers to a nucleic acid having at least a 5׳ regulatory region (e.g., promoter) operably linked to a nucleotide sequence that encodes a polypeptide, and optionally an untranslated 3^f termination region (e.g., stop codon and polyadenylation sequence). Under appropriate conditions, a polypeptide encoded by an expression cassette is produced by the expression cassette. An expression cassette may also have one or more nucleotide sequences that target integration of the expression cassette into a specific site in the genome of a host cell (for example, see Koduri et al., (2001) Gene 280:8795). For example, a CTLA4^A29YLI04E-Ig polypeptide expression cassette derived from a plasmid deposited as ATCC Accession No. PTA-2104, is one example of an expression cassette encoding a CTLA4 -1g.

[00165] As used herein, the term “substantially purified” refers to a composition comprising a CTLA4-Ig molecule or a selected population of CTLA4-Ig molecules that is removed from its natural environment (e.g., is isolated) and is at least 90% free, 91 /0 free, 92% free, 93% free, 94% free, 95% free, 96% free, 97% free, 98% free, 99% free, 99.5% free, or 99.9% free from other components, such as cellular material or culture medium, with which it is naturally associated. For example, with respect to a recombinantly produced . CTLA4-Ig protein molecule, the term “substantially purified” can also refer to.a composition comprising a CTLA4-Ig protein molecule that is removed from the production environment such that the protein molecule is at least 90% free, 91% free, 92% free, 93% free, 94/0 free, 95% free, 96% free, 97% free, 98% free, 99% free, 99.5% free, or 99.9% free from protein molecules which are not polypeptides.of SEQ ID NO: 2 or mutant polypeptides of SEQ ID NO: 2 which are of interest. “Substantially purified” does not exclude mixtures of CTLA4-Ig molecules (such as dimers) with other CTLA4-Ig molecules (such as tetramer).

“Substantially purified” does not exclude pharmaceutically acceptable excipients or carriers combined with CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have been taken out of their native environment.

[00166] As used herein, the term “large-scale process” is used interchangeably with the term “industrial-scale process”. The term “culture vessel is used interchangeably with “bioreactor”, “reactor” and “tank”.

[00167] A “liquid culture” refers to cells (for example, bacteria, plant, insect, yeast, or animal cells) grown on supports, or growing suspended in a liquid nutrient medium.

[00168] A “seed culture” refers to a cell culture grown in order to be used to inoculate larger volumes of culture medium. The seed culture can be used to inoculate larger volumes of media in order to expand the number of cells growing in the culture (for example, cells grown in suspension).

[00169] As used herein, “culturing” refers to growing one or more cells in vitro under defined or controlled conditions. Examples of culturing conditions which can be defined include temperature, gas mixture, time; and medium formulation

[00170] As used herein, “expanding” refers to culturing one or more cells in vitro for the purpose of obtaining a larger number of cells in the culture.

[00171] As used herein, “population” refers to a group of two or more molecules (“population of molecules”) or cells (“population of cells”) that are characterized by the presence or absence of one or more measurable or detectable properties. In a homogeneous population, the molecules or cells in the population are characterized by the same or substantially the same properties (for example, the cells of a clonal cell line). In a heterogeneous population,, the molecules or cells in the population are characterized by at least one property that is the same or substantially the same, where the cells or molecules may also exhibit properties that are not the same (for example, a population of CTLA4-Ig . molecules having a substantially similar average sialic content, but having non-similar . mannose content).

[00172] As used herein, “high molecular weight aggregate” is used interchangeably with “high molecular weight species” to refer to a CTLA4-lg molecule comprising at least three CTLA4-Ig monomers. For example, a high molecular weight aggregate may be a tetramer, a pentamer or a hexamer.

[00173] “Percent (%) yield” refers to the actual yield divided by the theoretical yield, and that value multipled by 100. The actual yield can be given as the weight in gram or in mol (for example, a molar yield). The theoretical yield can be given as the ideal or mathematically calculated yield.

[00174] As used herein, an amount of MCP-1 refers to (1) an amount of MCP-1 (Monocyte chemotactic protein-1, especially, hamster MCP-1) alone, or (2) an amount of MCP-1 like protein, wherein MCP-1 like protein includes MCP-1, together with proteins homologous to MCP-1, fragments of MCP-1, and/or fragments of proteins homologous to MCP-1 (for example, in each of the aforementioned instances, as may be cross-reactive with an antibody (e.g., polyclonal ELISA) assay for the detection of MCP-1). The absence of MCP-I (and/or proteins homologous to MCP-1, fragments of MCP-1, and/or fragments of proteins homologous to MCP-1) is contemplated where no lower limit is provided with regard to a range of amounts of MCP-1.

[00175] As used herein, “glycosylation content” refers to an amount of N-linked or Olinked sugar residues covalently attached to a protein molecule, such as a glycoprotein like a CTLA4-Ig molecule.

[00176] As used herein, the term “molar ratio of sialic acids to protein” is calculated and given as number of moles of sialic acid molecules per moles of protein (CTLA4-Ig molecules) or dimer.

[00177] As used herein, the term “glycoprotein” refers to a protein that is modified by the addition of one or more carbohydrates, including the addition of one or more sugar residues.

[00178] As used herein, the term “sialylation” refers to the addition of a sialic acid residue to a protein, including a glycoprotein.

[00179] As used herein, the term “glycoprotein isoform” refers to a molecule characterized by its carbohydrate and sialic acid content as determined by isoelectric focusing (IEF) gel electrophoresis or other suitable methods for distinguishing different proteins in a mixture by their molecular weight, charge, and/or other characteristics. For example, each distinct band observed on an IEF gel represents molecules that have a particular isoelectric point (pl) and thus the same net overall charge. A glycoprotein isoform can be a distinct band observed on an IEF gel where each band can be a population of molecules that have a particular pl.

[00180] “Immune tolerance” refers to a state of unresponsiveness to a specific antigen or group of antigens to which a person is normally responsive (for example, a state in which a T cell can no longer respond to antigen).

[00181] “Potency” refers to a measure of the response as a function of ligand concentration. For example, agonist potency is quantified as the concentration of ligand that produces half the maximal effect (EC50). A non-limiting pharmacological definition of potency includes components of affinity and efficacy, where, efficacy is the ability of a drug to evoke a response once bound. Potency is related to affinity, but potency and affinity are different measures of drug action.

[00182] As used herein, pharmaceutically acceptable carrier refers to a vehicle for a pharmacologically active agent. The carrier facilitates delivery of the active agent to the target site without terminating the function of the agent. Non-limiting examples of suitable

ו forms of the carrier include solutions, creams, gels, gel emulsions, jellies, pastes, lotions, salves, sprays, ointments, powders, solid admixtures, aerosols, emulsions (e.g., water in oil or oil in water), gel aqueous solutions, aqueous solutions, suspensions, liniments, tinctures, and patches suitable for topical administration.

[00183] As used herein, the phrase “pharmaceutically acceptable composition” (or pharmaceutical composition) refers to a composition that.is acceptable for pharmaceutical administration, such as to a human being. Such a composition can include substances that are impurities at a level not exceeding an acceptable level for pharmaceutical administration (such level including an absence of such impurities), and can include pharmaceutically acceptable excipients, vehicles, carriers and other inactive ingredients, for example, to formulate such composition for ease of administration, in addition to any active agent(s). For example, a pharmaceutically acceptable CTLA4-Ig composition can include MCP-1 or DNA, so long as those substances are at a level acceptable for administration to humans.

[00184] “Drug substance is the active pharmaceutical ingredient contained in a pharmaceutical composition. The term “drug substance” includes an active pharmaceutical ingredient in solution and/or in buffered form. Drug product is a pharmaceutical composition containing drug substance formulated for pharmaceutical administration. For purposes of the assays contained in the Examples and elsewhere herein, which may refer to drug substance and/or drug product, exemplary drug substances and drug products that may be assayed are as follows.

[00185] Exemplary drug substance for CTLA4Ig molecules comprising SEQ ID NO:s 2, 5, 6, 7, 8, 9, 10 or 18 isCLTA4-Ig protein at a concentration of 50 mg/ml, in a buffered aqueous solution (25 mM sodium phosphate, 50mM sodium chloride, pH of 7.5). ־

[00186] Exemplary drug product for CTLA4Ig molecules comprising SEQ ID NO:s 2, 5, 6, 7, 8, 9, 10 or 18 is, 250 mg lyophilized CTLA4-Ig protein, 500 mg maltose, 17.2 mg monobasic sodium phosphate, and 14.6 mg sodium chloride, pH 7.0-8.0; or

Composition of lyophilized CTLA4-Ig protein (250mg/vial) drug product

<td> Component</td><td> Amount (mg/vial)^a</td>

<td> CTLA4-Ig protein</td><td> 262.5</td>

<td> Maltose monohydrate</td><td> 525</td>

<td> Sodium phosphate monobasic, monohydrate”</td><td> 18.1</td>

<td> Sodium chloride</td><td> 15.3</td>

<td> Hydrochloric Acid</td><td> Adjust to pH 7.5</td>

<td> Sodium hydroxide</td><td> Adjust to pH 7.5</td>

buffered aqueous solution (25 mM sodium phosphate, •1 OmM sodium chloride, pH of 7.5).

Exemplary drug product for CTLA4Ig molecules comprising SEQ ID NO:s 4, 11, 12, 13, 14, 15, 16, or 24:

Composition of lyophilized CLTA4^A29VL104E-Ig lOOmg/vial drug product

<td> Component___________________ CLTA4^A29YL1U4E-Ig</td><td> Amount/Vial (mg) 110</td>

<td> Sucrose</td><td> 220</td>

<td> Sodium Phosphate Monobasic Monohydrate</td><td> 15.18</td>

<td> Sodium Chloride</td><td> 2.55</td>

<td> IN Sodium Hydroxide</td><td> Adjust to pH 7.5</td>

<td> IN Hydrochloric Acid</td><td> Adjust to pH 7.5</td>

[00188]

[00189] As used herein, the terms “culture medium” and “cell culture medium” and “feed medium” and “fermentation medium” refer to a nutrient solutions used for growing and or maintaining cells, especially mammalian cells. Without limitation, these solutions ordinarily provide at least one component from one or more׳ of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range. The nutrient solution can be supplemented electively with one or more components from any of the following categories: (1) hormones and other growth factors such as, serum, insulin, transferrin, and epidermal growth factor; (2) salts, for example, magnesium; calcium, and phosphate; .(3) buffers, such as HEPES; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (6) antibiotics, such as gentamycin; (7) cell protective agents, for example pluronic polyol; and (8) galactose.

[00190] The term “inoculation” as used herein refers to the addition of cells to culture medium to start the culture.

[00191] The term “growth phase” of the cell culture as used herein refers to the period of exponential cell growth (for example, the log phase) where cells are primarily dividing rapidly. During this phase, the rate of increase in the density of viable cells is higher thari at any other time point.

[00192] As used herein, the term “production phase” of the cell culture refers to the period of time during which cell growth is stationary or is maintained at a near constant level. The density of viable cells remains approximately constant over a given period of time.

Logarithmic cell growth has terminated and protein production is the primary activity during the production phase. ^,The medium at this time is generally supplemented to support continued protein production and to achieve the desired glycoprotein product.

[00193] As used herein, the terms expression or expresses are used to refer to transcription and translation occurring within a cell. The level of expression of a product gene in a host cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the product gene that is produced by the cell, or both.

[00194] As used herein, “glycosylation” refers to the addition of complex oligosaccharide structures to a protein at specific sites within the polypeptide chain. Glycosylation of proteins and the subsequent processing of the added carbohydrates can affect protein folding and structure, protein stability, including protein half life, and functional properties of a protein. Protein glycosylation can be divided into two classes by virtue of the sequence context where the modification occurs, O-linked glycosylation and N-linked glycosylation. O-linked polysaccharides are linked to a hydroxyl group, usually to the hydroxyl group of either a serine or a threonine residue. O-glycans are not added to every serine and threonine residue. O-linked oligosaccharides are usually mono or biantennary, i.e. they comprise one or at most two branches (antennas), and comprise from one to four different kinds of sugar residues, which are added one by ofle. N-linked polysaccharides are attached to the amide nitrogen of an asparagine. Only asparagines that are part of orie of two tripeptide sequences, either asparagine-X-serine or asparagine-X-threonine (where X is any amino acid except, proline), are targets for glycosylation. N-linked oligosaccharides can have from one to four branches referred to as mono-, bi-, tri- tetraantennary. The structures of and sugar residues found in N- and O-linked oligosaccharides are different. Despite that difference, the terminal residue on each branch of both N- and O-linked polysaccharide can be modified by a sialic acid molecule a modification referred as sialic acids capping: Sialic acid is a common name for a family of unique nine-carbon monosaccharides, which can be linked to other oligosaccharides. Two family members are N-acetyl neuraminic acid, abbreviated as Neu5Ac or NANA, and N-glycolyl neuraminic acid, abbreviated as Neu5Gc orNGNA. The most common form of sialic acid in humans is NANA. N-acetylneuraminic acid (NANA) is the primary sialic acid species present in CTLA4-Ig molecules. However, it should be noted that minor but detectable levels of N glycolylneuraminic acid (NGNA) are also present in CTLA4-Ig molecules. Furthermore, the method described herein can be used to determine the number of moles of sialic acids for both NANA and NGNA, and therefore levels of both NANA and NGNA are determined and reported for CTLA4-Ig molecules. N- and O-linked oligosaccharides have different number of branches, which provide different number of ' positions to which sialic acid molecules can be attached. N-linked ologosaccharides can provide up to four attachment positions for sialic acids, while O-linked oligosaccharides can provide two sites for sialic acid attachment.

[00195] As used herein, the term “large-scale process” can be used interchangeably with the term “industrial-scale process”. Furthermore, the term “culture vessel” can be used interchangeably with “bioreactor”, “reactor” and “tank”.

[00196] As used herein, the phrase “working solution(s)” refers to solutions that are used in a method. Non-limiting examples of working solutions include buffers.

[00197] As used herein, “reference material” refers to a material that is used as a standard in a method. For example,a reference material can be used as a standard to which experimental samples will be compared.

[00198] The absence of a substance is contemplated where no lower limit is provided with regard to a range of amounts of such substance.

[00199] As used herein, recited temperatures in reference to cell culture refers to the temperature setting on the instrument that regulates the temperature of the bioreactor. Of course, the temperature of the liquid culture itself will adopt the temperature set on the instrument regulating the temperature for the bioreactor. Where the temperature refers to a cell culture that is maintained on a shelf in an incubator, the temperature then refers to the shelf temperature of the incubator.

[00200] Non-limiting emobidments of the invention:

[00201] The invention provides for compositions of CTLA4-Ig molecules and compositions of mutant CTLA4-Ig molecules, such as CTLA4^A29YL104E-Ig. The invention provides for compositions with certain characteristics, such as certain amounts of bacterial endotoxin, bioburden, a pl within a certain range (or certain IEF bands within a pl of a certain range), a certain amount of monomer (single chain), dimer or high molecular weight species (such as tetramer), a certain tryptic peptide profile, a certain set of major bands on SDSPAGE, a certain DNA content, an amount of MCP-1 not exceeding a certain maximum, an amount of cell protein not exceeding a certain maximum, an amount of Triton X-l 00 not exceeding a certain maximum, an amount of Protein A not exceeding a certain maximum, a certain profile of N-linked carbohydrates, a certain amino monosaccharide composition (GlcNac, GalNAc), a certain neutral monosaccharide composition (galactose, fucose, mannose), a certain amount of B7 binding, a certain amount of activity in a IL-2 inhibition cell assay, and /or a certain sialic acid composition (NANA, NGNA), in each case where said certain amounts can be a range or ranges. The invention provides compositions with any one of the aforementioned characteristics, or more than one of the aforementioned characteristics, up to an including all of the aforementioned characteristics in any and all possible permutations or combinations. The invention includes all the compositions of the invention in isolated or substantially purified form, or not in isolated or substantially purified form. The invention provides for compositions which are pharmaceutical compositions.

[00202] In one aspect, the invention is directed to a method for obtaining a composition comprising an isolated population of CTLA4-Ig molecules from a liquid culture medium, the medium comprising an initial population of CTLA4-Ig molecules, wherein (1) CTLA4-Ig molecules of the initial population have one or more sialic acid residues, (2) the number of sialic acid residues per CTLA4-Ig molecule varies within the initial population, and (3) the initial population comprises CTLA4-Ig dimer and high molecular weight aggregate, and the method comprises (a) harvesting the liquid culture medium from a culture of mammalian cells expressing CTLA4-Ig molecules; (b)separating the CTLA4-Ig molecules from cellular components; (c) separating CTLA4-Ig dimers from CTLA4-Ig high molecular weight aggregates; and (d) separating the CTLA4-Ig molecules into two or more fractions, wherein at least one fraction has a greater molar ratio of sialic acid to CTLA4-Ig molecules compared to at least one other fraction, and wherein steps (b), (c) and (d) are carried out simultaneously or in any order, so as to obtain said composition.

[00203] In one embodiment of the method of the invention, the harvesting in step (a) comprises obtaining a soluble fraction of the liquid culture. In another embodiment, steps (c) and (d) of the method comprise the use of column chromatography so as to obtain fractions of CTLA4-Ig molecules having different sialic acid contents. In yet another embodiment, the method further comprises use of column chromatography to reduce MCP-1 content in the composition.

[00204] In some embodiments of the method of the invention, the CTLA4-Ig molecules comprise one or more polypeptides having SEQ ID NO:2, 5, 6, 7, 8, 9, or 10. In other embodiments, the CTLA4-Ig molecules comprise one or more polypeptides having SEQ ID NO:4, 11, 12, 13, 14, 15 or 16.

[00205] In some embodiment of the method of the invention, the fraction in. (d) having the greater molar ratio of sialic acid to CTLA4-Ig molecules exhibits an average molar ratio of sialic acid to CTLA4-Ig molecules from about 8 to about 14. In specific embodiments, the average molar ratio is from about 8 to about 11, from about 8 to about 10, or from about 8 to about 9.

[00206] The invention provides for a method for isolating CTLA4-Ig molecules, the method comprising: (i) obtaining a soluble fraction of a liquid culture comprising mammalian cells that produce composition comprising CTLA4-Ig molecules; (ii) subjecting the soluble fraction to anion exchange chromatography to obtain an eluted composition comprising CTLA4-Ig molecules; (iii) subjecting the composition of step (ii) to hydrophobic interaction chromatography so as to obtain an enriched composition comprising CTLA4-Ig molecules; (iv) subjecting the composition of (iii) to affinity chromatography to obtain a further enriched composition comprising CTLA4-Ig molecules; and (v) subjecting the composition of (iv) to anion exchange chromatography. In one embodiment, the composition obtained in step (ii) is characterized by: (a) an average of 6.0-10.1 moles of NANA per mole of CTLA4Ig molecule; and (b) less than or equal to 25.7 area percent CTLA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

In another embodiment, the composition obtained in step (iii) is characterized by: (a) an average of 6.8-11.4 moles of NANA per mole of CTLA4Ig molecule; and (b) less than or equal. to 2.5 area percent of CTLA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection. In a further embodiment, the composition obtained in step (iv) is characterized by: (a) an average of 8.0-11.0 moles of NANA per mole of CTLA4-Ig molecule; and (b) less than or equal to 2.5 area percent of CTLA4-Ig high molecular weight species. In another embodiment, the composition obtained in step (v) is characterized by: (a) an average of 8.0-11.9 moles of NANA per mole of CTLA4-Ig molecule; and (b) less than or equal to 2.0 area percent being CTLA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection (SPD). In one embodiment, an example of SPD can be at A 280 nm.

[00207] The present invention also provides a method for isolating a composition of .CTLA4־Ig molecules comprising: (i) obtaining a soluble fraction of a liquid culture comprising mammalian cells that produce CTLA4-Ig molecules, and, in any order, (ii) subjecting the soluble fraction to anion exchange chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules; (iii) subjecting the soluble fraction to hydrophobic interaction chromatography so as to obtain an enriched and eluted . composition comprising CTLA4-Ig molecules; (iv) subjecting the soluble fraction to affinity chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules; and (v) subjecting the soluble fraction to anion exchange chromatography so as to obtain an eriched and eluted composition comprising CTLA4-Ig molecules. In another aspect, the present invention provides a method for isolating a composition comprising CTLA4-Ig molecules, the method comprising: (i) obtaining a soluble fraction of a liquid culture comprising mammalian cells that produce CTLA4-Ig molecules; (ii) subjecting the soluble fraction to anion exchange chromatography to obtain an eluted composition comprising CTLA4-Ig molecules; (iii) subjecting the protein product of step (ii) to hydrophobic interaction chromatography so as to obtain an enriched composition comprising CTLA4-Ig molecules; (iv) subjecting the protein product of (iii) to affinity chromatography to obtain a further enriched composition comprising CTLA4-Ig molecules; and (v) subjecting the protein product of (iv) to anion exchange chromatography, so as to isolate a composition comprising CTLA4-Ig molecules.

[00208] In one embodiment, the composition comprising CTLA4-Ig molecules obtained in step (ii) of the method is characterized by: (a) an average molar ratio of NANA to

CTLA4Ig molecules of from 6.0 to 10.1, and (b) less than or equal to 2.5 area percent CTLA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection. In another embodiment, the composition comprising CTLA4-Ig molecules obtained in step (iii) of the method is characterized in that in that (a) CTLA4-Ig high molecular weight species is less than about 2.5 area % as determined by size exclusion chromatography and spectrophotometric detection, (b) cellular protein is less than about 95 ng/ml, and (c) MCP-1 is less than about 5 ppm. In an additional embodiment, the composition comprising CTLA4-Ig molecules obtained in step (iii) of the method is characterized by: (a) an average molar ratio of NANA to CTLA4-Ig molecules of from 6.8 to 11.4, and (b) less than or equal to 2.5 area percent CTLA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection. In a further embodiment, the composition comprising CTLA4-Ig molecules obtained in step (iv) of the method is characterized by: (a) an average molar ratio of NANA to CTLA4-Ig molecules of from 8.0 to 11.0, and (b) less than or equal to 2.5 area percent CTEA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection. In still another embodiment, the composition obtained in step (iii) of the invention is characterized in that CTLA4-Ig high molecular weight species is less than 2.5% area percent as determined by size exclusion chromatography and spectrophotometric detection. In yet another embodiment, the protein composition comprising CTLA4-Ig molecules in step (v) of the method is characterized by: (a) an average molar ratio of NANA to CTLA4-Ig molecules of from 8.0 to 11.9, and (b) less than or equal to 2.0 area percent CTLA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

[00209] The invention also provides, in another aspect, a method for isolating a composition of CTLA4-Ig molecules, comprising: (i) obtaining a soluble fraction of a liquid culture comprising mammalian cells that produce CTLA4-Ig molecules, and, in any order, (ii) subjecting the soluble fraction to anion exchange chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules; (iii) subjecting the soluble fraction to hydrophobic interaction chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules; (iv) subjecting the soluble fraction to affinity chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules; and (v) subjecting the soluble fraction to anion exchange chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules, wherein the composition obtained in step (iii) is characterized in that the percentage of CTLA4־Ig high molecular weight species is less than about 2.5 area %, cellular protein is less than 95 ng/ml, and MCP-1 is less than about 5 ppm.

[00210] In still another aspect, the invention provides a method for isolating a composition of CTLA4-Ig molecules, the method comprising: (i) obtaining a soluble fraction of a liquid culture comprising mammalian cells that produce CTLA4-Ig molecules, and, in any order, (ii) subjecting the soluble fraction to anion exchange chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules; (iii) subjecting the soluble fraction to hydrophobic interaction chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules; (iv) subjecting the soluble fraction to affinity chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules; and (v) subjecting the soluble fraction to anion exchange chromatography so as to obtain an enriched and eluted composition comprising CTLA4-Ig molecules, wherein the composition obtained in step (iii) is characterized in that the percentage of CTLA4-Ig high molecular weight species is less than about 2.5 area %, cellular protein is less than 95 ng/ml, MCP-1 is less than about 5 ppm, and the average molar ratio of NANA to CTLA4-Ig molecules is of from about 8.0 to about 12.

[00211] In one embodiment, the anion exchange chromatography of step (ii) of the method is carried out using a wash buffer comprising about 75 mM HEPES, and about 360 mM NaCI, and having a pH of about 8.0. In another embodiment, the anion exchange chromatography of step (ii) of the invention is carried out using an elution buffer comprising ' about 25 mM HEPES, and about 850 mM NaCI, and having a pH of about 7.0. In an additional embodiment, the hydrophobic interaction chromatography of step (iii) of the method is carried out using a single wash buffer comprising about 25 mM HEPES, and about 850 mM NaCI, and having a pH of about 7.0. In a further embodiment, the affinity chromatography of step (iv) of the method is carried out using a wash buffer comprising about 25 mM Tris, and about 250 mM NaCI, and having a pH of about 8.0. In still another embodiment, the affinity chromatography of step (iv) of the method is carried out using an elution buffer comprising about 100 mM glycine and having a pH of about 3.5. In yet another embodiment, the anion exchange chromatography of step (v) of the method is carried out using a wash buffer comprising about 25 mM HEPES, and from about 120 mM NaCI to about 130 mM NaCI, and having a pH of about 8.0. In still another embodiment, the anion exchange chromatography of-step (v) of the method is carried out using an elution buffer, comprising about 25 mM HEPES, and about 200 mM NaCl, and having a pH of about 8.0. In yet another embodiment, the anion exchange chromatography of step (ii) of the method is carried out using a column having an anion ex change resin comprising a primary, secondary, .tertiary, or quarternary amine functional group. In a specific embodiment, the resin comprises a quarternary amine functional group. In still another embodiment, the hydrophobic interaction chromatography of step (iii) of the method is carried out using a hydrophobic interaction resin comprising a phenyl, an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group. In a specific embodiment, the functional group comprises a phenyl functional group. In still another embodiment, the affinity chromatography of step (iv) of the method is carried out using an affinity chromatography resin comprising Protein A.

[00212] In yet another aspect, the invention provides a method for preparing a composition comprising CTLA4-Ig molecules, comprising purifying CTLA4-Ig molecules from a liquid cell culture, wherein the purified CTLA4-Ig composition comprises (a) a pharmaceutically acceptable amount of MCP-1 per mg of CTLA4-Ig molecules, and (b) less than 2.5 area % of CTLA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection. In one embodiment, the pharmaceutically acceptable amount of MCP-1 comprises from about 40 to about 0.5 ng/mg of CTLA4-Ig molecules. In another embodiment, the pharmaceutically acceptable amount of MCP-1 comprises from about 35 to about 0.5 ng/mg of CTLA4-Ig molecules. In an additional embodiment, the pharmaceutically acceptable amount of MCP-1 comprises from about 10 to about 0.5 ng/mg of CTLA4-Ig molecules. In a further embodiment, the affinity chromatography of step (iv) of the method is carried out using a column comprising a resin capable of reducing MCP-1 in the eluted protein product. In still another embodiment, the hydrophobic interaction chromatography of step (iii) of the method is carried out using a hydrophobic interaction resin, wherein the resin is capable of (a) separating CTLA4-Ig dimers from CTLA4-Ig high molecular weight species; (b) increasing sialic acid content of the eluted CTLA4-Ig molecules; or (c) both (a) and (b). In yet another embodiment, the ־ anion exchange chromatography of step (ii) or step (iv), or both, is carried out using an anion .exchange resin, wherein the resin is capable of (a) decreasing the CTLA4-Ig high molecular weight aggregate content of the eluted composition; (b) increasing the sialic content of the eluted composition; or (c) both (a) and (b).

[00213] . In another aspect, the invention provides a method for isolating a composition . comprising CTLA4-Ig׳-molecules, the method comprising: (i) obtaining a soluble fraction of a liquid culture comprising mammalian cells that produce CTLA4-Ig molecules, and in any order; (ii) subjecting the soluble fraction to affinity chromatography so as to obtain an eluted composition comprising CTLA4-Ig molecules; (iii) subjecting the soluble fraction to anion exchange chromatography so as to obtain an eluted and enriched composition comprising CTLA4-Ig molecules; and (iv) subjecting the soluble fraction to hydrophobic interaction chromatography so as to obtain an eluted and enriched composition comprising CTLA4-Ig molecules. In one embodiment, the affinity chromatography step is performed first. In another embodiment, the affinity chromatography of step (ii) of the method is carried out using a resin comprising Protein A. In an additional embodiment, the affinity chromatography of step (ii) is carried out using an elution buffer comprising guanidine. In a further embodiment, the affinity chromatography of step (ii) is carried out using an elution buffer comprising urea. In yet another embodiment, the affinity chromatography of step (ii) results in an increase in CTLA4-Ig dimers in the eluted composition comprising CTLA4-Ig molecules.

[00214] In yet another aspect, the invention provides a method for isolating composition comprising CTLA4-Ig molecules from liquid harvested from a mammalian cell culture, wherein the cells produce CTLA4-Ig molecules, the method comprising: (i) obtaining a soluble fraction of the harvested liquid; (ii) subjecting the soluble fraction to affinity chromatography to obtain an eluted composition comprising CTLA4-Ig molecules; (iii) subjecting the composition of step (ii) to anion exchange chromatography so as to obtain an eluted and enriched composition comprising CTLA4-Ig molecules; and (iv) subjecting the composition from step (iii) to hydrophobic interaction chromatography to obtain a further enriched composition comprising CTLA4-Ig molecules. In one embodiment, the composition obtained in step (iv) of the method is characterized in that the percentage of high molecular weight species is less than about 2.5 area % as determined by size exclusion chromatography and spectrophotometric detection, and the percentage of cellular protein is less than about 95 ng/ml, and the percentage of MCP-1 is less than about 5 ppm. In another embodiment, the anion exchange chromatography of step (iii) is carried out using a wash buffer comprising about 50 mM HEPES, and about 135 mM NaCl, and having a pH of about

7. In an additional embodiment, the anion exchange chromatography of step (iii) is carried out using an elution buffer comprising about 50 mM HEPES, and about 200 mM NaCl, and having a pH of about 7. In a specific embodiment, the hydrophobic interaction chromatography of step (iii) is carried out using a hydrophobic interaction resin comprising a phenyl, an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group. In a further embodiment, the hydrophobic interaction chromatography of step (iv) is carried out using a wash buffer comprising about 50 mM HEPES, and about 1.2 M (NH4)2SO4, and having a pH of about 7. In still another embodiment, the affinity chromatography of step (ii) is carried out using a wash buffer comprising about 25 mM NaH2PO4, and about 150 mM NaCl, .and. having a pH of about 7.5. In yet another embodiment, the affinity chromatography of step (ii) is carried out using an elution buffer-comprising about 250 mM glycine and having a pH of about 3. In another embodiment, the anion exchange chromatography of step (iii). is carried out using a column having an anion exchange resin comprising a primary, secondary, tertiary, or quarternary amine functional group. In a specific embodiment, the resin comprises a quarternary amine functional group.

[00215] In one embodiment, the hydrophobic interaction chromatography of step (iii) is carried out using a hydrophobic interaction resin comprising a phenyl, an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group. In one embodiment, the functional group comprises a phenyl functional group. In one embodiment, the affinity chromatography of step (ii) is carried out using a resin comprising Protein A. The invention provides for a composition comprising CTLA4-lg molecules obtained by any of the methods of the. invention. In one embodiment, the composition comprises one or more polypeptides having SEQ ID NO:2, 5, 6, 7, 8, 9 or 10. In one embodiment, the composition comprises one or more polypeptides having SEQ ID NO:4, 11, 12; 13, .14, 15 or 16. The invention provides for a . CTLA4-Ig expression plasmid having the nucleic acid sequence of SEQ ID NO: 17. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig protein of from about 5.5 to about 18. The invention provides for a substantially.purified . composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio, of sialic acid to CTLA4-Ig molecules of from about 5.5 to about 9.5.

[00216]

[00217] The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 5 to about 10. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 6 to about 18. The invention provides for a substantially purified composition comprising

CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic .

acid to CTLA4-Ig molecules of from about 8 to about 18.

[00218] The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 8 to about 12. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 8 to about 11. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 7 to about 12.

[00219] The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-lg molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 7 to about 11. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 11 to about 18. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 12 to about 18.

[00220] The invention provides for a substantially purified composition comprising

CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 13 to about 18. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules; wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4־Ig molecules of from about 14 to about 18. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 15 to about 17.

[00221] The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of about 16. The .invention provides, for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an .average molar ratio of sialic acid to CTLA4-Ig molecules of about 10. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules; wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of about 6. In one embodiment, the sialic acid is N-acetyl neuraminic acid (NANA). The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of NANA to CTLA4-Ig molecules of from about 8 to about 12. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio .of N-glycolyl neuraminic acid (NGNA) to CTLA4-Ig molecules of less than or equal to about 1.5.

[00222] The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of NGNA to CTLA4-Ig molecules of from about 0.5 to about 1.5. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of NGNA to CTLA4-Ig molecules of from about 1.0 . to about 1.5. The invention provides for a substantially purified composition comprising

CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 6 to about 18.

[00223] The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of sialic acid per mole of CTLA4-Ig molecules of from about 6 to about 12.

[00224] The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein each polypeptide of the molecule comprises the sequence of SEQ ID NO:11,12,13, 14, 15 or 16, and wherein the CTLA4-Ig molecules are characterized by an average molar ratio of sialic acid per mole of CTLA4-Ig molecules of from about 5.5 to about 9.5. In one embodiment, the molar ratio of sialic acid per mole of CTLA4-Ig • molecules is determined by acid hydrolysis and HPLC. In one embodiment, the CTLA4-Ig molecules comprise one or more polypeptides having SEQ ID NO:2, 5, 6, 7, 8, 9 or 10.

[00225J . In one embodiment, the CTLA4-Ig molecules comprise one or more polypeptides having SEQ ID NO:4, 11, 12,13,14,15 or 16. The invention provides for a substantially purified composition comprising CTLA4-Ig molecules, wherein greater than or equal to 95% of the CTLA4-Ig molecules are CTLA4-Ig dimers. In one embodiment, greater than or equal to 98% of the CTLA4-Ig molecules are CTLA4-Ig dimers. In one embodiment, greater than or equal to 99% of the CTLA4-Ig molecules are CTLA4-Ig dimers.

[00226] In one embodiment, greater than or equal to 99.5% of the CTLA4-Ig molecules are CTLA4-Ig dimers. In one embodiment, from about 95% to about 99.5% of the CTLA4-Ig molecules are CTLA4-Ig dimers and about 0.5 area percent to about 5 area percent of the molecules are CTLA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection. In one embodiment, about 98.6% of the molecules are CTLA4-Ig dimers and about 1.2 area percent of the molecules are CTLA4-Ig high molecular weight species and about less than 0.7 area percent of the molecules are CTLA4-Ig monomers as determined by size exclusion chromatography and spectrophotometric detection. In one embodiment, about less then about 0.3% of the molecules are multimers comprising five or more CTLA4-Ig monomers. The invention provides for a composition consisting essentially of CTLA4-Ig dimers. The invention provides for a composition consisting essentially of CTLA4-Ig molecules, wherein the population is substantially free of CTLA4-Ig monomers. The invention provides for a composition consisting essentially of CTLA4-Ig molecules, wherein the population is substantially free of CTLA4-Ig high molecular weight species. The invention provides for a composition consisting essentially of CTLA4-Ig monomers substantially free of CTLA4-Ig dimers and high molecular weight species. In one embodiment, each monomer of each CTLA4-Ig dimer has at least 3 sialic acid groups. In one embodiment, each monomer of each CTLA4-Ig dimer has at least 2.5 sialic acid groups. In one embodiment, each monomer of each CTLA4-Ig dimer has from at least 3 sialic acid groups to at least 8 sialic acid groups.

[00227] In one embodiment, each monomer of each CTLA4-Ig dimer has from at least

2.5 sialic acid groups to at least 5 sialic acid groups. In one embodiment, each dimer comprises two CTLA4-Ig polypeptides, wherein each polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOS:5-16. In one embodiment, the composition comprises one or more polypeptides having SEQ ID NO:2, 5, 6, 7, 8, 9 or 10. In one embodiment, the composition comprises one or more polypeptides having SEQ ID NO:4,

11, 12, 13,.14, 15 or״l 6. The invention provides for an isolated-composition comprising CTLA4-Ig tetramers, which is substantially free of CTLA4-Ig .dimers. The invention . provides for an isolated composition comprising CTLA4-Ig tetramers which is substantially free of CTLA4-Ig monomers. In one embodiment, the composition exists as an amount that is greater than about 100 grams. In one embodiment, each tetramer comprises two pairs of CTLA4-Ig polypeptides, wherein each polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NQS:5-10. In one embodiment, each tetramer comprises two pairs of CTLA4-Ig polypeptides, wherein each polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOS: 11 -16. In one embodiment, each tetramer is capable of binding to a CD80 or CD86. The invention provides for a pharmaceutically acceptable composition comprising CTLA4-Ig molecules, wherein the composition is substantially free of MCP-1. The invention provides for a pharmaceutically acceptable composition comprising CTLA4-Ig molecules, wherein the composition comprises no more than about 25 ppm MCP-1. In one embodiment, the composition comprises no more than 10 ppm MCP-1. In one embodiment, the composition comprises from about 0.2 ng/ml MCP-1 to about 10 ng / ml of MCP-1. In one embodiment,’ the invention provides for a pharmaceutically acceptable composition comprising CTLA4-Ig molecules, wherein the composition comprises (a) from about 0.2 ng/ml MCP-1 to about 10 ng / ml of MCP-1 and (b) no more than 25 ng/ml of CHO protein or no more than 10 ng/ml of CHO protein. In one embodiment, the composition comprises no more than about 20 pg/ml of DNA.

[00228] The invention provides for an isolated composition comprising CTLA4-Ig molecules, wherein, when administered to a subject at an intravenous dose of about 10 mg/kg, the CTLA4-Ig molecules are capable of exhibiting: an area under the curve (AUC) of about 44400 μg.h/ml; a volume of distribution of about 0.09 L/kg; a peak concentration (Cmax) of about 292 μg/ml; and a clearance rate of about 0.23 ml/h/kg. The invention . provides for an isolated composition comprising CTLA4-Ig molecules, wherein the composition comprises dominant isoforms of CTLA4-Ig molecules visualizable on an isoelectric focusing gel which have an isoelectric point, pl, less than or equal to 5.1± 0.2 as determined by isoelectric focusing. In one embodiment, the average pl of the composition increases after neuraminidase treatment. In one embodiment, at least 40% of the CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 5.1 ± 0.2 as determined by isoelectric focusing. In one embodiment, at least 70% of the CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 5.1 ± 0.2 as determined by isoelectric focusing. In one embodiment, at least 90% of the CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 5.1 ± 0.2 as determined by isoelectric focusing. The invention provides .for an isolated composition comprising CTLA4-Ig molecules having a pl of from about 3.0 ± 0.2 to about 5.0 ± 0.2. The invention provides for an isolated composition comprising CTLA4-Ig molecules having a pl from about 4.3 ± 0.2 to about 5.0 ± 0.2.

[00229] The invention provides for an isolated composition comprising CTLA4-Ig molecules having a pl of about 3.3 ± 0.2 to about 4.7 ± 0.2. In one embodiment, the composition is substantially purified. The invention provides for a method for preparing a composition, the composition comprising a CTLA4-Ig molecule with a pl of from about 3.0 ± 0.2 to about 5.0 + 0.2, the method comprising: (a) subjecting a mixture of CTLA4-Ig molecules to isoelectric focusing gel electrophoresis, wherein a single band on the gel represents a population of CTLA4-Ig molecules with a particular pl, and (b) isolating the population of CTLA4-Ig molecules having a pl of from about 3.0 ± 0.2 to about 5.0 + 0.2 so . as to prepare the composition. The invention provides for an isolated composition comprising CTLA4-Ig molecules, wherein the composition comprises dominant isoforms visualizable on an isoelectric focusing gel which have an isoelectric point, pl, less than or equal to 5.5 ± 0.2 as determined by isoelectric focusing. In one embodiment, the average pl of the composition increases after neuraminidase treatment. In one embodiment, at least 40% of the CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 5.3 ± 0.2 as determined by isoelectric focusing. In one embodiment, at least 70% of the CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 5.3 ± 0.2 as determined by isoelectric focusing. In one embodiment, at least 90% of the CTLA4-lg molecules exhibit an isoelectric point less than or equal to about 5.3± 0.2 as determined by isoelectric focusing. The invention provides for an isolated composition comprising CTLA4-Ig molecules having a pl of from about 3.0 ± 0.2 to about 5.2 ± 0.2.

[00230] The invention provides for an isolated composition comprising CTLA4-Ig molecules having a pl from about 4.5 ± 0.2 to about 5.2 ± 0.2. The invention provides for an isolated composition comprising CTLA4-Ig molecules having a pl of about 4.7 ± 0.2 to about 5.1 ± 0.2. In one embodiment, the composition is substantially purified.

[00231] The invention provides for a method for preparing, a composition, the־ composition comprising CTLA4-Ig molecules with a pl of from about 2.0 + 0.2 to about 5.2 ± 0.2, the method comprising: (a) subjecting a mixture of CTLA4-Ig molecules to isoelectric focusing gel electrophoresis, wherein a single band on the gel represents a population. of . . CTLA4-Ig molecules with a particular pl, and (b) isolating the population of CTLA4-Ig molecules having a pl of from about'3.0 ± 0.2 to about 5.2 ± 0.2 so as to prepare the composition. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of GlcNAc to CTLA4-Ig molecules of from about 17 to about 28. ׳The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by ah average molar ratio of GlcNAc to CTLA4-Ig molecules of from about 17 to about 25. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4Ig molecules are characterized by an average molar ratio of GlcNAc to CTLA4-Ig molecules of from about. 15 to about 35.

]00232] The invention provides for a composition comprising CTLA4-Ig molecules, wherein each polypeptide of the molecule comprises the sequence of SEQ IDNO:11, 12, 13, 14, 15 or 16, and wherein the CTLA4-Ig molecules are characterized by an average molar ratio of GlcNAc to CTLA4-Ig molecules of from about 24 to about 28. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of GalNAc to CTLA4-Ig molecules of from about 1.7 to about 3.6. The invention provides for a composition comprising CTLA4Ig molecules, wherein each polypeptide of the molecule comprises the sequence of SEQ ID NO: 11,12, 13, 14,15 or 16, and wherein the CTLA4-Ig molecules are characterized by an average molar ratio of GalNAc to CTLA4-Ig molecules of from about 2.7 to about 3.6.

[00233] The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of galactose to CTLA4-Ig molecules of from about 8 to about 17. The invention provides for a composition comprising CTLA4-Ig molecules, wherein each polypeptide of the molecule comprises the • sequence of SEQ ID NO:11, 12, 13, 14, 15 or 16, and wherein the CTLA4-Ig molecules are characterized by an average molar ratio of galactose to CTLA4-Ig molecules of from about 11 to about 13. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-lg molecules ’are characterized by an average molar ratio of fucose to

CTLA4-Ig molecules of from about 3.5 to about 8.3.

[00234] The invention provides for a composition comprising CTLA4-Ig molecules, wherein each polypeptide of the molecule comprises the sequence of SEQ ID NO:11, 12, 13, 14, 15 or 16, and wherein the CTLA4-Ig molecules are characterized by an average molar ratio of fucose to CTLA4-Ig molecules of from about 6.4 to about 7.0. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of mannose to CTLA4-Ig molecules of from about 7.7 to about 22. The invention provides for a composition comprising CTLA4-Ig molecules, wherein each polypeptide of the molecule comprises the sequence of SEQ ID NO:11, 12, 13, 14, 15 or 16, and wherein the CTLA4-Ig molecules are characterized by an average molar ratio of mannose to CTLA4-Ig molecules of from about 14 to about 16.

[00235] In one embodiment, the molar ratio of GlcNAc to CTLA4-Ig molecules is determined by capillary electrophoresis. In one embodiment, the molar ratio of GalNAc to CTLA4-Ig molecules is determined by capillary electrophoresis. In one embodiment, the molar ratio of galactose to CTLA4-Ig molecules is determined by capillary electrophoresis.

[00236] In one embodiment, the molar ratio of fucose to CTLA4-Ig molecules is determined by capillary electrophoresis. In one embodiment, the molar ratio of mannose to CTLA4-Ig molecules is determined by capillary electrophoresis. In one embodiment, the CTLA4-Ig molecules are obtained by enzymatic attachment of one or more carbohydrates to the molecule. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the molecules comprise carbohydrate residues attached to the molecules enzymatically in vitro. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 15 to about 35; and (b) an average molar ratio of sialic acid to CTLA4-Ig molecules from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 15 to about 35; (b) an average molar ratio of GalNAc to CTLA4-Ig molecules from about 1.7 to about 3.6; and (c) an average molar ratio of sialic acid to CTLA4-Ig molecules from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 15 to about 35; (b) an average molar ratio of GalNAc to

CTLA4-Ig molecules from about 1.7 to about 3.6; (c) an average molar ratio of galactose to CTLA4-Ig molecules from about 8 to about 17; and (d) an average molar ratio of sialic acid to CTLA4-Ig molecules from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from. about 15 to about 35; (b) an average molar ratio of GalNAc to CTLA4-Ig molecules from about 1.7 to about 3.6; (c) an average molar ratio of galactose to CTLA4-Ig molecules from about 8 to about 17; (d) an average molar ratio of fucose to CTLA4-Ig molecules from about 3.5 to about 8.3; and (e) an average molar ratio of sialic acid to CTLA4-lg molecules from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 15 to about 35; (b) an average molar ratio of GalNAc to CTLA4-Ig molecules from about 1.7 to about 3.6; (c) an average molar ratio of galactose to CTLA4-Ig molecules from about 8 to about 17; (d) an average molar ratio of fucose to CTLA4-Ig molecules from about 3.5 to about 8.3; (e) an average molar ratio of mannose to CTLA4-Ig molecules from about 7.2 to about 22; and (f) an average molar ratio of sialic acid to CTLA4-Ig molecules from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 24 to about 28; and (b) an average molar ratio ofsialic acid to CTLA4-Ig molecules from about 5.5 to about 9.5. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 24 to about 28; (b) an average molar ratio of GalNAc to CTLA4־lg molecules from about 2.7 to about 3.6; and (c) an average molar ratio of sialic acid to CTLA4-Ig molecules from about 5.5 to about 9.5. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 24 to about 28; (b) an average molar ratio of GalNAc to CTLA4-Ig molecules from about 2.7 to about 3.6; (c) an average molar ratio of galactose to CTLA4-Ig molecules from about 11 to about 13; and (d) an average molar ratio of sialic acid to CTLA4-Ig molecules from about 5.5 to about 9.5. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 24 to about 28; (b) an average molar ratio of GalNAc to CTLA4-Ig molecules from about 2.7 to about 3.6; (c) an average molar ratio of galactose to CTLA4-Ig molecules from about 11 to about 13; (d) an average molar ratio of fucose to CTLA4-Ig molecules from about 6.4 to about 7.0; and (e) an average molar ratio of sialic acid to CTLA4-Ig molecules from about 5.5 to about 9.5. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 24 to about 28; (b) an average molar ratio. o.f GalNAc to CTLA4-Ig molecules from about 2.7 to about 3.6; (c) an average molar ratio of galactose to CTLA4-Ig molecules from about 11 to about 13; (d) an average molar ratio of fucose to CTLA4-Ig molecules from about 6.4 to about 7.0; (e) an average molar ratio of mannose to CTLA4-Ig protein from about 14 to about 16; and (f) an average molar ratio of sialic acid to CTLA4-Ig protein from about 5.5 to about 9.5.

[00237] The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are glycosylated at an asparagine amino acid residue at position 102 of SEQ ID NO:2 or 4, an asparagine amino acid residue at position 134 of SEQ ID NO:2 or 4, an asparagine amino acid residue at position 233 of SEQ ID NO:2 or 4, a serine amino acid residue at position 155 of SEQ ID NO:2 or 4, or a serine amino acid residue at position 165 of SEQ ID NO:2 or 4. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are glycosylated, and wherein at least about 2 % of total mass of glycosylation is O-linked glycosylation.

[00238] The invention provides for a composition comprising CTLA4-Ig molecules, wherein the composition exhibits an NGNA chromatogram peak of about 9.6 ± 0.3 and an NANA chromatogram peak of about 10.5 ± 0.3. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules exhibit a carbohydrate profile substantially the same as Figure 68. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA-Ig molecules exhibit a carbohydrate profile as shown in Figure 68. The invention provides for a composition consisting essentially of CTLA4-Ig molecules, wherein the CTLA4-Ig molecules exhibit a carbohydrate profile of Domains I - IV, wherein Domain I comprises peaks which represent a-sialylated oligosaccharides, Domain II comprises peaks which represent mono-sialylated oligosaccharides, Domain III comprises peaks which represent di-sialylated oligosaccharides, Domain IV comprises peaks which represent tri-sialylated oligosaccharides, and Domain V comprises peaks which represent tetra-sialyated oligosaccharides, and wherein the profile is a chromatogram of oligosaccharides released from CTLA4-Ig. ' In one embodiment, the difference in retention times of N-linked oligosaccharides between a first peak in Domain I and a main peak in Domain II is from about 10 to about 12 minutes. In one embodiment, the difference in retention times of N-linked oligosaccharides between a first peak in Domain I and a main peak in Domain II is from about 11 to about 13 minutes. In one embodiment,־ glycosylation of Domains III and IV comprises about 25% to about 36% of N-linked. glycosylation as measured by HPAEC. In one embodiment, glycosylation of Domain I comprises about 24.5% to about 35.2% of N-linked glycosylation as measured by HPAEC. In one embodiment, glycosylation of Domain II comprises about 26.3% to about 34.1% of N. linked glycosylation as measured by HPAEC. In one embodiment, glycosylation of Domain

III comprises about 21.9% to about 31.5% of N-linked glycosylation as measured by HPAEC. In one embodiment, glycosylation of Domain IV and Domain V comprises about 7.9% to about 18.6% of N-linked glycosylation as measured by HPAEC.

[00239] In one embodiment: (a) Domain I exhibits an area percentage of at least about 31; (b) Domain II exhibits an area percentage of at least about 33; (c) Domain III exhibits an area percentage of at least about 24; (iv) Domain IV exhibits an area percentage of at least about 9.4, (v) Domain V exhibits an area percentage of at least about 67; or wherein the area is measured from a chromatogram of oligosaccharides released from CTLA4-Ig.

' [00240] In one embodiment: (a) Domain I exhibits at least about 5 peaks; (b) Domain II exhibits at least about 5 peaks; (c) Domain III exhibits at least about’ 5 peaks; (d) Domain

IV exhibits at least about 6 peaks, or (e) Domain V exhibits at least about 6 peaks, and wherein the peaks are exhibited on a chromatogram. A composition wherein Domain I exhibits'at.least two peaks, wherein a first peak has a minimum area of about 4.5% and a maximum area of about 11.2%, and wherein a second peak has a minimum area of about 8.7% and a maximum of about 11.8%.

[00241] In one embodiment, Domain III and IV exhibit an area percentage of about 25% to about 36% as measured by HPAEC. In one embodiment, Domain I exhibits an area percentage of about 24.5% to about 35.2% as measured by HPAEC. In one embodiment, Domain II exhibits'an area percentage of about 26.3% to about 34.1% as measured by HPAEC. In one embodiment, Domain III exhibits an area percentage of about 21.9% to about 31.5% as measured by HPAEC. In one embodiment, Domain IV exhibits an area percentage of about 7.9% to about 18.6% as measured by HPAEC. . .

[00242] The invention provides for a composition comprising CTLA4-Ig polypeptides, wherein: (a) about 80 % of the polypeptides have biantennary N-linked glycosylation; (b) about 14 % of the polypeptides have triantennary N-linked glycosylation; and (c) about 6 % of the polypeptides have tetraantennary N-linked glycosylation. In one embodiment, the Nlinked glycosylation is !determined by high pH anion exchange chromatography.w.ith pulsed amperometric detection (HPEAC-P AD). The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of galactose to־ CTLA4-Ig molecules of from about 8 to about 17; and (b) an average molar ratio of NANA to CTLA4-Ig molecules of from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by:

[00243] (a) an average molar ratio of galactose to CTLA4-Ig molecules of from about to about 17; (b) an average molar ratio of NANA to CTLA4-Ig molecules of from about 6 to about 12; and (c) a CTLA4-Ig high molecular weight species area percent of less than about 3% as determined by size exclusion chromatography and spectrophotometric detection. The invention provides for a composition comprising CTLA4-Ig molecules characterized by:

(a) an average molar ratio of galactose to CTLA4-Ig molecules of from about 8 to about 17;

(b) an average molar ratio of NANA to CTLA4-Ig molecules offrom about 6 to about 12; and (c) an average molar ratio of NGNA to CTLA4-Ig molecules of less than or equal to about 1.5.

[00244] The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of galactose to CTLA4-Ig molecules of from. . about 8 to about 17; (b) an average molar ratio of NANA to CTLA4-Ig molecules of from about 6 to about 12; (c) a CTLA4-Ig high molecular weight aggregate content less than about 3 area percent as determined by size exclusion chromatography and spectrophotometric detection; and (d) a carbohydrate profile substantially the same as that of Figure 68. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of galactose to CTLA4-Ig molecules offrom about 8 to about 17; (b) an average molar ratio of NANA to CTLA4-Ig molecules of from about 6 to about 12; (c) a CTLA4-Ig high molecular weight aggregate content less than about 3 area percent as . determined by size exclusion chromatography and spectrophotometric detection; and (d) a glycosylation content in Domains III, IV and V of at least about 29.8% to about 50.1% of Nlinked glycosylation as determined by HPAEC. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of galactose to CTLA4-Ig molecules of from about 8 to about 17; (b) an average molar ratio of NANA to CTLA4-Ig molecules of from about 6 to about 12; and (c) a CTLA4-Ig high molecular weight species, of les.s than about 3 area percent as determined by size exclusion chromatography and spectrophotometric detection. In one embodiment, the molecules׳ are further characterized by an average molar ratio of NANA to CTLA4-Ig molecules from about

8to about 12.

[00245] In one embodiment, the molecules are further characterized by: (a) about 80 % biantennary N-linked glycosylation; (b) about 14 % triantennary N-linked glycosylation; and (c) about 6 % tetraantennary N-linked glycosylation. In one embodiment, the molecules further comprise any combination of one or more of: (a) the amino acid sequence of SEQ ID NO: 10 (methionine at amino acid position 27 and glycine at amino acid position 382 of SEQ ID NO:2); (b) the amino acid sequence of SEQ ID NQ:7 (methionine at amino acid position 27 and lysine at amino acid position 383 of SEQ ID NO:2); (c) the amino acid sequence of SEQ ID NO:9 (alanine at amino acid position 26 and glycine at amino acid position 382 of SEQ ID NO:2); and (d) the amino acid sequence of SEQ ID NO:6 (alanine at amino acid position 26. and lysine at amino acid position 383 of SEQ ID NO:2). In one embodiment, (a) about 90% of the molecules comprise the amino acid sequence of SEQ ID NO:2 beginning with the methionine at residue 27; (b) about 10% of the molecules comprise the amino acid sequence of SEQ ID NO:2 beginning with the alanine at residue number 26; (c) about 4% of the molecules comprise the amino acid sequence of SEQ ID NO:2 ending with the lysine at residue number 383; and (d) about 96% of the molecules comprise the amino acid sequence of SEQ ID NO:2 ending with the glycine at residue number 382. The invention provides for a composition comprising CTLA4-Ig polypeptides, wherein: (a) about 80 % of the polypeptides have biantennary N-linked glycosylation; (b) about 14 % of the polypeptides have triantennary N-linked glycosylation; (c) about 6 % of the polypeptides have tetraantennary N-linked glycosylation; and(d) an average molar ratio of NGNA to CTLA4-Ig molecules of less than or equal to 1.5. The invention provides for a composition comprising CTLA4-Ig polypeptides, wherein: (a) about 80 % of the polypeptides have biantennary Nlinked glycosylation; (b) about 14 % of the polypeptides have triantennary N-linked glycosylation; (c) about 6 % of the polypeptides have tetraantennary N-linked glycosylation; and(d) an average molar ratio of GlcNAc to CTLA4-Ig molecules of from about 15 to about 35. The invention provides for a composition comprising CTLA4-Ig polypeptides, wherein: (a) about 80 % of the polypeptides have biantennary N-linked glycosylation; (b) about 14 % of the polypeptides have triantennary N-linked glycosylation; (c) about 6 % of the polypeptides have tetraantennary N-linked glycosylation; and(d) an average molar ratio of

GalNAc to CTLA4-Ig molecules of from about 1.7 to about 3.6. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of galactose to CTLA4-Ig molecules of from about 11 to about 13; and (b) an average molar ratio of sialic to CTLA4-Ig molecules of from about 5.5 to about 9.5.

[00246] The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of galactose to CTLA4-Ig molecules of from about 11 to about 13; (b) an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 5.5 to about 9.5; and (c) a CTLA4-Ig high molecular weight species of less than about 5 area percent as determined by size exclusion chromatography and spectrophotometric detection. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of galactose to CTLA4-Ig molecules of from about 11 to about 13;

[00247] (b) an average molar ratio of sialic acid to CTLA4-Ig molecules of from about

5.5 to about 9.5; (c) a CTLA4-Ig high molecular weight species content less than about 5 area percent as determined by size exclusion chromatography and spectrophotometric detection; and (d) a carbohydrate profile substantially the same as that of Figure 68. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of galactose to CTLA4-Ig molecules of from about 11 to about 13, (b) an average molar ratio of sialic acid to CTLA4-Ig molecules of from 5.5 to about 9.5, (c) a CTLA4-Ig high molecular weight species content less than about 5 area percent as determined by size exclusion chromatography and spectrophotometric detection; and (d) a glycosylation content in Domains III, IV and V of at least about 29.8% to about 50.1% of Nlinked glycosylation as determined by HPAEC.

[00248] The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of galactose to CTLA4-Ig molecules of from about 11 to about 13; (b) an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 5.5 to about 9.5; and (c) a CTLA4-Ig high molecular weight species content less than about 5 area percent as determined by size exclusion chromatography and spectrophotometric detection. In one embodiment, the molecules are further characterized by: (a) about 80 % biantennary N-linked glycosylation; (b) about 14 % triantennary N-linked glycosylation; and (c) about 6 % tetraantennary N-linked glycosylation. In another embodiment, the molecules further comprise any combination of one or more of: (a) the amino acid sequence of SEQ ID NO: 16 (methionine at amino acid position 27 and glycine at amino acid position 382 of SEQ ID NO:4); (b) the amino acid sequence of SEQ ID NO: 13 (methionine at amino acid position 27 and lysine at amino acid position 383 of SEQ ID NO:4); (c) the amino acid sequence of SEQ ID NO:15 (alanine at amino acid position 26 and glycine at amino acid position 382 of SEQ ID NO:4); and (d) the amino acid sequence of SEQ ID NO: 12 (alanine at amino acid position 26 and lysine at amino acid position 383 of SEQ ID NO:4). In another embodiment, (a) about 90% of the molecules comprise the amino acid sequence of SEQ ID NO:4 beginning with the methionine at residue 27; (b) about 10% of the molecules comprise the amino acid sequence of SEQ ID NO:4 beginning with the alanine at residue number 26; (c) about 4% of the molecules comprise the amino acid sequence of SEQ ID NO:4 ending with the lysine at residue number 383; and (d) about 96% of the molecules comprise the amino acid sequence of SEQ ID NO:4 ending with the glycine at residue number 382. The invention provides for a composition comprising CTLA4-Ig polypeptides, wherein:(a) about 80 % of the polypeptides have biantennary N-linked glycosylation; (b) about 14 % of the polypeptides have triantennary N-linked glycosylation; (c) about 6 % of the polypeptides have tetraantennary N-linked glycosylation; and(d) an average molar ratio of GlcNAc per mole of CTLA4-Ig protein of from about 24 to about 28. The invention provides for a composition comprising CTLA4-Ig polypeptides, wherein:(a) about 80 % of the polypeptides have biantennary N-linked glycosylation; (b) about 14 % of the polypeptides have triantennary N-linked glycosylation; (c) about 6 % of the polypeptides have tetraantennary N-linked glycosylation; and(d) an average molar ratio of GalNAc to CTLA4-Ig molecules of from about 2.7 to about 3.6. In another embodiment, the composition is a substantially purified composition. The invention provides for a composition comprising CTLA4-Ig molecules, wherein less than or equal to about 2.5 % of the CTLA4-Ig molecules are oxidized. The invention provides for a composition comprising CTLA4-Ig molecules, wherein less than or equal to about 2.0 % of the CTLA4-Ig molecules are deamidated. The invention provides for a composition comprising CTLA4-Ig dimer molecules, wherein at least 0.5 % of the CTLA4-Ig dimer molecules are cysteinylated. In one embodiment, at least 1.0% of the CTLA4-Ig dimer molecules are cysteinylated. The invention provides for a population of CTLA4-Ig molecules, wherein the population exhibits a mass spectrometry profile substantially the same as Figure 64, 65 or 67. The invention provides for a population of CTLA4-Ig molecules, wherein the population exhibits a capillary electrophoresis profile substantially the same as Figure 48.

[00249] The invention provides for a composition comprising CTLA4-Ig molecules, wherein the composition is characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 15 to about 35; (b) an average molar ratio of GalNAc to CTLA4-Ig molecules from about 1.7 to about 3.6; (c) an average molar ratio of galcatose to CTLA4-Ig molecules from about 8 to about 17; (d) an average molar ratio of fucose to CTLA4-Ig molecules from about 3.5 to about 8.3; (e) an average molar ratio of mannose to CTLA4-Ig molecules from about 7.2 to about 22; (f) an average molar ratio of sialic acid to CTLA4-Ig molecules from about 6 to about 12; (g) a pl as determined from visualization on an isoelectric focusing gel in a range from about 2.4 ± 0.2 to about 5.0 ± 0.2; (h) MCP-1 of less than or equal to 3 ppm; (i) less than 2.5 area percent of high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection; (j) less than 0.5 area percent of monomer as determined by size exclusion chromatography and spectrophotometric detection; (k) CTLA4-Ig polypeptides having an amino acid at least 95% identical to any of SEQ ID NOS:5-10; (1) CTLA4-Ig molecules capable of binding to CD80 and CD86. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the population of molecules is characterized by: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules from about 15 to about 35; (b) an average molar ratio of GalNAc to CTLA4-Ig molecules from about 1.7 to about 3.6; (c) an average molar ratio of galcatose to CTLA4-Ig molecules from about 8 to about 17; (d) an average molar ratio of fucose to CTLA4-Ig molecules from about 3.5 to about 8.3; (e) an average molar ratio of mannose to CTLA4-Ig molecules from about 7.2 to about 22; (f) an average molar ratio of sialic acid to CTLA4-Ig molecules from about 6 to about 12; (g) a pl as determined from visualization on an isoelectric focusing gel in a range from about 3.4 ± 0.2 to about 5.0 + 0.2; (h) MCP-1 of less than or equal to 5 ppm; (i) less than 2.5 area percent of high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection; (j) less than 0.5 area percent of monomer as determined by size exclusion chromatography and spectrophotometric detection; (k) CTLA4-Ig polypeptides having an amino acid at least 95% identical to any of SEQ ID NOS:5-10; (1) CTLA4-Ig molecules capable of binding to CD80 and CD86; or pharmaceutical equivalents thereof.

[00250] The invention provides for an isolated composition comprising CTLA4-Ig molecules having an incidence of immunogenicity of less than or equal to 7.4%. In one embodiment, the incidence of immunogenicity is from about 2.1% to about 7.4%. In one embodiment, the incidence of immunogenicity is less than or equal to 3.7%. In one embodiment, the incidence of immunogenicity is less than or equal to 3.0%. In one embodiment, the incidence of immunogenicity is from about 2.8% to about 3.0%. The invention provides for an isolated composition comprising CTLA4-Ig molecules, wherein, following administration of the composition to humans, production of antibodies that bind to the CTLA4-Ig molecules occurs at an incidence in the humans of less than or equal to 7.4%. In one embodiment, the incidence is from about 2.1% to about 7.4%. In one embodiment, the incidence is less than or equal to 3.7%. In one embodiment, the incidence is less than or equal to 3.0%. In one embodiment, the incidence is from about 2.8% to about 3.0%. The invention provides for an isolated composition comprising CTLA4-Ig molecules, wherein, following administration of the composition to humans, production of antibodies that bind to the CTLA4 portions of the CTLA4-Ig molecules occurs in the humans at an incidence of less than or equal to 4.9%. In one embodiment, the incidence is from about 0.5% to about 4.9%. In one embodiment, the incidence is less than or equal to 1.2%. In one embodiment, the incidence is less than or equal to 1.0%. In one embodiment, the incidence is from about 0.9% to about 1.0%. In one embodiment, the incidence is measured in an enzyme-lined immunosorbent assay (ELISA). In one embodiment, wherein the incidence is measured in an an electrochemoluminescence assay (ECL).

[00251] The invention provides for an isolated composition comprising CTLA4-Ig molecules, wherein, following administration of the composition to humans, production of antibodies that neutralize the CTLA4-Ig molecules occurs at an incidence of less than or equal to 75% of the humans having antibodies that bind to the CTLA4 portion of the CTLA4Ig molecule. In one embodiment, the incidence is 40-75%. In one embodiment, the incidence isless than or equal to 40%. In one embodiment, the incidence is measured in a cell-based luciferase reporter assay.

[00252] The invention provides for a method for producing CTLA4-Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4-Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4-Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture; and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs when the liquid culture exhibits greater than or equal to about 6.0 moles of NANA per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule, as determined by assessing an aliquot of the liquid culture. The method also provides for a method for producing CTLA4-Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4-Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture; and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs when the liquid culture exhibits from about 5.2 to about 7.6 moles of sialic acid per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule, as determined by assessing an aliquot of the liquid culture. The method also provides for a method for producing CTLA4-Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4-Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4-Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture; and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs when the liquid culture has a cell density of from about 33 x 105 viable cells per mL of liquid culture to about 79 x 105 cells per mL of liquid culture. The invention also provides a method for producing CTLA4-Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4-Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4-Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture; and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs when cell viability in the liquid culture is not less than about 38%. The invention also provides for a method for producing CTLA4-Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4־Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4-Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture; and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs when cell viability in the liquid culture is not less than about 37%. The method also provides for a method for producing CTLA4-Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4-Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4-Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture;

and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs when endotoxin is less than or equal to about 76.8 EU per mL of liquid culture, as determined by assessing an aliquot of the liquid culture. The method also provides for a method for producing CTLA4-Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4-Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4-Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture; and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs when endotoxin is less than or equal to about 4.8 EU per mL of liquid culture, as determined by assessing an aliquot of the liquid culture. The invention also provides for a method for producing CTLA4Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4-Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4-Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture; and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs only when bioburden is less than 1 colony forming unit per mL of liquid culture, as determined by assessing an aliquot of the liquid culture. The invention also provides for a method for producing CTLA4-Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4-Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4-Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture; and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs when at least two of the following conditions are met: (i) the liquid culture contains greater than or equal to about 6.0 moles of NANA per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule; (ii) the liquid culture has a cell density of from about 33 x 105 viable cells per mL of liquid culture to about 79 x 105 viable cells per mL of liquid culture; (iii) the cell viability in the liquid culture is not less than about 38%; or (iv) the yield of CTLA4-Ig protein is greater than about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, wherein NANA concentration in (i) and yield in (iv) are determined by assessing an aliquot of the liquid culture. The invention also provides for a method for producing CTLA4-Ig protein, the method comprising: (a) expanding mammalian cells that produce CTLA4-Ig protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L until the CTLA4-Ig protein is produced at a yield of at least about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determined by assessing an aliquot of the liquid culture; and (b) isolating the CTLA4-Ig protein from the at least 10,000 L liquid culture, wherein the isolating occurs when at least two of the following conditions are met: (i) the liquid culture contains from about 5.2 to about 7.6 moles of sialic acid per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule; (ii) the cell viability in the liquid culture is not less than about 37%; or (iii) the yield of CTLA4-Ig protein is greater than about 0.5 grams of CTLA4-Ig protein per liter of liquid culture, wherein the sialic acid content in (i) and yield in (iii) are determined by assessing an aliquot of the liquid culture.

[00253] Sequences:

SEQ ID NO:1 [CTLA4-Ig nucleotide sequence, See Fig. 1]

SEQ ID NO:2 [CTLA4־Ig amino acid sequence, See Fig. T]

SEQ ID NO:3 [CTLA4^A29YLI04E-Ig nucleotide sequence comprises nucleotides 79 to 1149 of the nucleic acid sequence shown in Fig. 2]

SEQ ID NO: 23 is the foil nucleotide sequence shown in Fig. 2. This nucleotide sequence includes the coding sequence for the prosequence.

SEQ ID NO:4 [CTLA4^A29YL104E-Ig amino acid sequence, Fig. 3, without the pro-sequence]

SEQ ID NO:5 [amino acids 25-383 of SEQ ID NO:2]

MAMHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGT QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

SEQ ID NO:6 [amino acids 26-383 of SEQ ID NO:2]

AMHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

SEQIDNO:7 [amino acids 27-383 of SEQ ID NO:2]

MHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAAIYMMG

NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK

EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

SEQ ID NO:8 [amino acids 25-382 of SEQ ID NO:2]

MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAAIYM

MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGT

QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT

CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSIYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPG

SEQ ID NO:9 [amino acids 26-382 of SEQ ID NO:2]

AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM

GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI

YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV

VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG

SEQ ID NO: 10 [amino acids 27-382 of SEQ ID NO:2]

MHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG

NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCW

VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG

SEQ ID NO: 11 [amino acids 25-383 of SEQ ID NO:4]

MAMHVAQPAWLASSRGIASF.V.CEYASPGKYTEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGT QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

SEQ ID NO: 12 [amino acids 26-383 of SEQ ID NO:4]

AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMM GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

SEQ ID NO:13 [amino acids 27-383 of SEQ ID NO:4]

MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG NELTFLDDS1CTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIY VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

SEQ ID NO:14 [amino acids 25-382 of SEQ ID NO:4]

MAMHVAQPAWLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGT QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG

SEQ ID NO: 15 [amino acids 26-382 of SEQ ID NO:4]

AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMM GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG

SEQ ID NO:16 [amino acids 27-382 of SEQ ID NO:4]

MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIY VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG

SEQ ID NO: 17

SEQ ID NO: 18 [CTLA4 extracellular domain sequence]

MHVAQPAWLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI YVIDPEPCPDSD

SEQ ID NO: 19

5’-AGAAAAGGGGCTGGAGAGATGGCTCAGTGGTTAAG AGCA-3 ’

SEQ ID NOS: 20-22

SEQ ID NO: 20 - 5’-GTACTCAGG

SEQ ID NO:21 ״ AGTCAGAGAC

SEQ ID NO: 22 - CGGCAGATCTCTGTGAGTTTGAGGCCAGCCTGG TCTACAA AGO AAGTT-3 ’

[00254] CTLA4-Ig Monomers and Multimers

[00255] In certain embodiments, the invention provides cell lines having an expression cassette that comprises SEQ ID NO:1 (FIG. 1 A). Such an expression cassette when expressed in mammalian cells, including CHO cells, can result in the production of N- and Cterminal variants, such that the proteins produced from the expression cassette can have the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2; (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382 of SEQ ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2 (FIG. 1 A). These proteins can be referred to herein as “SEQ ID NO:2 monomers,” or monomers “having a SEQ ID NO:2 sequence.” These SEQ ID NO:2 monomers can dimerize, such that dimer combinations can include, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii) and (ii);

(ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii) and (v);

(iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); and, (vi) and (vi). These different dimer combinations can also associate with each other to form tetramer CTLA4-Ig molecules. These monomers, dimers, teramers, and other multimers can be referred to herein as “SEQ ID NO:2 proteins” or proteins ‘‘having a SEQ ID NO.2 sequence. While the cell lines can produce these variants immediately upon translation, the variants can more typically be a product of post-translational actions in the cells. The cell line also secretes CTLA4-Ig molecules. Abatacept refers to SEQ ID NO:2 proteins.

[00256] CTLA4-Ig molecules can include, for example, CTLA4-Ig proteins in monomer, dimer, trimer, tetramer, pentamer, hexamer, or other multimeric forms. CTLA4-Ig molecules can comprise a protein fusion with at least an extracellular domain of CTLA4 and an immunoglobulin constant region. CTLA4-Ig molecules can have wild-type or mutant sequences, for example, with respect to the CTLA4 extracellular domain and immunoglobulin constant region sequences. CTLA4-Ig monomers, alone, or in dimer, tetramer or other multimer form, can be glycosylated.

[00257] In some embodiments, the invention provides populations of CTLA4-Ig molecules that have at least a certain percentage of dimer or other multimer molecules. For example, the invention provides CTLA4-Ig molecule populations that are greater than 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% CTLA4-ig dimers. In one embodiment, the invention provides a CTLA4-Ig molecule population that comprises from about 95% to about 99.5% CTLA4-Ig dimer and from about 0.5% to about 5% of CTLA4-Ig tetramer. In another embodiment, the CTLA4-Ig molecule population comprises about 98 % CTLA4-Ig dimer, about 1.5% CTLA4-Ig tetramer and about 0.5% CTLA4-Ig monomer.

[00258] In one embodiment, the invention provides a population of CTLA4-Ig molecules wherein the population is substantially free of CTLA4-Ig monomer molecules. Substantially free of CTLA4-Ig monomer molecules can refer to a population of CTLA4-Ig molecules that have less than 1 %, 0.5%, or 0.1 % of monomers.

[00259] In one embodiment, the invention provides a population of CTLA4-Ig molecules wherein the population is substantially free of CTLA4-Ig multimers that are larger than dimers, such as tetramers, hexamers, etc. (e.g., high molecular weight species). Substantially free of CTLA4-Ig multimer molecules larger than dimers can refer to a population of CTLA4-Ig molecules that have less than 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of CTLA4-Ig multimers (e.g., high molecular weight species) larger than dimeric form.

[00260] A CTLA4-Ig monomer molecule can have, for example, the amino acid sequence • of: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2 (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382 of SEQ ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2. When an expression cassette comprising the nucleic acid sequence of SEQ ID NO:1 is expressed in CHO cells, the predominant monomer form expressed has the Nterminus amino acid residue of methionine (residue 27 of SEQ ID NO:2), which corresponds to the N-terminus amino acid residue of wild-type human CTLA4. However, because SEQ ID NO:1 also includes the coding sequence for an Oncostatin M Signal Sequence (nucleotides 11-88 of SEQ ID NO:1), the expressed protein from SEQ ID NO:1 contains an Oncostatin M Signal Sequence. The signal sequence is cleaved from the expressed protein during the process of protein export from the cytoplasm, or secretion out of the cell. But cleavage can result in N-terminal variants, such as cleavage between amino acid residues 25 and 26 of SEQ ID NO. 2 (resulting in an N-terminus of residue 26, i.e., the “Ala variant”), or between amino acid residues 24 and 25 of SEQ ID NO. 2 (resulting in an N-terminus of residue 25, i.e., the “Met-Ala variant”), as opposed to cleavage between amino acid residues 26 and 27־ of SEQ ID NO. 2 (resulting in an N-terminus of residue 27). For example, the Met-Ala variant can be present in a mixture of CTLA4-Ig molecules at about 1%, and the Ala variant can be present in a mixture of CTLA4-Ig molecules at about 8-10%. In addition, the expressed protein from SEQ ID NO:1 can have C-terminus variants due to incomplete processing. The predominant C-terminus is the glycine at residue 382 of SEQ ID NO:2. In a mixture of CTLA4-Ig molecules, monomers having lysine at the C-terminus (residue 383 of SEQ ID NO:2) can be present, for example, at about 4-5%.

[00261] In one embodiment, a CTLA4-Ig molecule has the amino acid sequence of SEQ ID NO: 5 as follows (which is the same as amino acids 25-383 of SEQ ID NO:2):

[SEQ ID NO: 5]

MAMHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGT QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK;

[00262] In another embodiment, a CTLA4-Ig molecule has the amino acid sequence of SEQ ID NO: 6 as follows (which is the same as amino acids 26-383 of SEQ ID NO:2):

[SEQ ID NO: 6]

AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK;

[00263] In another embodiment, a CTLA4-Ig molecule has the’ amino acid sequence of

SEQ ID NO: 7 as follows (which is the same as amino acids 27-383 of SEQ ID NO:2):

[SEQ ID NO: 7]

MHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG

NELTFLDDSICTGTSSGNQVNLT1QGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK

EYKCKVSNKALP.APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK;

[00264] In another embodiment, a CTLA4-Ig molecule has the amino acid sequence of

SEQ ID NO: 8 as follows (which is the same as amino acids 25-382 of SEQ ID NO:2):

[SEQ ID NO: 8]

MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM

MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGT QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL

NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG;

[00265] In one embodiment, a CTLA4-Ig molecule has the amino acid sequence of SEQ ID NO: 9 as follows (which is the same as amino acids 26-382 of SEQ ID NO:2):

[SEQ ID NO: 9]

AMHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM

GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQI YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG;

[00266] In one embodiment, a CTLA4-Ig molecule has the amino acid sequence of SEQ .

ID.NO: .10 as follows (which is the same as amino acids 27-382 of SEQ ID NO:2):

[SEQ ID NO: 10]

MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMG NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG.

[00267] A CTLA4-Ig monomer molecule can comprise an extracellular domain of human CTLA4. In one embodiment, the extracellular domain can comprise the nucleotide sequence of nucleotides 89-463 of SEQ ID NO:1 that code for amino acids 27-151 of SEQ ID NO:2. In another embodiment, the extracellular domain can comprise mutant sequences of human CTLA4. In another embodiment, the extracellular domain can comprise nucleotide changes to nucleotides 89-463 of SEQ ID NO:1 such that conservative amino acid changes are made. In another embodiment, the extracellular domain can comprise a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% ideritical to nucleotides 89-463 ofSEQIDNOrl.

[00268] A CTLA4-Ig monomer molecule can comprise a constant region of a human immunoglobulin. This constant region can be a portion of a constant region, this constant region can have a wild-type or mutant sequence. The constant region can be from human IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD or IgE. The constant region can be from a light chain or a heavy chain of an immunoglobulin. Where the constant region is from an IgG, IgD, or IgA molecule, the constant region can comprise one or more of the following constant region domains: Cl, CHI, hinge, CH2, or CH3. Where the constant region is from IgM or IgE, the constant region can comprise one or more of the following constant region domains: Cl, ChI, Ch2, Ch3, or Ch4. In one embodiment, the constant region can comprise on or more constant region domains from IgG, IgD, IgA, IgM or IgE.

[00269] In one embodiment, CTLA4-Ig dimers are comprised of two monomers, wherein each monomer can have the same or different amino acid sequence, and where the sequence can be the amino acid sequence of: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2. Such CTLA4-Ig monomers can dimerize through the extracellular domain of the human CTLA4 sequence via a cysteine amino acid residue at position 146 of SEQ ID NO:2.

[00270] A CTLA4-Ig molecule can multimerize through the interaction of IgM or IgA constant region domains with a׳ J chain protein. IgM and IgA are usually produced as multimers in association with an additional polypeptide chain, the J chain. In pentameric IgM, the monomers are crosslinked by disulfide bonds to each other in the CH3 domain and to the J chain through the CH4 domain. IgM can also form hexamers that lack a J chain where multimerization is achieved through disulfide bonds to each. In dimeric IgA, the monomers have disulfide bonds to the J chain via their CH3 domain and not each other.

Thus, in one embodiment, the invention provides CTLA4-Ig multimers, including dimers, pentamers, and hexamers, wherein the Ig portion comprises an IgM constant region or portion thereof or an IgA constant region or portion thereof. Such CTLA4-Ig multimers based on IgM or IgA can include the J chain.

[00271] In one embodiment,- a CTLA4-Ig monomer molecule (CTLA4 GenBank Accession No. 113253) comprises a modified human IgGl hinge region (nucleotides 464-508 of SEQ ID NO:1; amino acids 152-166 of SEQ ID NO:2) wherein the serines at amino acid residues 156, 162, and 165 of SEQ ID NO:2 have been engineered from cysteines present in the wild-type sequence.

[00272] In one embodiment, a CTLA4-lg monomer molecule comprises a modified human IgGl CH2 region and a wild-type CH3 region (the modified human IgGl Ch2 domain having nucleotides 509-838 of SEQ ID NO:1 and amino acids 167-276 of SEQ ID NO:2; the human IgGl C_h3 domain having nucleotides 839-1159 of SEQ ID NO:1 and amino acids 277-383 of SEQ ID NO:2).

!00273] In one embodiment, a CTLA4-Ig molecule population comprises monomers having a sequence shown in any one or more of Figures 7, 8, or 9 of the U.S. patent application published as Publication No. US 2002/0182211 Al, and in U.S. patent applications published as Publication Nos. US20030083246 and US20040022787, each of which is hereby incorporated by reference in its entirety.

[00274] In one embodiment, a CTLA4-Ig tetramer molecule comprises two pairs or two dimers of CTLA4-Ig polypeptides, wherein each.polypeptide has one of the following amino acid sequences: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2. Each member of the pair of polypeptides or dimer is covalently linked to the other member, and the two pairs of polypeptides are non-covalently associated with one another thereby forming a tetramer. Such tetramer molecules are capable of binding to CD80 or CD86. In another embodiment, such tetramer molecules can bind to CD80 or CD86 with an avidity that is at least 2-fold greater than the binding avidity of a CTLA4-Ig dimer (whose monomers have one of the above amino acid sequences) to CD80 or CD86. In another embodiment, such tetramer molecules can bind to CD80 or CD86 with an avidity that is at least 2-fold greater than the binding affinity or avidity of wild-type CTLA4 to CD80 or CD86. Such greater avidity can contribute to higher efficacy in treating immune disorders and other diseases as described below, as well as in inhibiting tissue and/or solid organ transplant rejections. In addition, greater or improved avidity can produce the result of higher potency of a drug. For example, a therapeutic composition comprising CTLA4-Ig tetramer would have a higher avidity and therefore higher potency than the same amount of a therapeutic composition having CTLA4-Ig monomer. In another embodiment, such tetramer molecules can have at least a 2-fold greater inhibition on T cell proliferation as compared to a CTLA4-Ig dimer (whose monomers have one of the above amino acid sequences). In another embodiment, such tetramer molecules can have at least a 2-fold greater inhibition on T cell proliferation as compared to a wild-type CTLA4 molecule.

CTLA4^A29YL104E-Ig Monomers, Dimers, and Multimers

[00275] CTLA4^A29YL104E-Ig are modified forms of CTLA4-Ig (FIG. 1 A; SEQ ID NOS: 1 2). The modification consists of point mutations that result in two amino acid substitutions (L104E and A29Y) as shown in FIG. 2 (corresponding to amino acid positions 55 and 130 in FIG. 3; SEQ ID NO: 4). Relative to CTLA4-Ig, CTLA4^A29YL104E-Ig (for example, SEQ ID NOS:5-10) bind CD80 (B7-1) with approximately 2-fold increased avidity, and binds CD86 (B7-2). with approximately 4-fold increased avidity. CTLA4^A29YL104E-Ig are approximately 10-fold more effective than CTLA4-Ig at inhibiting T cell proliferation, cytokine production, and CD28-dependent killing of target cells by natural killer cells. CTLA4^A29YLI04E-Ig cause modest inhibition of B7-1 mediated T cell proliferation but are markedly more potent than CTLA4-Ig at blocking B7-2 mediated T cell proliferation. The increased potency is comparable, whether blocking B7-2 alone or blocking both B7-1 and B7-2, suggesting that the enhanced immunomodulatory activity of CTLA4^A29YL104E-Ig can most likely be attributed to the enhanced potency for blocking B7-2.

[00276] CTLA4^A29YL104E-Ig is a genetically engineered fusion protein, which consists of the functional binding domain of modified human CTLA-4 and the Fc domain of human immunoglobulin of the IgGl class (FIG. 3A-B). Two amino acid substitutions were made in the B7 binding region of the CTLA-4 domain (L104E and A29Y) to generate a CTLA4^A29YLI04E-Ig molecule. A CTLA4^A29YLI04E-Ig dimer is comprised of two glycosylated CTLA4^A29YL104E-Ig chains. It exists as a covalent dimer linked through an inter-chain disulfide bond. A molecular model of a CTLA4^A29YL104E-Ig molecule is shown in FIG. 4. A CTLA4^A29YL104E-Ig molecule has an average mass of approximately 91,800 Da as determined by matrix-assisted laser desorption-ionizationtime-of-flight (MALDI-TOF) mass spectrometry.

[00277] In certain embodiments, the invention provides cell lines having an expression cassette that comprises SEQ ID NO:3. Such an expression cassette when expressed in mammalian cells, for example CHO cells, can result in the production of N- and C- terminal variants, such that the polypeptides produced from the expression cassette can have the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4; (iii) 27383 of SEQ ID NO:4, or (iv) 27-382 of SEQ ID NO:4, or optionally (v) 25-382 of SEQ ID NO:4, or (vi) 25-383 of SEQ ID NO:4. These polypeptides can be referred to herein as “SEQ ID NO:4 monomers,” or monomers “having a SEQ ID NO:4 sequence.”

[00278] These SEQ ID NO:4 monomers can dimerize, such that dimer combinations can include, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi);

(ii) and (ii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv);

(iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v);(iv) and (vi); (v) and (v); (v) and (vi); and, (vi) and (vi). These different dimer combinations can also associate with each other to form tetramer CTLA4^A29YL104E-Ig molecules. These monomers, dimers, teramers, and other multimers can be referred to herein as “SEQ ID NO:4 polypeptides” or polypeptides “having a SEQ ID NO:4 sequence.” While the cell lines can produce these variants immediately upon translation, the variants can more typically be a product of post-translational actions in the cells. Dimers can be covalently joined together, non-covalently joined together, or both. The invention provides for compositions that consist essentially of dimers that are covalently bound together. For example, the invention provides for compositions where at least 50% of the CTLA4-Ig dimers are made up of monomers joined covalently. The invention also provides for compositions where at least 60%, 70%, 80%, 85%, 90%, 95%,98% ,97% ,96% ׳, 99% or 100% of the CTLA4-Ig dimers are made up of monomers joined covalently. The invention also provides for compositions of CTLA4-Ig molecules, wherin the molecules are predominantly in dimer form, and the dimers are predominantly formed by covalent linkages. For example, the invention provides for a composition wherein the majority of the CTAL4-Ig dimers are joined covalently. It is possible that some fraction of the CTLA4-Ig dimers in the composition are joined non-covalently.

[00279] CTLA4^A29YL104E-Ig molecules can include, for example, CTLA4^A29YLI04E־Ig in monomer, dimer, trimer, tetramer, pentamer, hexamer, or other multimeric forms. CTLA4^A29YL104E-Ig molecules can comprise a protein fusion with at least an extracellular domain of modified CTLA4 (SEQ ID NO: 18) and an immunoglobulin constant region. CTLA4^A29YLI04E-Ig molecules can have mutant sequences, for example, with respect to the modified CTLA4 extracellular domain and immunoglobulin constant region sequences. CTLA4^A29YL104E-Ig monomers, alone, or in dimer, tetramer or other multimer form, can be glycosylated.

[00280] In some embodiments, the invention provides populations of CTLA4^A29YL104E-Ig molecules that have at least a certain percentage of dimer or other multimer molecules. For example, the invention provides CTLA4^A29YL104E-Ig molecule populations that are greater than 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of CTLA4^A29YL104E-Ig dimers. In one embodiment, the invention provides a CTLA4^A29YL104E-Ig molecule population that comprises from about 95% to about 99.5% of CTLA4^A29YLI04E-Ig dimer and from about 0.5% to about 5% of CTLA4^A29YL104E-Ig tetramer. In a further embodiment, the invention provides a CTLA4^A29YLI04E-Ig molecule population that comprises from about 95% to about 99.5% of CTLA4^A29YLI04E-Ig dimer, from about 0.5% to about 2.5% of CTLA4^A29YL104E-Ig monomer, and from about 0.5% to about 5% of CTLA4^A29YLI04E-Ig tetramer. In another embodiment, the CTLA4^A29YL104E-Ig molecule population comprises about 96% of CTLA4^A29YL104E-Ig dimer, about 2.5% of CTLA4^A29YL104E-Ig tetramer, and about 0.5% of CTLA4^A29YLI04E-Ig monomer.

[00281] In one embodiment, the invention provides a population of CTLA4^A29YL104E-Ig molecules wherein the population is substantially free of CTLA4^A29YL!04E-Ig monomers.

Substantially free of CTLA4^A29YL104E-Ig monomers can refer to a population of

CTLA4^A29YL104E-Ig molecules that have less than 1%, 0.5%, or 0.1% of monomers.

[00282] In another embodiment, the invention provides a population of CTLA4^A29YL104E-Ig molecules wherein the population is substantially free of CTLA4^A29YL104E-Ig multimers that are larger than dimers, such as tetramers, hexamers, etc. Substantially free of CTLA4^A29YL104E-Ig multimers larger than dimers can refer to a population of CTLA4^A29YL104E-Ig molecules that have less than. 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of CTLA4^A29YLI04E-Ig multimers larger than dimers.

[00283] A CTLA4^A29YLI04E-Ig monomer can have, for example, the amino acid sequence of: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4 (iii) 27-383 of SEQ ID NO:4, or (iv) 27-382 of SEQ ID NO:4, or optionally (v) 25-382 of SEQ ID NO:4, or (vi) 25-383 of SEQ ID NO:4. When an expression cassette comprising the nucleic acid sequence of SEQ ID NO:3 or 23 is expressed in CHO cells, the predominant monomer form expressed has the Nterminus amino acid residue of methionine (residue 27 of SEQ ID NO:4), which corresponds to the N-terminus amino acid residue of human CTLA4. However, because SEQ ID NO:23 also includes the coding sequence for an Oncostatin M Signal Sequence (nucleotides 11-88 of SEQ ID NO:23), the expressed protein from SEQ ID NO:23 contains an Oncostatin M Signal Sequence.

[00284] The signal sequence is cleaved from the expressed protein during the process of protein export from the cytoplasm, or secretion out of the cell. But cleavage can result in Nterminal variants, such as cleavage between amino acid residues 25 and 26 of SEQ ID NO:4 (resulting in an N-terminus of residue 26, i.e., the “Ala variant”), or between amino acid residues 24 and 25 SEQ ID NO:4 (resulting in an N-terminus of residue 25, i.e., the “Met-Ala variant”), as opposed to cleavage between amino acid residues 26 and 27 SEQ ID NO:4 (resulting in an Ν-terminus beginning with the Met residue at amino acid position 27). For example, the. Met.Ala variant can be. present in a mixture of CTLA4^A29YLI04E-Ig molecules at about.1%, and.the Ala variant can be present in a mixture of CTLA4^A29YLI04E-Ig molecules at about 10-20%.

[00285] In addition, the expressed protein from a nucleic acid comprising SEQ ID NO:3 can have C-terminus variants due to incomplete processing. The predominant C-terminus is the glycine at residue 382 of SEQ ID NO:4. In a mixture of CTLA4^A29YLI04E-Ig molecules, monomers having lysine at the C-terminus (residue 383 of SEQ ID NO :4) can be present, for example, at about 4-8%

[00286] In one embodiment, a CTLA4^A29YL104E-Ig molecule comprises the amino acid sequence of SEQ ID NO: 11 as follows (which is the same as amino acids 25-383 of SEQ ID NO:4):

[SEQ ID NO: 11]

MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGT QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL

NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK.

[00287] In another embodiment, a CTLA4^A29YL104E-Ig molecule comprises the amino acid sequence of SEQ ID NO: 12 as follows (which is the same as amino acids 26-383 of SEQ ID NO:4):

[SEQ ID NO: 12]

- AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMM GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.

[00288] In a further embodiment, a CTLA4^A29YL104E-Ig molecule comprises the amino acid sequence of SEQ ID NO: 13 as follows (which is the same as amino acids 27-383 of SEQ ID NO:4):

[SEQ ID NO: 13]

MHVAQPAWLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIY

VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVV

VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK

EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS

DIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK.

[00289] In another embodiment, a CTLA4^A29YL104E-Ig molecule comprises the amino acid sequence of SEQ ID NO: 14 as follows (which is the same as amino acids 25-382 of SEQ ID NO:4):

[SEQ ID NO: 14]

MAMHVAQPAWLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGT QIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG.

[00290] In one embodiment, a CTLA4^A29YL104E-Ig molecule has the amino acid sequence of SEQ ID NO: 15 as follows (which is the same as amino acids 26-382 of SEQ ID NO:4):

[SEQ ID NO: 15]

AMHVAQPAWLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMM GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQI YVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG.

[00291] In a further embodiment, a CTLA4^A29YL104E-Ig molecule has the amino acid sequence of SEQ ID NO: 16 as follows (which is the same as amino acids 27-382 of SEQ ID NO:4):

[SEQ ID NO: 16]

MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMG NELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIY VIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYK.TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG.

[00292] A CTLA4^A29YLI04E-Ig monomer comprises an extracellular domain of human CTLA4, wherein two amino acid substitutions were made in the CTLA-4 domain (L104E and A29Y) (FIG. 5). In one embodiment, the extracellular domain can comprise the nucleotide sequence of nucleotides 89-463 of SEQ ID NO:23 that code for amino acids 27-151 of SEQ ID NO:4. In another embodiment, the extracellular domain can comprise mutant sequences of human CTLA4 (such as single, double, and triple site mutants in amino acids 27-151 of SEQ ID NO:4). In another embodiment, the extracellular domain can comprise nucleotide changes to nucleotides 89-463 of SEQ ID NO:23 such that conservative amino acid changes are made. In a further embodiment, the extracellular domain can comprise nucleotide changes to nucleotides 89-463 of SEQ ID NO:23 such that non-conservative amino acid changes are made. In another embodiment, the extracellular domain can comprise a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 89-463 of SEQ ID NO:23.

[00293] A CTLA4^A29YL,04E-Ig monomer can comprise a constant region of a human immunoglobulin. This constant region can be a portion of a constant region. This constant region also can have a wild-type or mutant sequence. The constant region can be from human IgG!, IgG₂, IgG3, IgG4, IgM, IgA!, IgA₂, IgD or IgE. The constant region can be from a light chain or a heavy chain of an immunoglobulin. Where the constant region is from an IgG, IgD, or IgA molecule, the constant region can comprise one or more of the following constant region domains: Cl, ChI, hinge, Ch2, or Ch3. Where the constant region is from IgM or IgE, the constant region can comprise one or more of the following constant region domains: Cl, ChI, Ch2, Ch3, or Ch4. In one embodiment, the constant region can comprise on or more constant region domains from IgG, IgD, IgA, IgM or IgE.

[00294] In one embodiment, CTLA4^A29YL104E-Ig dimers are.'comprised of two monomers, wherein each monomer can have the same or different amino acid sequence, and where the sequence can be the amino acid sequence of: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4. Such CTLA4^A29YL104E-Ig monomers can dimerize through the extracellular domain of the human CTLA4 sequence via a cysteine amino acid residue at position 146 of SEQ ID NO:4 (or cysteine amino acid residue at position 120 of FIG. 5).

[00295] A CTLA4^A29YL104E-Ig molecule can multimerize through the interaction of IgM or IgA constant region domains with a J chain protein. IgM and IgA are usually produced as multimers in association with an additional polypeptide chain, the J chain. In pentameric IgM, the monomers are crosslinked by disulfide bonds to each other in the Ch3 domain and to the J chain through the C_H4 domain. IgM can also form hexamers that lack a J chain where multimerization is achieved through disulfide bonds to each. In dimeric IgA, the monomers have disulfide bonds to the J chain via their Ch3 domain and not each other. Thus, in one embodiment, the invention provides CTLA4^A29YLI04E-Ig multimers, including dimers, pentamers, and hexamers, wherein the Ig portion comprises an IgM constant region or portion thereof or an IgA constant region or portion thereof. Such CTLA4^A29YLI04E-Ig multimers based on IgM or IgA can include the J chain.

[00296] In one embodiment, a CTLA4^A29YL104E-Ig monomer comprises a modified human IgGl hinge region (nucleotides 464-508 of SEQ ID NO:23; amino acids 152-166 of SEQ ID NO:4) wherein the serine residues at positions 156, 162, and 165 of SEQ ID NO:4 have been engineered from cysteine residues present in the wild-type sequence.

[00297] In one embodiment, a CTLA4^A29YL104E-Ig monomer comprises a modified human IgGl CH2 region and a wild-type CH3 region (the modified human IgGl Ch2 domain having nucleotides 509-838 of SEQ ID NO:1 and amino acids 167-276 of SEQ ID NO:2; the human IgGl Ch3 domain having nucleotides 839-1159 of SEQ ID NO:1 and amino acids 277-383 of SEQ ID NO:2).

[00298] In one embodiment, a CTLA4^A29YL104E-Ig molecule population comprises monomers having a sequence shown U.S. Patent Application Publication Nos. U.S. 2002/0039577, U.S. 2003/0007968, U.S. 2004/0022787, U.S. 2005/0019859 and U.S.

2005/0084933, and U.S. Patent No. 7,094,874, each of which is hereby incorporated by ״reference in its entirety.

[00299] In one embodiment, a CTLA4^A29YL104E-Ig tetramer comprises two pairs or two dimers of CTLA4^A29YLI04E-Ig molecules, wherein each polypeptide has one of the following amino acid sequences: (i) 26-383 ofSEQ ID NO:4, (ii) 26-382 ofSEQ ID NO:4, (iii) 27-383 ofSEQ ID NO:4, (iv) 27-382 ofSEQ ID NO:4, (v) 25-382 ofSEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4. Each member of the pair of polypeptides or dimer is covalently linked to the other member, and the two pairs of polypeptides are non-covalently associated with one another thereby forming a tetramer. Such tetramer molecules are capable of binding to CD80 or CD86. In another embodiment, such tetramer molecules can bind to CD80 or CD86 with an avidity that is at least 2-fold greater than the binding avidity of a CTLA4^A29YL104E-Ig dimer (whose monomers have one of the above amino acid sequences) to CD80 or CD86.

[00300] Such greater avidity can contribute to higher efficacy in treating immune disorders and other diseases as described below, as well as in inhibiting tissue and/or solid organ transplant rejections. In addition, greater or improved avidity can produce the result of higher potency of a drug. For example, a therapeutic composition comprising CTLA4^A29YL104E-Ig tetramer would have a higher avidity and therefore higher potency than the same amount of a therapeutic composition having CTLA4^A29YL104E-Ig monomer. In another embodiment, such tetramer molecules can have at least a 2-fold greater inhibition on T cell proliferation as compared to a CTLA4^A29YLI04E-Ig dimer (whose monomers have one of the above amino acid sequences). In another embodiment, such tetramer molecules can have at least a 2-fold greater inhibition on T cell proliferation as compared to a CTLA4-Ig tetramer molecule.

[00301] Characterization of CTLA4-lg and CTLA4^A29YL104E־Ig molecules

[00302] T cell proliferation can be measured using standard assays known in the art. For example, one of the most common ways to assess T cell proliferation is to stimulate T cells via antigen or agonistic antibodies to TCR and to measure, for example, the incorporation of tritiated thymidine (³H-TdR) in proliferating T cells or the amount of cytokines released by proliferating T cells into culture. The inhibitory effect of CTLA4-Ig or CTLA4^A29YLI04E-Ig molecules upon T cell activation or proliferation can thereby be measured.

[00303] The affinity of a CTLA4-Ig molecule is the strength of binding of the molecule to a single ligand, including CD80, CD86, or CD80Ig or CD86Ig fusion proteins. The affinity of CTLA4-Ig or CTLA4^A29YLl04E-Ig to .ligands can be measured, for example, by using binding interaction analysis (BIA) based on surface plasmon technique. Aside from measuring binding strength, it permits real time determination of binding kinetics, such as association and dissociation׳rate constants. A sensor chip, consisting of a glass slide coated with a thin metal film, to.which a surface matrix is covalently attached, is coated with one of the interactants, i.e, CTLA4-Ig, CTLA4^A29YLI04E-Ig, or one of the ligands. A solution containing the other interactant is allowed to flow over its surface. A continuous light beam is directed against the other side of the surface, and its reflection angle is measured. On binding of CTLA4-Ig or CTLA4^A29YL104E-Ig to the ligand, the resonance angle of the light beam changes (as it depends on the refractive index of the medium close to the reactive side of the sensor, which in turn is directly correlated to the concentration of dissolved material in the medium). It is subsequently analyzed with the aid of a computer.

[00304] In one embodiment, CTLA4-Ig binding experiments can be performed by surface plasmon resonance (SPR) on a BIAcore instrument (BIAcore AG, Uppsala, Sweden). CTLA4-Ig can be covalently coupled by primary amine groups to a carboxymethylated dextran matrix on a BIAcore sensor chip, thereby immobilizing CTLA4-Ig to the sensor chip. Alternatively, an anti-constant region antibody can be used to.immobilize CTLA4-Ig indirectly to the sensor surface via the Ig fragment. Thereafter, ligands are added to the chip to measure CTLA4-Ig binding to the ligands. Affinity measurements can be performed, for example, as described in van der Merwe, P. et al., J. Exp. Med. (1997) 185 (3):393-404, which is hereby incorporated by reference in its entirety. In another embodiment, CTLA4^A29YLI04E-Ig binding experiments can be performed using surface plasmon resonance (SPR) technology as described above (FIG. 6; see EXAMPLE 21).

[00305] The avidity of CTLA4-Ig or CTLA4^A29YL104E-Ig molecules can also be measured. Avidity can be defines as the sum total of the strength of of binding of two molecules or cells to one another at nrultimple sites. Avidity is distinct from affininty, which is the strength of binding one site on a molecule to its ligand. Without being bound by theory, higer avidity of CTLA4-Ig or CTLA4^A29YL104E-Ig molecules can lead to increased potency of inhibiton by CTLA4-Ig or CTLA4^A29YL104E-Ig molecules on T-cell proliferation and activation. Avidity can be measured, for example, by two categories of solid phase assays: a) competitive inhibition assays, and b) elution assays. In both of them the ligand is attached to a solid support. In the competitive inhibition assay, CTLA4-Ig or CTLA4^A29YL104E-Ig molecules are then added in solution at a fixed concentration, together with free ligand in different concentrations, and the amount of ligand, which inhibits solid phase binding by 50%, is determined. The less ligand needed, the stronger the avidity. In elution assays, the ligand is added in solution. After obtaining a state of equilibrium, a chaotrope or denaturant agent (e.g. isothiocyanate, urea, or dieth-ylamine) is added in different concentrations to disrupt CTLA4Ig/ligand interactions or CTLA4^A29YL104E-Ig /ligand interactions. The amount of CTLA4-Ig or CTLA4^A29YL104E-Ig resisting elution is determined thereafter with an ELISA. The higher the avidity, the more chaotropic agent is needed to elute a certain amount of CTLA4-Ig or CTLA4^A29YLI04E־lg. The relative avidity of a heterogeneous mixture of CTLA4-Ig molecules or CTLA4^A29YL,04E-Ig can be expressed as the avidity index (AI),־ equal to the concentration of eluting agent needed to elute 50% of the bound CTLA4-Ig or CTLA4^A29YL104E-Ig molecules. Refined analysis of data can be performed by determining percentages of eluted CTLA4-Ig or CTLA4^A29YL104E-Ig at different concentrations of the eluting agent.

[00306] A Phenyl Sepharose 4 Fast Flow column chromatography, Hydrophobic Interaction Chromatography (HIC), process can be used to reduce the amount of CTLA4-Ig high molecular weight species eluted in a HIC purification step (see Example 15). Therefore, the cleaning peak from the HIC column is enriched in CTLA4-Ig HMW species. For example, preparative single or tandem column SEC HPLC can be employed to purify dimer, tetramer and multimer subpopulations from HIC cleaning peak material. In one embodiment, the purified components are CTLA4-Ig dimer, tetramer, and hexamer. Characterization of high molecular weight components of CTLA4-Ig present in the HIC cleaning peak can be done by static and dynamic light scattering techniques. Samples taken at the hydrophobic interaction chromatography (HIC) process step chase revealed the presence of dimer, tetramer, and multimers at various sampling points. Hexamer species can be detected only in samples corresponding to the start of the cleaning peak and cleaning peak maximum OD. Decamer species were detected in the cleaning peak maximum OD only. Molar mass and hydrodynamic radius formation can be determined by fractionation via size exclusion chromatography (SEC) employing MultiAngle light scattering (MALS) coupled with quasi elastic light scatter (QELS) detection.

[00307] With respect to CTLA4-Ig molecules produced from the cell line, SEC shows the Protein A eluate is a mixture of multimer, tetramer, and dimer components. Fractionation of this mixture on preparative tandem SEC column enables isolation of quantities of multimer, tetramer and dimer species. The area percent recovery for each component in SEC analysis of the isolated fractions results in 93-98% homogeneity for each fraction. In one aspect, purification of the individual components enables comparison of the physicochemical .properties of components of CTLA4-Ig HMW material to those of CTLA4-Ig dimer. FIG. 7 shows the apparent molecular weights, which correspond to multimer, tetramer, and dimer fractions of CTLA4-Ig HIC cleaning peak, as determined by SEC with dynamic light scattering detection (DSL) and retention time on SEC. In one embodiment, the biospecific binding activity of purified components from the HIC cleaning peak is comparable to the binding activity of determined by the BIAcore based immobilized B7.-lIg binding assay. In another aspect, sialic acid molar ratio for components isolated from HIC cleaning peak are in the range of 4.9 to 7.6 whereas the sialic acid molar ratio of CTLA4-Ig molecules or dimer (not in the HIC cleaning peak) is in the range of 8- 10. Analysis by IEF gel indicates reduced mobility CTLA4-Ig isoforms purified from HIC cleaning peak compared to the migration of CTLA4-Ig dimer. This is consistent with lower sialic acid molar ratios observed for the CTLA4-Ig HIC cleaning peak fractions (FIG. 8).

[00308] The choice of cell culture conditions can influence the formation of single chain (i.e., monomer) and high molecular weight species (i.e, dimers, tetramers, etc.) of a recombinant protein product. Growth conditions, also including but not limited to media composition, are factors that can affect the formation of single chain, and the level of cysteinylation. This is likely the result of presence of agents that lead to disulfide bond reduction. The supplementation of cysteine directly or cysteine containing media to cells secreting CTLA4-Ig or CTLA4^A29YL104E-Ig can result in a rapid formation of single chain and high molecular weight species. The rate is proportional to amount of cysteine added. In another embodiment, the supplementation of iodo acetamide, a compound that reacts with free sulfhydryls, blocks the formation of high molecular weight species of CTLA4-Ig or CTLA4^A29YLI04E-Ig that are dependent upon disulfide bonds.

[00309] For example, the iodoacetamide sensitive and non-sensitive high molecular weight pathway highlight two major and distinctly different mechanisms by which high molecular weight species can form in CTLA4-Ig. The supplementation of high salt concentrations (0.5M) to־CTLA4-Ig solutions results in a sustained, rapid rate of high molecular weight formation. EDTA, ConAcidSol II, and yeastolates modestly increase single chain formation (see Example 5).

[00310] In certain embodiments, the invention provides methods for generating high molecular weight CTLA4-Ig populations, wherein mixtures containing predominantly monomers or dimers of CTLA4-Ig are supplemented with high salt such that the mixture has׳ . ׳ a salt concentration greater than about 0.3,0.4, 0.45, 0.5, or 0.6M. In one embodiment, such methods generate a mixture comprising a CTLA4-Ig population that has at least 50%, 55%, 60%, 65%, •70%; 75%,•80%, 85%, 90%, or 95% CTLA4-Ig tetramer molecules.

[00311] In one embodiment, the invention provides a population of CTLA4-Ig single chain species containing a modification on Cys¹⁴⁶ such that it is cysteinylated (see Example 4). Cysteinlyation is a posttranslational modification wherein a cysteine within a polypeptide chain is modified by the attachment of another cysteine via a disulfide bond. Cysteinylation of proteins have been implicated in modifying protein bioactivity including immunogenicity and antigenicity of MHC Class-I restricted viral determinants. In one embodiment, the invention provides a composition that comprises at least 1, 5, 10, 15, 20, 25, 50, 75, 90,or 95% of cysteinylated single chain CTLA4-Ig molecules. In another embodiment of the invention, a CTLA4-Ig population has no more than about 1 % CTLA4-Ig monomer molecules, or in another embodiement, less than 0.6% CTLA4-Ig monomer.

[00312] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of sialic acid to CTLA4-Ig molecules of from about 5 to about 18. In some embodiments the average molar ratio of sialic acid to CTLA4-Ig molecules is between from about X to about Y, inclusive of X and Y, where X is about 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, or 17, and Y is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. In other embodiments the average molar ratio of sialic acid to CTLA4-Ig molecules is between from about X to about Y, inclusive of X and Y, where X is about 4.0, 4.5, 5.0, 5.5 or 6.0, and Y is about 8.0, 8.5, 9.0, 9.5, or 10.0. In other embodiments the average molar ratio of sialic acid to CTLA4-Ig molecules is between from about X to about Y, inclusive of X and Y, where X is about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0 and Y is about 11.0, 11.5, 12.0,12.5 or 13.0. In other embodiments the average molar ratio of sialic acid to CTLA4-Ig molecules is from about 6׳ to about 14, from about 7 to about 13, from about 8 to about 12, or from about 9 to about 11. In other embodiments the average molar ratio of sialic acid to CTLA4-Ig molecules is from about 5 to about 9, from about 5.5 ־ to about 9.5, from about 6 to about 9, from about 6 to about 10, or from about 7 to about 10. In other embodiments the average molar ratio of sialic acid to CTLA4-Ig molecules is greater than or equal to 5, or greater than or equal to 8. In certain embodiments, the sialic acid is Nacetyl neuraminic acid (NANA).

[00313] Hie present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of N-glycolyl neuraminic acid (NGNA) to CTLA4-Ig molecules of less than or equal to 2.5, less than or equal to 2.0, less than or equal to 1.5, less than or equal to 1.0, or less than or equal to 0.5.

[00314] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are greater than or equal to 93.0 area percent, greater than or equal to 93.5 area percent, greater than or equal to 94.0 area percent, greater than or equal to 94.5 area percent, greater than or equal to 95.0 area percent, greater than or equal to 95.5 area percent, greater than or equal to 96.0 area percent, greater than or equal to 96.5 area percent, or greater than or equal to 97.0 area percent CTLA4-Ig dimers as determined by size exclusion chromatography and spectrophotometric detection. In some embodiments, the composition comprises CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are greater than or equal to 95.0 area percent CTLA4-Ig dimers, and less than or equal to 4.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection. In some embodiments, the composition comprises CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are greater than or equal to 95.0 area percent CTLA4-Ig dimers, and less than or equal to 5.0 area percent high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

[00315] י The present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are of less than or equal to 2.0 area percent, less than or equal to 1.5 area percent, less than or equal to 1.0 area percent; or less than or equal to 0.5 area percent area percent CTLA4-Ig monomers (i.e., single chain) as determined by size exclusion chromatography and spectrophotometric detection.

[00316] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are of less than or equal to 5.0 area percent, less than or equal to 4.5 area percent, less than or equal to 4.0 area percent, less than or equal to 3.5 area percent, less than or equal to 3.0 area percent, less than or equal to 2.5 area percent, less than or equal to 2.0 area percent, less than or equal to 1.5 area percent, less than or equal to 1.0 area percent, or less than or equal to 0.5 area percent CTLA4-Ig high molecular weight species (e.g., tetramer) as determined by size exclusion chromatography and...

spectrophotometric detection. In some embodiments, especially those involving concentrated compositions comprising CTLA4-Ig molecules, (such as, for example, those for subcutaneous administration) the CTLA4-Ig molecules are of less than or equal tolO area percent, less than or equal to 9 area percent, less than or equal to 8 area percent, less than or equal to 7 area percent, less than or equal to 6 area percent CTLA4-Ig high molecular weight species as determined by size exclusion chromatography and spectrophotometric detection.

[00317] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises an amount of MCP-1 or MCP-l-like material less than or equal to 50 ppm, less than or equal to 40 ppm, less than or equal to 38 ppm, less than or equal to 30 ppm less than or equal to 20 ppm, less than or equal to 10 ppm, 5 ppm, less than or equal to 4 ppm, less than or equal to less than or equal to 3 ppm, less than or equal to 2 ppm or less than or equal to 1 ppm. The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises MCP-1 or MCP-l-like material at less than or equal to 50 ng/mg CTLA4-Ig molecules, less than or equal to 40 ng/mg CTLA4Ig molecules, less than or equal to 38 ng/mg CTLA4-Ig molecules, less than or equal to 30 ng/mg CTLA4-Ig molecules, less than or equal to 20 ng/mg CTLA4-Ig molecules, less than or equal to 10 ng/mg CTLA4-Ig molecules, less than or equal to 5 ng/mg, less than or equal to' 4 ng/mg CTLA4-Ig molecules, less than or equal to 3 ng/mg CTLA4-Ig molecules, less than or equal to 2 ng/mg CTLA4-Ig molecules or less than or equal to 1 ng/mg CTLA4-Ig molecules. The present invention provides a composition comprising CTLA4-Ig molecules and an amount of MCP-1 (including the absence of MCP-1) wherein said composition is a .pharmaceutically acceptable composition.

[00318] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of galactose to CTLA4-Ig molecules of from about 6 to about 19. In some embodiments the average molar ratio of sialic acid to CTLA4-Ig molecules is from about X to about Y, where X is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, and Y is about 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18 or

19. In other embodiments the average molar ratio of galactose to CTLA4-Ig molecules is between from about X to Y, inclusive of X and Y, where X is about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9;0, 9.5, or 10.0 and Y is about 12.0, 12.5,13.0,13.5,14;0,14.5, 15.0,15.5, or 16.0. In other embodiments the average molar ratio of galactose to CTLA4-Ig molecules is between from about X־to about Y, inclusive of X and Y, wherein X is about 6.0, 6.5, 7.0, 7.5 or 8.0 and Y is about 15.0,15.5,16.0, 16.5,17.0,17.5,18.0,18.5, or 19.0. In other embodiments the average molar ratio of galactose to CTLA4-Ig molecules is from about from about 7 to about 15, from about 8 to about 14, from about 9 to about 13, from about 10 to about 12. In other embodiments the average molar ratio of galactose to CTLA4-Ig molecules is from about 7 to about 18, from about 8 to about 17, from about 9 to about 17, from about 9 to about 16, or from about 10 to about 15. In other embodiments the average molar ratio of galactose to CTLA4-Ig molecules is greater than or equal to 8.

[00319] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of fucose to CTLA4-Ig molecules of from about 0.5 to about 12. In some embodiments the average molar ratio of sialic acid to CTLA4-Ig molecules is between from about X to about Y, inclusive of X and Y, where X is about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.5, and Y is about 7.0, 7.5, 8.0,

8.5, 9.0, 9.5,10.0 or 10.5. In other embodiments the average molar ratio of fucose to CTLA4-Ig molecules is between from about X to Y, inclusive of X and Y, where X is about

2.9, 3.1, 3.3, 3.5, 3.7, 3.9, or 4.1 and Y is about 7.9, 8.1, 8.3, 8.5, 8.7, 8.9, or 9.1. In other embodiments the average molar ratio of fucose to CTLA4-Ig molecules is between from about X to Y, inclusive of X and Y, wherein X is about 1.0, 1.5, 1.7, 1.9, 2.1,2.3, or 2.5, and Y is about 8.7, 8.9, 9.1, 9.3, 9.6, 9.9, 10.1,10.3 or 10.5. In other embodiments the average molar ratio of fucose to CTLA4-Ig molecules is from about from about 3.3 to about 8.5, from about 3.5 to about 8.3, from about 3.7 to about 8.1, from about 3.9 to about 7.9. In other embodiments the average molar ratio of fucose to CTLA4-Ig molecules is from about 1.5 to about 9.5, from about 1.7 to about 9.3, from about 1.9 to about 9.1, or from about 2.1 to about

8.9. In other embodiments the average molar ratio of fucose to CTLA4-Ig molecules is greater than or equal to 1.7.

[00320] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of mannose to CTLA4-Ig molecules of from about 5 to about 25. In some embodiments the average molar ratio of sialic acid to CTLA4-Ig molecules is between from about X to about Y, inclusive of X and Y, where X is about 5, 6, 7, 8, 9,10, 11, 12, 13, 14,15,16, 17, 18, 19,20 or 21, and Y is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,21,22,23 or 24. In other embodiments the average molar ratio of mannose to CTLA4-Ig molecules is between from about X to Y, inclusive of X and Y, where X is about 6.5, 7.0, 7.5, 7.7, 7.9, 8.1, 8.3, 8.5, 9.0,

9.5, 10.0,10.5,11.0,11.5 or 12.0 and Yisabout 17.0, 17.5,18.0,18.5, 19.0,19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23, 23.5 or 24.0. In other embodiments the average molar ratio of mannose to CTLA4-Ig molecules is between from about X to Y, inclusive of X and Y, where Xis about 8, 8.5, 9.0, 9.5 10.0 or 11.0 and Y is about 17.0, 17.5, 18.0,18.5, 19.0, 19.5 or 20.0. In other embodiments the average molar ratio of mannose to CTLA4-Ig molecules is from about from about 6 to about 23, from about 7 to about 22, from about 7.7 to about 22, from about 8 to about 21, from about 9 to about 20, from about 10 to about 19, from about 11 to about 19, and from about 11 to about 17. In other embodiments the average molar ratio of mannose to CTLA4-Ig molecules is from about 8 to about 19, from about 9 to about 18, from about 10 to about 17, or from about 11 to about 16. In other embodiments the average molar ratio of mannose to CTLA4-Ig molecules is greater than or equal to 7.

[00321] The present .invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are of less than or equal to 5.0 area percent, less than or equal to 4.5 area percent, less than or equal to 4.0 area percent, less than or equal to 3.5 area percent, less than or equal to 3.0 area percent, less than or equal to 2.5 area percent, less than or equal to 2.0 area percent, less than or equal to 1.5 area percent, less than or equal to 1.0 area percent, or less than or equal to 0.5 area percent oxidized species. The present invention provides a.composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are less than or equal to 5.0 area percent, less than or equal to 4.5 area percent, less than or equal to 4.0 area percent, less than or equal to 3.5 area percent, less than or equal to 3.0 area percent, less than or equal to 2.5 area percent, less than or equal to 2.0 area percent, less than or equal to 1.5 area percent, less than or equal to 1.0 area percent, or less than or equal to 0.5 area percent deamidated species. In some embodiments the composition comprises CTLA4Ig molecules, wherein the CTLA4-Ig molecules are less than or equal to 3.5 area percent oxidized species and less than or equal to 2.5 area percent deamidated species.

[00322] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises bacterial endotoxins LAL at less than or equal to 0.7 EU/mg CTLA4-Ig molecules, less than or equal to 0.6 EU/mg CTLA4-Ig molecules, less than or equal to 0.5 EU/mg CTLA4-Ig molecules, less than or equal to 0.42 EU/mg CTLA4Ig molecules, less than or equal to 0.4 EU/mg CTLA4-Ig molecules, less than or equal to 0.35 EU/mg CTLA4-Ig molecules, less than or equal to 0.3 EU/mg CTLA4-Ig molecules, less than or equal to 0.25 EU/mg CTLA4-Ig molecules, less than or equal to 0.20 EU/mg CTLA4Ig molecules, less than or equal to 0.15 EU/mg CTLA4-Ig molecules, or less than or equal to

0.05 EU/mg CTLA4-Ig molecules.

[00323] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises bioburden at less than or equal to 2 CFU/lOmL, less than or equal to 1.5 CFU/lOmL, less than or equal to 1 CFU/lOmL, or less than or equal to 0.5 CFU/lOmL.

[00324] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises DNA at less than or equal to 25 pg/mg CTLA4-Ig molecules, less than or equal to 20 pg/mg CTLA4-Ig molecules, less than or equal to 15 pg/mg CTLA4-Ig molecules, less than or equal to 10 pg/mg CTLA4-Ig molecules, less than or equal to 5.0 pg/mg CTLA4-Ig molecules, less than or equal to 4.0 pg/mg CTLA4-Ig molecules, less than or equal to 3.5 pg/mg CTLA4-Ig molecules, less than or equal to 3.0 pg/mg CTLA4-Ig molecules, less than or equal to 2.5 pg/mg CTLA4-Ig molecules, less than or equal to 1.5 pg/mg CTLA4-Ig molecules, less than or equal to 1.0 pg/mg CTLA4-Ig molecules, or less than or equal to 0.5 pg/mg CTLA4-Ig molecules, or less than or equal to 0.20 pg/ml CTLA4-Ig molecules.

[00325] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises cellular protein (e.g., CHO protein or CHOP) at less than or equal to 200 ng/mg CTLA4-Ig molecules, less than or equal to 150 ng/mg CTLA4-Ig molecules, less than or equal to 125 ng/mg CTLA4-Ig molecules, less than or equal to-100 ng/mg CTLA4-Ig molecules, less than or equal to 90 ng/mg CTLA4-Ig molecules, less than or equal to 80 ng/mg CTLA4-Ig molecules, 70 ng/mg CTLA4-Ig molecules, less than or equal to 60 ng/mg CTLA4-Ig molecules, less than or equal to 50 ng/mg CTLA4-Ig molecules, less than or equal to 40 ng/mg CTLA4-Ig molecules, less than or equal to 30 ng/mg CTLA4-Ig molecules, less than or equal to 25 ng/mg CTLA4-Ig molecules, less than or equal to 20 ng/mg CTLA4-Ig molecules, less than or equal to 15 ng/mg CTLA4-Ig molecules, less than or equal to 10 ng/mg CTLA4-Ig molecules, or less than or equal to 5 ng/mg CTLA4-Ig molecules. The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises cellular protein at less than or equal to 200 ppm, less than or equal to 150 ppm, less than or equal to 125 ppm, less than or equal to 100 ppm, less than or equal to 90 ppm, less than or equal to 80 ppm, 70 ppm, less than or equal to 60 ppm, less than or equal to 50 ppm, ess than or equal to ppm, less than or equal to 30 ppm, less than or equal to 25 ppm, less than or equal to 20 ppm, less than or equal to 15 ppm, less than or equal to 10 ppm, or less than or equal to 5 ppm.

[00326] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises Triton-X (e.g., Triton X-100) at less than or equal to 4.0 ng/mg CTLA4-Ig molecules, less than or equal to 3.5 ng/mg CTLA4-Ig molecules, less than or equal to 3.0 ng/mg CTLA4-Ig molecules, less than or equal to 2.5 ng/mg CTLA4-Ig molecules, less than or equal to 2.0 ng/mg CTLA4-Ig molecules, less than or equal to 1.5 ng/mg CTLA4-Ig molecules, less than or equal to 1.0 ng/mg CTLA4-Ig molecules, or less than or equal to 0.5 ng/mg CTLA4-Ig molecules. The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises Triton-X at less than or equal to 4.0 ppm, less than or equal to 3.5 ppm, less than or equal to 3.0 ppm, less than or equal to 2.5 ppm, less than or equal to 2.0 ppm, less than or equal to 1.5 ppm, less than or equal to 1.0 ppm, or less than or equal to 0.5 ppm.

[00327] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises Protein A at less than or equal to 8.0 ng/mg CTLA4-Ig molecules, less than or equal to 7.5 ng/mg CTLA4-Ig molecules, less than or equal to 7.0 ng/mg CTLA4-Ig molecules, less than or equal to 6.5 ng/mg CTLA4-Ig molecules, less than or equal to 6.0 ng/mg CTLA4-Ig molecules, less than or equal to 5.5 ng/mg CTLA4-Ig molecules, less than or equal to 5.0 ng/mg CTLA4-Ig molecules, less than or equal to 4.5 ng/mg CTLA4-Ig molecules, less than or equal to 4.0 ng/mg CTLA4-Ig molecules, less than or equal to 3.5 ng/mg CTLA4-Ig molecules, less than or equal to 3.0 ng/mg CTLA4-Ig molecules, less than or equal to 2.5 ng/mg CTLA4-Ig molecules, less than or equal to 2.0 ng/mg CTLA4-Ig molecules, less than or equal to 1.5 ng/mg CTLA4-Ig molecules,, less than or equal to 1.0 ng/mg CTLA4-Ig molecules, or less than or equal to 0.5 ng/mg CTLA4-Ig molecules. The present invention provides a composition comprising CTLA4-Ig molecules, wherein the composition comprises Protein A at less than or equal to 8.0 ppm, less than or equal to 7.5 ppm, less than or equal to 7.0 ppm, less than or equal to 6.5 ppm, less than or equal to 6.0 ppm, less than or equal to 5.5 ppm, less than or equal to 5.0 ppm, less than or equal to 4.5 ppm, less than or equal to 4.0 ppm, less than or equal to 3.5 ppm, less than or equal to 3.0 ppm, less than or equal to 2.5 ppm, less than or equal to 2.0 ppm, less than or equal to 1.5 ppm, less than or equal to 1.0 ppm, or less than or equal to 0.5 ppm.

[00328] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of GlcNAc to CTLA4-Ig molecules of from about 10 to about 40. In some embodiments the average molar ratio of GlcNAc to CTLA4-Ig molecules is between from about X to about Y, inclusive of X and Y, where X is any whole number between 10 and 39 and Y is any whole number between 11 and 40. In other embodiments the average molar ratio of GlcNAc to CTLA4-Ig molecules is between .from about X to Y, inclusive of X and Y, where X is about 12, 14, 14, 15, 16 or 17, and Y is about 32, 33, 34, 35, 36 or 37. In other embodiments the average molar ratio of GlcNAc to CTLA4-Ig molecules is from about 12 to about 35, from about 13 to about 35, from about 14 to about 35, from about 15 to about 35.

[00329] The present invention provides a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molar ratio of GalNAc to CTLA4-Ig molecules of from about 0.5 to about 7.0. In some embodiments the average molar ratio of GalNAc to CTLA4-Ig molecules is between from about X to about Y, inclusive of X and Y, where X is 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, and Y is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2,4.3, 4.4,

4.5, .46, 4.7, 4.8, 4.9, 5.0, 6.0, 7.0 or 8.0. In other embodiments the average molar ratio of GalNAc to CTLA4-lg molecules is between from about X to Y, inclusive of X and Y, where X is about 0.6, 0.7, 0.8, 0.9, or 1.0, and Y is about 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1 or 4.2. In other embodiments the average molar ratio of GalNAc to CTLA4-Ig molecules is from about 0.7 to about 4.1, from about 0.8 to about 4.0, from about 0.9 to about 3.9, or about 1.0 to about 3.8, or about 1.1 to about 3.7. In other embodiments the average molar ratio of GalNAc to CTLA4-Ig molecules is from about 1.6 to about 3.7, from about 1.7 to about 3.6, from about 1.8 to about 3.5, or about 1.9 to about 3.4.

[00330] The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig composition exhibits bands in pl ranges as determined on an isoelectric focusing gel (IEF gel) as follows: from about 10 to about 22 bands in the pl range of from about 4.3 to about 5.6; cumulative bands intensity of from about 90% to about 110% in a pl range from about 4.3 to about 5.3 and about 3 major bands in a pl range from about

4.5 to about 5.2. In one embodiment, the bands in the range of from about 4.3 to about 5.6 is from about 5 to about 30, from about 6 to about 29, from about 7 to about 28, from about 8 to about 27, from about 9 to about 26, from about 10 to about 25, from about 11 to about 24, from about 12 to about 23, from about 13 to about 22, from about 14 to about 21, from about to about 20, from about 16 to about 19, from about 17 to about 20, from about 18 to about

19.

[00331} Glycosylated CTLA4־Ig and CTLA4^AZ9YL104E-Ig Molecules and Populations . Thereof

[00332] Without limitation, glycosylation can refer to the addition of complex oligosaccharide structures to a protein at specific sites within the polypeptide chain. Glycosylation of proteins and the subsequent processing of the added carbohydrates can affect protein folding and structure, protein stability, including protein half life, and functional properties of a protein. Protein glycosylation can be divided into two classes by virtue of the sequence context where the modification occurs, O-linked glycosylation and Nlinked glycosylation. O-linked polysaccharides are linked to a hydroxyl group, usually to the hydroxyl group of either a serine or a threonine residue. O-glycans are not added to every serine and threonine residue. O-linked oligosaccharides are usually mono or biantennary, i.e. they comprise one or at most two branches (antennas), and comprise from one to four different kinds of sugar residues, which are added one by one.

[00333] N-linked polysaccharides are attached to the amide nitrogen of an asparagine. Only asparagines that are part of one of two tripeptide sequences, either asparagine-X-serine or asparagine-X-threonine (where X is any amino acid except proline), are targets for glycosylation. N-linked oligosaccharides can have from one to four branches referred to as mono-, bi-, tri- tetraantennary. The structures of and sugar residues found in N- and O-linked oligosaccharides are different. Despite that difference, the terminal residue on each branch of both N- and O-linked polysaccharide can be modified by a sialic acid molecule a modification referred as sialic acids capping. Sialic acid is a common name for a family of unique nine-carbon monosaccharides, which can be linked to other oligosaccharides. Two family members are N-acetyl neuraminic acid, abbreviated as Neu5Ac or NANA, and Nglycolyl neuraminic acid, abbreviated as Neu5Gc or NGNA.

[00334] The most common form of sialic acid in humans is NANA. N-acetylneuraminic acid (NANA) is the primary sialic acid species present in CTLA4-Ig molecules. However, it should be noted that minor but detectable levels of N-glycolylneuraminic acid (NGNA) are also present in CTLA4-Ig molecules. Furthermore, the method described herein can be used to determine the number of moles of sialic acids for both NANA and NGNA, and therefore levels of both NANA and NGNA are determined and reported for CTLA4-Ig molecules. Nand O-linked oligosaccharides have different number of branches, which provide different number of positions to which sialic acid molecules can be attached. N-linked ologosaccharides can provide up to four attachment positions for sialic acids, while O-linked oligosaccharides can provide two sites for sialic acid attachment.

[00335] Glycosylated proteins, (glycoproteins), many of which have been produced by recombinant DNA technology methods, are o.f great interest as diagnostic and therapeutic agents. Many eukaryotic transmembrane proteins destined for the cell surface and secreted proteins are post-translationally modified to incorporate N-linked and O-linked carbohydrate groups. N-linked oligosaccharides are attached to asparagine residues when they are part of the peptide motif Asn-X-Ser/Thr, where X can be any amino acid except proline. O-linked oligosaccharides are attached to serine or threonine residues. The structures of N-linked and O-linked oligosaccharides as well as the sugar residues found in each can be different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (NANA; hereafter referred to as sialic acid). Usually, sialic acid is the terminal residue of both N-linked and Olinked oligosaccharides. The glycoprotein, because of its negative charge, can exhibit acidic properties.

[00336] Glycosylated proteins are purported to play roles in augmenting protein folding, regulating cell sorting and trafficking, preventing protein aggregation, mediating cell-cell adhesion, and increasing resistance to proteolysis. In eukaryotic organisms, the nature and extent of glycosylation can have a profound impact on the circulating half-life and bioactivity of glycoprotein therapeutics by processes which involve receptor mediated endocytosis and clearance. Receptor-mediated systems are thought to play a major role in clearing serum glycoproteins by recognizing the various sugar components of the oligosaccharide. A glycoprotein’s terminal sialic acid group can affect absorption, half-life, and serum clearance. Thus, glycoprotein production strategies, which maintain the terminal sialic acid component of the glycosylated protein, can better increase the protein’s bioavailability and serum halflife. Several production process parameters have been investigated pertaining to recombinant glycoprotein synthesis, especially the effect of media composition and temperature shifts in various production strategies.

[00337] CTLA4-Ig dimers composed of monomers having the amino acid sequence of residues (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID . NO:2, (iv) 27-382 of SEQ IDNO:2, (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2, can have a predicted theoretical MW of about 78,000 to about 79,000 Daltons. However, the MW for such dimers obtained by MALDI-TOF is approximately 91,000 Daltons. This difference in MW of approximately 13,000-:14,000 Daltons is due at least in part to glycosylation, which in one embodiment, accounts for approximately 15% of the mass of this particular CTLA4-Ig monomer molecule. The above specified monomers have three N-linked glycosylation sites that have been confirmed by peptide mapping to occur at asparagines at residues 102, 134, and 233 of SEQ ID NO:2. Carbohydrate molecules that are linked through asparagine can be cleaved selectively using the enzyme Peptide-N Glycosidase F (PNGase F). In one instance, treatment of the monomer having the sequence 27-383 of SEQ ID NO:2 with PNGase F resulted in a species with a MW of approximately 80,200 Daltons, and because the theoretical MW of this monomer is about 80,200, the treatment suggested that the unaccounted ’1,400 Daltons (80,200 - 78,800 = 1,400) can be due to O-linked glycosylation. Although there are numerous serine and threonine residues that have the potential of being glycosylation sites, only two O-linked sites were identified: Ser¹⁵⁵and Ser¹⁶⁵ of SEQ ID NO:2. In one embodiment, the predominant glycan attached to these two sites is HexNAc-Hex-NeuAc.

[00338] For example, FIG. 9 presents an overall view of the N-linked and O-linked carbohydrate structures on CTLA4-Ig molecules comprised of monomers having a sequence from SEQ ID NO :2 (i.e, a monomer having one of the following sequences: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2), (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2 wherein in one embodiment such molecules with the shown carbohydrate characteristics are produced by the cell-line of the invention or progeny thereof according to the method of production described in Examples 14-15 and also shown in FIG. 10. The major structures listed for each site are based on the orthogonal techniques (see herein). For each structure there is an estimated percentage of that structure observed during these experiments. These percentages represent best estimates from the orthogonal techniques.

[00339] CTLA4^A29YU04E-lg dimers composed of monomers having the amino acid sequence of residues (1)-26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, or (vi) 25-383 of SEQ ID NO:4, can have a predicted theoretical MW of about 78,000 to about 79,000 Daltons. However, the MW for such dimers obtained by MALDI-TOF is approximately 91,500 Daltons. This difference in MW of approximately 12,000-13,000 Daltons is due at least in part to glycosylation. The above specified monomers have three N-linked glycosylation sites that have been confirmed by peptide mapping to occur at asparagines at residues 102,134, and 233 of SEQ ID NO:4 (N76, N108, and N207 of FIG. 4). Carbohydrate molecules that are linked through asparagine can be cleaved selectively using the enzyme Peptide-N Glycosidase F (PNGase F). Although there are numerous serine and threonine residues that have the potential of being glycosylation sites, only three O-linked sites were identified: Seri 49, Seri 55, and Seri 65 of SEQ ID NO:4 (See Table 25 in EXAMPLE 22) In one embodiment, the predominant glycan attached to these sites is HexNAc-Hex-NeuAc.

[00340] In certain embodiments, CTLA4.-Ig or CTLA4^A29YLI04E-Ig molecules are glycoproteins that can be produced by the culture methods of the invention. In one embodiment, CTLA4-Ig glycoproteins are modified with oligosaccharides that represent approximately 15% (w/w) of the molecule. These oligosaccharides can play an important role in the pharmacokinetic (PK) parameters of a CTLA4-Ig or CTLA4^A29YLI04E-Ig glycoprotein. In addition, different oligosaccharide profiles can influence the stability and degradation of proteins. For example, O-linked oligosaccharides may enhance the stability of CTLA4^A29YL104E-Ig molecules by preventing autolysis in the hinge region of the immunoglobulin constant region.

[00341]. . The oligosaccharide distribution on apopulation of CTLA4-Ig or CTLA4^A29YL104E-Ig molecules can be heterogeneous in nature due to the complexity of cell culture and processes. The heterogeneity can be present due to glycosylation sites being completely occupied to unoccupied, and the fact that any specific site can be populated with many different oligosaccharide structures, which can further display variation in the pattern of sialic acid modification.

[00342] In one embodiment, the primary sialic acid moiety on CTLA4-Ig or CTLA4^A29YL104E-Ig molecules is N-acetyl neuraminic acid (NeuAc, NANA), and the secondary sialic acid moiety is N-glycolyl neuraminic acid (NGNA). The charged nature of sialic acid and the complex sialic acid-containing structures can result in multiple isoforms of

CTLA4-Ig or CTLA4^A29YLI04E-Ig, respectively, where such isoforms can be evident in an isoelectric focusing (IEF) profile. For example, see FIG. 11 and Example 3 for IEF profile of

CTLA4-Ig. Additionally, see FIG. 12 and EXAMPLE 22 for IEF profile of CTLA4^a29yl104eIg.

[00343] In one embodiment, the invention provides a population of CTLA4-Ig molecules ’ that have a dominant CTLA4-Ig isoform having an isoelectric point (pl) that is less than or equal to 5.1 or 5.0, which can be determined for example by IEF. In another embodiment, a population of CTLA4^A29YL104E-Ig molecules, is provided that has dominant CTLA4^a29yl104eIg isoforms having an isoelectric point (pl) that is less than or equal to 5.5, which can be determined for example by IEF (FIG. 12).

[00344] In one embodiment, the invention provides a population of CTLA4-Ig molecules that have a pl of from about 4.2 to about 5.7, from about 4.25 to about 5.5, from about 4.3 to about 5.3, or from about 4.5 to about 5.2. In another embodiment, the invention provides a population of CTLA4-Ig molecules that have a pl of from about 4.45 to about 5.30. In a further embodiment, the invention provides a population of CTLA4-Ig molecules that have a pl of from about 4.3 to about 5.1. In a particular embodiment, the invention provides a population of CTLA4- Ig molecules that have a pl of from about 4.45 to about 5.0. In one embodiment, the invention provides a population of CTLA4-Ig molecules where at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the population exhibit an isoelectric point less than or equal to about 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7,

4.6, 4.5, 4.4. 4.3, 4.2, 4.1,4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6,

2.5, 2.4, 2.3, 2.2, 2.1, or 2.1 as determined by IEF (these values can have a Standard Deviation of ± 0.2). In one embodiment, the invention provides a method for preparing a population of CTLA4-Ig molecules having a pl of from about 4.45 to about 5.30, or from about 4.45 to about 5.1, or from about 4.45 to about 5.0, wherein the methods involves subjecting a population of CTLA4-Ig molecules to IEF gel electrophoresis, wherein a single band on the gel represents a sub-population of CTLA4-Ig molecules having a particular pl, and isolating the sub-population of CTLA4-Ig molecules having the particular pl by excising the band from the gel and subsequent purification of the proteins from the excised gel band.

[00345] In further embodiments, the invention provides a population of CTLA4^a29yli04eIg molecules that have a pl of from about 4.5 to about 5.2. In other embodiments, the invention provides a population of CTLA4^A29YL104E-Ig molecules that have a pl of from about 4.7 to about 5.1. In another embodiment, the invention provides a population of CTLA4^A29YLI04E-Ig molecules that have a pl of from about 2.0 to about 5.2. In one embodiment, the invention provides a population of CTLA4^A29YL104E-Ig molecules where at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the population exhibit an isoelectric point less than or equal to about 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4. 4.33.1 ,3.2 ,3.3 ,3.4 ,3.5 ,3.6 ,.3.7 ,3.8 ,3.9 ,4.2,4.1,4.0 _־, or 3.0 as determined by IEF (these values can have a Standard Deviation of ± 0.2). In one embodiment, the invention provides a method for preparing a population of CTLA4^A29YL104E-Ig molecules having a pl of from about 4.5 to about 5.2; from about 4.7 to about 5.1; from about 2.0 to about 5.2, wherein the method involves subjecting a population of CTLA4^A29YL104E-Ig molecules to IEF gel electrophoresis, wherein a single band on the gel represents a sub-population of CTLA4^A29YLI04E-Ig molecules having a particular pl, and isolating the sub-population of CTLA4^A29YL104E-Ig molecules having the particular pl by excising the band from the gel and subsequent purification of the proteins from the excised gel band.

[00346] In certain embodiments, the invention provides populations of CTLA4-Ig molecules having an average molar ratio of moles sialic acid groups to moles CTLA4-Ig moleculesof from: about 6 to about 32, about 8 to about 32, about 11 to about 30, about 12 to about .20, about 13 to about 19, about 14 to about 18, about 15 to about 17, about 6 to about 16, about 8 to about 16, about 8 to about 14, about 8 to about 12.

[00347] In some embodiments, a maximum allowable CHO host cell protein of &lt;25 ppm to &lt;10 ng/mg characterizes the composition of CTLA4-Ig molecules. In another embodiment, the composition of CTLA4-Ig molecues is characterized by host cell DNA at a level of &lt;2.5 pg/mg to &lt;1.0 pg/mg. In another embodiment, the composition of CTLA4-Ig molecues is characterized by Triton X-100 at a level of &lt;1.0 ng/mg or &lt;1.0 ppm. The concentration of Triton X-100 can be determined by extraction of the Triton X-l 00 using Waters OASIS-HLB solid-phase extraction followed by washing with water to remove residual protein. The bound Triton X-100 is removed by elution with acetonitrile. The acetonitrile eluate is analyzed by reversed-phase chromatography using a SAS Hypersil 5pm column and a mobile phase consisting of acetonitrile:water (80:20). Detection is by UV absorbance at 225 nm. In one embodiment, the composition of CTLA4-Ig molecules is characterized by &lt;2.5 area % oxidation and &lt;2.0 area % deamidation. In another embodiment, the composition of CTLA4-Ig molecules is characterized by &lt;3.0 area % oxidation and. &lt;2.5 area % deamidation. The tryptic peptide mapping method was used for quantitation of oxidation and deamidation. The percent oxidation data was determined by the use of an RP-HPLC tryptic mapping assay that quantitates־ the area percent oxidation of . Met85 in the CTLA4-Ig protein to methionine sulfoxide. Percent oxidation in the method is obtained by measuring UV peak areas in the RP-HPLC tryptic map for the T6 tryptic peptide, comprised of residues 84-93 containing Met85, and the corresponding oxidized tryptic peptide, T6ox, containing Met(O)85. The area percent oxidation of Met85 to Met(O)85 is proportional to the area percent of the T60x peak: Percent Oxidation = 100 * AT60x/ (AT60x + AT6), where, AT6 = peak area for T6 tryptic peptide, (84-93). AT6ox = peak area for־ ־ . T6ox tryptic peptide, Met(O)85(84-93). The percent deamidation data, acquired by using a RP-HPLC tryptic mapping assay that quantitates the area percent oxidation and deamidation, ־ is obtained by measuring UV peak areas in the RP-HPLC tryptic map for the T26 tryptic peptide, comprised of residues 281-302_containing Asn294, and the corresponding deamidated tryptic peptide, T26deaml, containing isoAsp294. The area percent deamidation of Asn294 to isoAsp294, then, is proportional to the area percent of the T26deaml peak: where, AT26 = peak area for T26, (281-302), AT26deaml = peak area for T26deaml, isoAsp294(281-302). AT26deam2 = peak area for T26deam2, Asp299(281-302).

AT26deam3 = peak area for T26deam3, Asp294(281 -302). AT26deam4 = peak area for T26deam4, Asu294(281-302).

[00348] In another embodiment, the composition of CTLA4-Ig molecules is characterized by N-Acetylglucosamine (GlcNAc) of from 15 to 35 Moles:Mole CTLA4-Ig Protein, orNAcetylgalactosamine (GalNAc) of from 1.7 to 3.6 Moles:Mole CTLA4-Tg Protein. The amino monosaccharides are quantitated by capillary electrophoresis (CE) following release from the protein by acid hydrolysis. The released amino monosaccharides are re-acetylated, and fluorescently labeled with aminopyrene trisulfonic acid (APTS) to facilitate their detection and quantitation. N-Acetylmannosamine is added to a sample and amino monosaccharide standards to serve as an internal standard. The peak areas of the amino monosaccharides in the samples are normalized using the internal standard and quantified by comparing with their respective normalized amirio monosaccharides peak areas in the standard. The molar ratio of each monosaccharide relative to the CTLA4-Ig molecule is then calculated.

[00349] In one embodiment, the composition of CTLA4-Ig molecues is characterized by the following N-linked oligosaccharide profile specifications:

________________N-Linked Oligosaccharide Profile Specifications_________________

<td></td><td> % Difference ׳ Domain I .</td><td> % Difference Domain II</td><td> % Difference Domain III</td>

<td> % Difference</td><td> 19-3.1</td><td> 7-19</td><td> - -18</td>

<td> Standard Deviation (% Difference from above specification)</td><td> ±29</td><td> ±27</td><td> ±25</td>

[00350]

[00351] In one embodiment, the composition of CTLA4-Ig molecules is characterized by neutral monosaccharide where the composition has ratios of about:

[00352] Galactose: 8.0 to 17 MolescMole CTLA4-Ig Protein

[00353] Fucose: 3.5 to 8.3 Moles:Mole CTLA4-Ig Protein

[00354] Mannose: 7.7 to 22 Moles:Mole CTLA4-Ig Protein, or

[00355] Galactose: 9.0 to 17 MoleszMole CTLA4-Ig Protein

[00356] Mannose: 11 to 19 M01es:M01e CTLA4-Ig Protein.

Illustrative Neutral Monosaccharide Composition: Moles:Mole Protein of Galactose, Fucose and Mannose

<td rowspan="2"> Neutral Monosaccharide</td><td colspan="2"> Process W (n=34)</td><td colspan="2"> Process CD-CHO1 (n=109)</td>

<td> Mean (SD)</td><td> Min, Max</td><td> Mean (SD)</td><td> Min, Max</td>

<td> Galactose</td><td> 13.9(1.1)</td><td> 12.0,16.0</td><td> 12.6(1.0)</td><td> 10.0,16.0</td>

<td> Fucose</td><td> 5.8 (1.0)</td><td> 4.2, 7.7</td><td> 5.6 (0.7)</td><td> 4.5, 7.6</td>

<td> Mannose</td><td> 15.3 (1.0)</td><td> 13.0,17.0</td><td> 15.4(1.0)</td><td> 13.0, 18.0</td>

Illustrative Sialic Acid (NANA:Mole Protein)

<td rowspan="2"> Sialic Acid</td><td colspan="2"> Process W (n=34)</td><td colspan="2"> Process CD-CHO1 (n=109)</td>

<td> Mean (SD)</td><td> Min, Max</td><td> Mean (SD)</td><td> Min, Max</td>

<td> NANA</td><td> 10.2 (0.6)</td><td> 9.3, 11.6</td><td> 9.7 (0.6)</td><td> 8.2, 11.5</td>

. [00357]

[00358] In another embodiment, the monosaccharide molar ratio range for a CTLA4^A29YL104E-Ig composition is as follows: mannose from about 10-20 moles/mole protein; fucose from about 4.2 -7.0 moles/mole protein; and galactose from about 9.2 —17 moles/mole protein. In another embodiment, the CTLA4^A29YL104E-Ig composition is characterized by a NANA molar ratio of from about 5.0 - 10.0.mole of NANA/mole protein. In another embodiment, the CTLA4^A29YLI04E-Ig composition is characterized by a NGNA molar ratio of &lt; 1.5 mole NGNA/mole protein. In some embodiment, the % deviation of molar ratio for sialic acids is &lt; 15% or &lt; 20% or &lt; 30%.

[00359] In one embodiment, a population of CTLA4-Ig molecules can comprise CTLA4Ig monomers that each have at least 3 sialic acid groups. In another embodiment a population of CTLA4-Ig molecules comprises CTLA4-Ig monomers that each have from 3 to 8 sialic acid groups.

[00360] In one embodiment, the invention provides a population of CTLA4-Ig molecules where at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the population exhibit an isoelectric point less than or equal to about 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4:4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, or 3.0.

[00361] In some embodiments, the invention provides populations of CTLA4-Ig molecules having an average molar ratio of moles NANA to moles CTLA4-Ig molecules or dimer of from: about 6 to about 16, about 6 to about 14, about 6 to about 12, about 8 to about 12, about 8 to about 14, about 8 to about 16.

[00362] In other embodiments, the invention provides populations of CTLA4-Ig molecules having an average molar ratio of moles NGNA to moles CTLA4-Ig molecules or dimer of less than or equal to about 2, 1.8, 1.6, 1.5, 1.4, 1.0, 0.8, or 0.5

[00363] In particular embodiments, the invention provides populations of CTLA4^A29YL104E-Ig molecules having an average molar ratio of moles sialic acid groups to moles CTLA4^A29YL104E-Ig molecules or dimer of from about 5.5 to about 8.5. In another embodiment, the invention provides populations of CTLA4^A29YL104E-Ig molecules having an average molar ratio of moles sialic acid groups to moles CTLA4^A29YLI04E-Ig molecules or dimer of from about 5 to about 10.

[00364] In one embodiment, a population of CTLA4^A29YLI04E-Ig molecules can comprise CTLA4^A29YL104E-Ig monomers that each have at least 2.5 sialic acid groups. In another embodiment a population of CTLA4^A29YL104E-Ig molecules comprises CTLA4^A29YLI04E-Ig monomers that each have from 2.5 to 5 sialic acid groups.

[00365] In other embodiments, the invention provides populations of CTLA4-Ig molecules that are distinguished by the population’s average molar ratio of moles amino monosaccharides and/or neutral monosaccharides and/or sialic acids to moles CTLA4-Ig molecules or dimer. In particular embodiments, the invention provides populations of CTLA4^A29YL104E-Ig molecules that are distinguished by the population’s average molar ratio of moles amino monosaccharides and/or neutral monosaccharides and/or sialic acids to moles CTLA4^A29YLI04E-Ig moleculesor dimer. Amino monosaccharides include N-acetyl galactosamine (GalNAc) and N-acetyl glucosamine (GlcNAc). Neutral monosaccharides include mannose, fucose, and galactose. Sialic acids include N-acetyl neuraminic acid (NANA) and N-glycolyl neuramininc acid (NGNA).

[00366] In one embodiment, the invention provides a population of CTLA4-Ig molecules that are characterized by an average molar ratio of moles GlcNAc per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is from about 10 to about 40, from about 15 to about 35, from about 15 to about 25, or from about 15 to about 20. In another embodiment, the invention provides a population of CTLA4-Ig molecules where at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the population are characterized by an average molar ratio of moles GlcNAc per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is less than or equal to about 40, 38, 35, 30, 25, 20, 18, or 15.

[00367] In another embodiment, the invention provides a population of CTLA4-Ig molecules that are characterized by an average molar ratio of moles GalNAc per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is from about 1.5 to about 8.5, from about 1.7 to about 3.0, from about 1.7 to about 4.0, from about 1.7 to about 5.0, from about 1.7 to about 6.0, from about 1.7 to about 7.0־, from about 1.7 to about 8.0, or from about 1.7 to about 8.3. In another embodiment, the invention provides a population of CTLA4-Ig molecules where at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the population are characterized by an average molar ratio of moles GalNAc per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is less than or equal to about 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4.0, 3.8, 3.6, 3.5, 3.0, 2.5, 2.0, 1.7, or 1.5.

[00368] In a further embodiment, the invention provides a population of CTLA4-Ig molecules that are characterized by an'average molar ratio of moles galactose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is from about 7.5 to about 20.0, from about 8.0 to about 19.0, from about 8 to about 18.0, from about 8.0 to about 17.0, from about 8.5 to about 1-7.0, or from about 9.0 to.about 17.0,- .In another embodiment, the invention provides a population of CTLA4-Ig molecules where at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the population characterized by an average molar ratio of moles galactose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is less than or equal to about 20.0, 19.0, 18.0,17.0, 16.0,15.0, 14.0, 13.0,12.0, 11.0, 10.0, 9.0, 8.5, 8.0, or 7.5.

[00369] In a further embodiment, the invention provides a population of CTLA4-Ig molecules that are characterized by an average molar ratio of moles fucose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is from about 3 to about 8.5, from about 3.5 to about 8.5, from about 3.5 to about 8.3, from about 3.5 to about 8.0, from about 3.5 to about

7.5, or from about 3.5 to about 7.0. In another embodiment, the invention provides a population of CTLA4-Ig molecules where at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the population characterized by an average molar ratio of moles fucose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is less than or equal to about 8.5, 8.3, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.2, or 3.0.

[00370] In a further embodiment, the invention provides a population of CTLA4-Ig molecules that are characterized by an average molar ratio of moles mannose per mole of CTLA4-Ig dimer or to CTLA4־Ig molecule that is from about 7 to about 23, from about 7.5 to about 23, from about 7.7 to about 23, from about 7.7 to about 22.5, from about 7.7 to about 22, from about 7.7 to about 20, from about 7.7 to about 18, from about 7.7 to about 16, from about 8.0 to about 16.0, from about 9.0 to about 17.0, from about 10 to about 19.0, or from about 11 to about 19.0. In another embodiment, the invention provides a population of CTLA4-Ig molecules where at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the population characterized by an average molar ratio of moles mannose per mole of CTLA4-Ig molecules or dimer or to CTLA4-Ig molecule that is less than or equal to about 23, 22.5, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9.5, 9, 8.5, 8, 7.7, 7.5, 7.3, or 7.

[00371] In one embodiment, the invention provides a glycosylated CTLA4-Ig population that exhibits increased PK. values, such as increased exposure as measured by area under the curve (AUC), such as resulting from or as־demonstrated by decreased clearance from the serum while retaining bioactivity. In another embodiment, the invention provides a glycosylated CTLA4^A29YL104E-Ig population that exhibits increased pharmacokinetic (PK) values as demonstrated by decreased clearance from the serum while retaining bioactivity. .

[00372] In some embodiments, the invention provides analogs of soluble CTLA4-Ig molecules, which have additional glycosylation sites. In other embodiments, the invention provides analogs of soluble CTLA4^A29YL104E-Ig molecules, which have additional glycosylation sites. Additional glycosylation sites provide attachment points for additional carbohydrate structures that can be sialylated. Increased sialic content can be lead to increased PK values, and/or increased glycoprotein stability. Higher sialic acid content is beneficial. In vitro post-purification methods that use enzymes to add more sialic acids can be performed to produce further embodiments of the CTLA4-Ig or CTLA4^A29YL104E-Ig molecules of the invention.

[00373] The embodiments of the invention include any one range disclosed herein in combination with any one-or more ranges disclosed herein. The embodiments of the invention include any one characteristic or property of CTLA4-Ig disclosed herein in combination with any one or more characteristics or properties of CTLA4-Ig disclosed herein.

[00374] Methods for Analyzing and Isolating CTLA4-Ig and CTLA4^A29YL104E-Ig Glycoproteins

[00375] The following methods described herein can be used to distinguish, identify, or isolate particular CTLA4-Ig or CTLA4^A29YL104E-Ig molecule populations on the basis of various sugar profiles, including but not limited to a population’s average molar ratio of moles amino monosaccharides and/or neutral monosaccharides and/or sialic acids per mole CTLA4-Ig or CTLA4^A29YL104E-Ig molecules or dimer.

[00376] A glycoprotein that is secreted from cultured cells can be isolated from the culture medium or supernatant. The glycoprotein produced by the cells is collected, recovered, isolated, and/or purified, or substantially purified, as desired, at the end of the total cell culture period using isolation and purification methods as known and practiced in the art or as described herein. In one embodiment, a glycoprotein of the invention, which is expressed by the cell but not secreted by the cell, can still be recovered from the cells, e.g., via making cell lysates and isolating the glycoprotein, and/or using methods that are known and'practiced in the art, and as further described below.

[00377] The glycoprotein produced by the cell culture processes of this invention comprises complex carbohydrates that can be analyzed by various techniques of carbohydrate analysis. For example, techniques such as lectin blotting, well-known in the art, reveal proportions of terminal mannose, or other sugars such as galactose. Termination of mono-, bi-, tri-, or tetra-antennary oligosaccharide by sialic acids can be confirmed by release of sugars from the protein using anhydrous hydrazine or enzymatic methods and fractionation of oligosaccharides by ion-exchange chromatography, size exclusion chromatography, or other methods that are known in the- art.

[00378] There are two main types of glycosidic linkages found in glycoprotiens, N- and Olinked, N-glycosylations are created by a covalent link ofthe glycan to the amide nitrogen of an asparagine residue. O-glycosidic linkages are created by the covalent linkage of the hydroxyl group of serine, threonine, hydroxylysine or hydroxyproline to the glycan. The carbohydrate moieties of glycoproteins are involved in numerous molecular recognition phenomena, including host-pathogen interactions, clearance from serum and targeting of different tissues. With respect to CTLA4-Ig and CTLA4^A29YL104E-Ig molecules, carbohydrate moieties can at least affect binding between CTLA4-Ig molecules and CD80 or CD86, or between CTLA4^A29YLI04E-Ig molecules and CD80 or CD86.

[00379] Carbohydrate structures typically occur on the expressed protein as N-linked or Olinked carbohydrates. The N-linked and O-linked carbohydrates differ primarily in their core structures. N-linked glycosylation refers to the attachment of the carbohydrate moiety via GlcNAc to an asparagine residue in the peptide chain. In one embodiment, the N-linked carbohydrates all contain a common Manl-6(Manl-3)Manpl-4Glc- NAcpl-4GlcNAcp-R core structure, where R in this core structure represents an asparagine residue. The peptide sequence of the protein produced will contain an asparagine-X-serine, asparagine-Xthreonine, and asparagine-X-cysteine, wherein X is any amino acid except proline.

[00380] In contrast, O-linked carbohydrates are characterized by a common core structure, which contains GalNAc attached to the hydroxyl group of a threonine or serine. Of the Nlinked and O-linked carbohydrates, the most important are the complex N- and O-linked carbohydrates. Such complex carbohydrates contain several antennary structures. The mono־-, bi-, tri, -־, and tetra-, antennary structures are important for the addition of terminal sialic acids. Such outer chain structures provide for additional sites-for the specific, sugars and linkages that comprise the carbohydrates of the protein products.

[00381] Therapeutic glycoproteins are often produced using recombinant DNA cell culture techniques. Protein glycosylation distributions in cell culture can be affected by variations in pH, cell density, nutrient concentrations; and metabolite concentrations. The sensitivity of glycan distributions to environmental effects makes it necessary to carefully monitor the glycan distribution during product development and production in order to ensure that a reproducible product is manufactured.

[00382] The development of recombinant-derived glycoproteins for therapeutic use has led to an increasing demand for methods to characterize and profile their carbohydrate structures. Oligosaccharide mapping has been used during initial characterization of recombinant proteins for comparison to the native protein, to identify oligosaccharide structures present, to monitor consistency of oligosaccharide composition, to evaluate changes that can result from alteration in cell culture or production process, and to identify changes in glycosylation that occur as a result of expression in different cell lines.

[00383] A variety of techniques are available to evaluate carbohydrate structural distributions. These include gel-filtration, chromatographic and electrophoretic separation techniques coupled with a wide range of detection techniques. If sample amounts are limited, the glycoproteins are often derivatized with fluorescence reagents such as 2aminobenzoic acid and 2-aminopyridine in order to improve detection. However, derivatization and purification of the derivatives can be time consuming. When sample size is not an issue, direct evaluation of carbohydrate structural distributions is possible.

[00384] Analysis of oligosaccharide content of a glycoprotein

[00385] A particular glycoprotein can display heterogeneity of carbohydrates. Heterogeneity can be seen at several levels: glycosylation sites can vary from completely occupied to unoccupied, and any specific site can be populated with many different oligosaccharide structures, wherein each structure can be modified by sialic acid molecules, such as NANA or NGN A.

[00386] The carbohydrate content of the protein of the present invention can be analyzed by methods known-in the art, including methods described in the.Examples herein. Several methods are known in the art for glycosylation analysis and are useful in the context of the present invention. These methods provide information regarding the identity and the composition of the oligosaccharide attached to the produced peptide. Methods for carbohydrate analysis useful in connection with the present invention include, but are not limited to, lectin chromatography; high performance anion-exchange chromatography combined with pulsed amperometric detection (HPAEC-PAD), which uses high pH anion exchange chromatography to separate oligosaccharides based on charge; NMR; Mass spectrometry; HPLC; porous graphitized carbon (GPC) chromatography.

[00387] Methods for releasing oligosaccharides are known. These methods include 1) enzymatic methods, which are commonly performed using peptide-N-glycosidase F/endo-agalactosidase; 2) β-elimination methods, using a harsh alkaline environment to release mainly O-linked structures; and 3) chemical methods using anhydrous hydrazine to release both Nand O-linked oligosaccharides. Methods for analysis can comprise the following steps: 1. Dialysis of the sample against deionized water to remove all buffer salts, followed by lyophilization. 2. Release of intact oligosaccharide chains with anhydrous hydrazine. 3. Treatment of the intact oligosaccharide chains with anhydrous methanolic HCI to liberate individual monosaccharides as O-methyl derivatives. 4. N-acetylation of any primary amino groups. 5. Derivatization to yield per-O-trimethylsilyl methyl glycosides. 6. Separation of these derivatives by capillary gas-liquid chromatography (GLC) on a CP-SIL8 column. 7. Identification of individual glycoside derivatives by retention time from the GLC and mass spectroscopy, compared to known standards. 8. Quantification of individual derivatives by FID with an internal standard (13-O-methyl-D-glucose). .

[00388] The presence of neutral and amino sugars can be determined by using high performance anion-exchange chromatography combined with pulsed amperometric detection (HPAEC-PAD Carbohydrate System; Dionex Corp.). For instance, sugars can be released by hydrolysis in 20% (v/v) trifluoroacetic acid at 100°C for 6 hours. Hydrolysates are then dried by lyophilization or with a Speed-Vac (Savant Instruments). Residues are then dissolved in 1% sodium acetate trihydrate solution and analyzed on an HPLC-AS6 column (as described by Anumula et al., 1991, Anal. Biochem,, 195:269-280).

[00389] Alternatively, immunoblot carbohydrate analysis can be performed. In this . procedure protein-bound carbohydrates are detected using a commercial glycan detection system (Boehringer), which is based on the oxidative immunoblot procedure described by Haselbeck et al. (1993, Glycoconiugate J., 7:63). The staining protocol recommended by the manufacturer is followed except that the protein is transferred to a polyvinylidene difluoride membrane instead of a nitrocellulose membrane and the blocking buffers contain 5% bovine serum albumin in 10 mM Tris buffer, pH 7.4, with 0.9% sodium chloride. Detection is carried out with anti-digoxigenin antibodies linked with an alkaline phosphate conjugate (Boehringer), 1:1000 dilution in Tris buffered saline using the phosphatase substrates, 4nitroblue tetrazolium chloride, 0.03% (w/v) and 5-bromo-4 chloro-3-indoyl-phosphate 0.03% (w/v) in 100 mM Tris buffer, pH 9.5, containing 100 mM sodium chloride and 50 mM magnesium chloride. The protein bands containing carbohydrate are usually visualized in about 10 to 15 minutes.

[00390] Carbohydrates associated with protein can also be cleaved by digestion with peptide-N-glycosidase F. According to this procedure the residue is suspended in 14 μΕ of a buffer containing 0.18% SDS, 18 mM beta-mercaptoethanol, 90 mM phosphate, 3.6 mM EDTA, at pH 8.6, and heated at 100°C for 3 minutes. After cooling to room temperature, the reaction mixture is divided into two approximately equal parts. One part, which is not treated further, serves as a control. The other part is adjusted to about 1% NP-40 detergent followed by the addition of 0.2 units of peptide-N-glycosidase F (Boehringer). Both parts are warmed at 37°C for 2 hours and then analyzed by SDS-polyacrylamide gel electrophoresis.

[00391] Glycan mapping of glycoproteins is becoming increasingly accepted. The methodology described herein allows for rapid characterization of oligosaccharides in terms of glycan type, extent of sialylation and number of branches on the non-reducing end of the carbohydrates. Thus, in certain embodiments, the invention provides CTLA4-Ig populations characterized by particular oligosaccharide profiles. Oligosaccharide profiling is typically done by chromatographic separation of oligosaccharides followed by detection and relative quantitation. An alternative to chromatographic profiling is the direct analysis of oligosaccharides by ESI infusion after online desalting.

[00392] Oligosaccharide profiling by PGC can be been used to characterize the N-linked oligosaccharides from CTLA4-Ig molecules. There are 31 structural classes of oligosaccharides identified from CTLA4-Ig molecules (SEQ ID NO:2), including a structural class containing O-acetylated sialic acid groups. Structural class verification is achieved through the use of MS/MS and positive ion mode MS. Relative quantitation of structural classes is possible through integration of the UV trace at 206 nm. Comparison of the subpopulation profiles from individual N-link sites is known, revealing significant population differences between N-link sites. Oligosaccharide profiling using PGC provides a convenient information rich alternative to the more traditional profiling methods such as HPAEC.

[00393] N-linked Structures in CTLA4-Ig molecules comprising monomers of SEQ ID

NO:2

[00394] There are three N-linked glycosylation sites per chain (i.e., per monomer) on a CTLA4-Ig multimer or dimer, wherein the monomer has a sequence from SEQ ID NO:2, for example: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2). The variations in glycosylation by site are analyzed by isolating peptide fragments containing N-linked glycans from a tryptic digest of the protein. The N-linked glycosylation sites on the protein are located at Asn¹⁰², Asn¹³⁴ and Asn²³³, contained in tryptic fragments 5, 7, and 14, respectively. Enzymatic release of N-linked oligosaccharides from the isolated peptide fragments, followed by PGC profiling of the released oligosaccharides results in the profiles shown in FIG. 13. It is clear from the profile of the glycans released from Asn²³³ (Tryptic fragment 14, T14) that the oligosaccharide population is enriched in the asialo structures (structures have no sialic acids). Oligosaccharide profiles from the glycans attached at Asn¹⁰² and Asn¹³⁴ (T5 and T7) contain the bulk of the sialylated structures

[00395] . Isolated oligosaccharides released from glycoprotein are directly injected into the porous graphitized carbon LC/UV/MS system. FIGS. 14 and 15 show the TIC and UV chromatograms of a typical PGC profiles generated by acetonitrile gradients containing acidic and basic additives. In most cases, the mass spectra from a single chromatographic peak contain mass peaks for a single oligosaccharide. Thirty oligosaccharide structural classes are identified from the TFA containing elution profile. Only sixteen oligosaccharide structural classes are identified from the NH4OH containing elution profile. Within each structural class there are variant structures containing substitution of N-glycolylneuraminic acid (NGNA) in place ofN-acetylneuraminic acid (NANA) as well as differing degrees of sialic acid acetylation. Although only qualitative information can be gained from comparison of the ion counts for the oligosaccharide classes, it is apparent that the major structural classes within each of the four domains are P2100, P2111, P2122, and P3133. This is consistent with the integration values obtained from the UV trace at 206 nm. Further structural verification can be obtained from the positive ion mass spectrogram. Positive ion mode ionization promotes in source fragmentation of oligosaccharides, mainly at the glycosidic bonds. Because there is good separation of oligosaccharides, as determined by . the negative, ion mass spectra, the fragmented spectra from the positive ion mode mimic the positive ion MS/MS spectra. Domain III (di-sialylated structures) contains a significant amount of the O-acetylated structure P2122-Ac. Positive ion m/s data .supports O-acetylation of one of the sialic acids on the structure. The most common O-acetylation site of sialic acid residues are at the C-7 and C-9 positions (Shi WX, Chammas R., Varki A., J. Biol. Chem. 271 (1996) 15130-15188). At physiologic extracellular pH, O-acetyl esters at C-7 spontaneously migrate to C-9. The most likely O-acetlylation site is therefore C-9.

[00396] Analysis of N-linkedOligosaccharide Content'. The analytical techniques can comprise cleavage and isolation of N-linked oligosaccharides by column chromatography, which in a non-limiting embodiment uses a Hypercarb column. Glycans subjected to Hypercarb chromatography are isolated and can be analyzed by HPAEC-PAD which analysis determines the types of carbohydrates that modify a particular glycoprotein. Analytical characterization of the N-linked oligosaccharides can also be achieved by Liquid Chromatography/Mass Spectrometry (LC/MS ) using a Porous Graphitized Carbon (PGC). Carbohydrate analysis can also include trypsin, Asp-N, and Trypsin/Chymotrypsin peptide mapping to determine the peptides, which comprise carbohydrate structures.

[00397] N-linked oligosaccharide structures can be analyzed using a series of orthogonal mass spectrometry and HPAEC-PAD techniques (see Examples). These techniques include several endopeptidase cleavages followed by LC/MS/MS analysis. With respect to CTLA4-Ig monomers having a sequence from SEQ ID NO:2, the three major sites of Nlinked glycosylation were characterized using LC/MS and LC/MS/MS electrospray ionization and the major structures at each N-link site were determined. These data are summarized in FIG. 9. There are at least three major attachment points for N-linked oligosaccharides at Asn¹⁰², Asn¹³⁴, and Asn²³³. In addition, Asn²³³ is found to contain a population of N-linked structures that contained no sialic acid groups occurring about 80% of the time.

[00398] N-linked oligosaccharide structures of CTLA4-Ig determined by LC/MS of the glycopeptides, LC/MS of the oligosaccharides, and HPAEC-PAD: The N-linked

- carbohydrates are associated with a consensus sequence motif of Asn - X - Ser/Thr. This sequence appears three times on CTLA4-Ig monomer chains having one of the following sequences: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv)-27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2. The consensus sequence motif appears in SEQ ID NO:2 at: Asn¹⁰² Leu¹⁰³ Thr¹⁰⁴; Asn¹³⁴Gly¹³⁵ Thr¹³⁶; and Asn²³³ Sei²³⁴ Thr²³⁵. Based on the consensus sequence, there are six N-linked carbohydrate sites per dimer molecule that is formed of any one or two of the following monomer sequences: (i) 26-383 of SEQ IDNO:2, (ii) 26-382 of SEQ IDNO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2.

[00399] N-linked carbohydrates can be of three general varieties: high-mannose, hybrid and/or complex. A LC/MS technique for the glycopeptide analysis was developed. Trypsin endoproteolytic cleavage of monomers (having one of the sequences (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2) result in three peptides that contain N-linked glycosylation. All three N-linked sites are populated with carbohydrate structures. Tryptic fragment T5 corresponding to amino acids 65-109 of SEQ ID NO:2 contains a glycosylation on Asn¹⁰². Tryptic fragment T7 corresponding to amino acids 120154 of SEQ ID NO:2 contains a glycosylation on Asn¹³⁴. Tryptic fragment T14 corresponding to amino acids 229-237 of SEQ ID NO:2 contains a glycosylation on Asn²³³.

[00400] In order to determine the specific types of glycosylation on each site, carbohydrates were obtained from each specific site by increasing the scale of protein digestion and separation followed by collection of the T5, T7, and T14 peptides. The tryptic peptide peaks of interest were treated with PNGase F and processed for analysis by LC/MS on a Hypercarb column. Results showed that a heterogeneous population of complex bi-, tri-, and tetra-antennary structures at each site. These can be seen in FIG. 13 where the chromatography separates the sugars into five domains: asialo, mono-sialo, di-sialo, tri-sialo, and tetra-sialo structures (referred to as Domains I, II, III, IV, and V, respectively). A chromatogram (FIG. 13 panel A) for the Asn¹⁰² (T5) carbohydrates illustrates a series of mono- and di-sialo structures at the site. A chromatogram (FIG. 13 panel B) for Asn¹³⁴ (T7) illustrates two main di-sialo structures with a population of mono-sialo structures. A chromatogram (FIG. 13 panel C) for Asn²³³ (T14) illustrates little sialylation. For each of the N-linked carbohydrate sites, a MS spectrum and corresponding structure is shown for the major peak in each chromatogram.(see FIG. 13,־ panels E, F, H). In FIG. 13, panel D, the total N-linked carbohydrate profile of CTLA4-Ig is shown in the chromatogram. The mass and structures of selected peaks are listed in Table 1. The oligosaccharide LC/MS data were supported by in-depth analysis of the peptide map. Asn¹⁰² (T5 peptide) has the greatest degree of carbohydrate heterogeneity ranging from bi-antennary, nomsialylated structures to .tetra-antennary, tetra-sialylated structures. Asn¹³⁴ (T7 peptide) contains primarily biantennary structures. This site contains much less heterogeneity than the Asn¹⁰² site. The Asn²³³ (T14 peptide) site contains little sialylation. A third analytical technique, HPAEC-PAD, was also employed to support the two orthogonal LC/MS findings.

Table 1: The major N-linked structures and selected minor complex structures observed using LC/MS methods

<td> Structure</td><td> Theoretical Mass</td><td> Actual Deconvoluted Mass</td>

<td> (GlcNAc)4 (Puc)1 (Man)3</td><td> 1462</td><td> 1575*</td>

<td> (G1cNAc)₄ (Fuc)l (Man)₃ (Gal),</td><td> 1624</td><td> 1737*</td>

<td> (G1cNAc)₄ (Fuc)l (Man)₃ (Gal)2</td><td> 1786</td><td> 1899*</td>

<td> (GlcNAc)4 (Fuc)l (Man)₃ (Gal), (NeuAc),</td><td> 1916</td><td> 1916</td>

<td> (GlcNAc)4 (Fuc)l (Man)3 (Gal)2 (NeuAc),</td><td> 2077</td><td> 2077</td>

<td> (GlcNAc)5 (Fuc)l (Man)₃ (Gal)₃ (NeuAc),</td><td> 2443</td><td> 2442</td>

<td> (GlcNAc)4 (Fuc)l (Man)3 (Gal)2 (NeuAc)2</td><td> 2369</td><td> 2368</td>

<td> (GlcNAc)5 (Fuc)l (Man)3 (Gal)₃ (NeuAcfe</td><td> 2734</td><td> 2734</td>

<td> (G1cNAc)5 (Fuc)l (Man)₃ (Gal)₃ (NeuAc)₃</td><td> 3025</td><td> 3025</td>

<td> (G1cNAc)6 (Fuc)l (Man)₃ (Gal)₃ (NeuAc)₃</td><td> 3388</td><td> 3388</td>

<td> (GlcNAc)6 (Fuc)l (Man)₃ (Gal)₃ (NeuAc)4</td><td> 3680 .</td><td> 3680</td>

<td> *The asialo species are detected as TFA adducts.</td><td></td><td></td>

[00401] The population of total N-linked carbohydrates was analyzed using HPAEC-PAD. The data obtained by this method are listed in Tables 2 and 3. In Table 2, the relative area percentages of asialo to tri-sialo domains are listed within each site (Asn¹⁰², Asn¹³⁴, and Asn²³³ of SEQ ID NO:2). In Table 3, the oligosaccharide domain area percentages are listed as a fraction of the entire population of oligosaccharides.

Table 2: The area percentages for each domain observed by the HPAEC-PAD

N linked

Asialo

Mono

Di Tri

<td> N^1uz</td><td> . 27</td><td> 37</td><td> 25</td><td> 11</td>

<td> N^1J4</td><td> 25</td><td> 38</td><td> 28</td><td> • 8</td>

<td> N^2JJ</td><td> 82</td><td> 12</td><td> 5</td><td> 1</td>

Table 3: The area percentages for each domain expressed as weighted average on Table 2

<td colspan="5"> data set.</td>

<td> N linked</td><td> Asialo</td><td> Mono ’</td><td> Di</td><td> Tri</td>

<td> N¹⁰²</td><td> 9</td><td> 12</td><td> 8</td><td> 4</td>

<td> N‘³⁴</td><td> 8</td><td> 13</td><td> 9</td><td> 3</td>

<td> N^2jj</td><td> 28</td><td> 4</td><td> 2</td><td> 0</td>

<td> Total / Molecule</td><td> 45</td><td> 29</td><td> 19</td><td> 7</td>

Assuming full glycosylation.

[00402]

[00403] N-linked oligosaccharide structures of CTLA4^A29YLI04E-Ig molecules determined by LC/MS of the glycopeptides, LC/MS of the oligosaccharides, and HPAEC-PAD: The Nlinked carbohydrates are associated with a consensus sequence motif of Asn - X - Ser/Thr. This sequence appears three times on CTLA4^A29YL104E-Ig monomer chains having one of the following sequences: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4. The consensus sequence motif appears in SEQ ID NO:4 at: Asn¹⁰² Leu¹⁰³Thr¹⁰⁴; Asn¹³⁴ Gly¹³⁵ Thr¹³⁶; and Asn²³³ Ser²³⁴ Thr²³⁵. Based on the consensus sequence, there are six N-linked carbohydrate sites per dimer molecule that is formed of any one or two of the following monomer sequences: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4.

[00404] N-linked carbohydrates can be of three general varieties: high-mannose, hybrid and/or complex. A LC/MS technique for the glycopeptide analysis was developed. Trypsin endoproteolytic cleavage of monomers (having one of the sequences (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4) result in three peptides that contain N-linked glycosylation (See Table 25 in EXAMPLE 22). All three N-linked sites are populated with carbohydrate structures. Tryptic fragment T5 corresponding to amino acids 65-109 of SEQ ID NO:4 contains a glycosylation on Asn¹⁰². Tryptic fragment T7 . corresponding to amino acids 120-154 of SEQ ID NO:4 contains a glycosylation on Asn¹³⁴.

Tryptic fragment T14 corresponding to amino acids 229-237 of SEQ ID NO:4 contains a glycosylation on Asn²³³ (See Table 25 in EXAMPLE 22).

[00405] In order to determine the specific types of glycosylation on each site, carbohydrates were obtained from each specific site by increasing the scale of protein digestion and separation followed by collection of the T5, T7, and T14 peptides. The tryptic peptide peaks of interest were treated with PNGase F and processed for analysis by LC/MS on a Hypercarb column. Results showed that a heterogeneous population of complex bi-, tri-, and tetra-antennary structures at each site. These can be seen in FIG. 16 where the chromatography separates the sugars into four domains: asialo, mono-sialo, di-sialo, and trisialo structures (referred to as Domains I, II, III, and IV respectively). The characteristics of a carbohydrate profile that can be analyzed and compared between glycosylated molecules, or populations or compositions comprising glycosylated molecules include peak area percent, domain area percent, valley-to-valley distance, or peak-to-peak distance.

[00406] LC/MS Characterization of CTLA4-Ig N-Linked Oligosaccharides

[00407] LC/MS porous graphitic carbon (PGC) chromatography is a method for profiling N-linked oligosaccharides can provide several advantages over the High pH Anion Exchange Chromatography (HPAEC). Some of these advantages include: Direct profiling from digest mixtures which minimizes sample lossand degradation; direct MS interface provides a method for rapid characterization and analysis of oligosaccharides; increased resolution ^Kthrough PGC chromatography permits both the inter-domain comparisons as well as more subtle intra-domain analysis.

[00408] The LC/MS PGC method allows for rapid profiling and characterization of oligosaccharides in terms of glycan type, and determining the extent of sialylation and branching on the non-reducing end of the carbohydrates. Then negative ion mode MS spectra produce data that is simple to interpret, with minimal oligosaccharide fragmentation, while positive mode ionization allows for structural class verification. The method described here can be applied to whole digest mixtures of glycoproteins, as well as to previously isolated oligosaccharide samples without the need for derivatization. The chromatographic mobile phases used allow for collection of peaks from the profiles and concentration to dryness, without further manipulation for more detailed characterization. In one embodiment the methodjs used to characterize CTLA4-Ig

N-linked oligosaccharides. Using the LC/MS PGC method, thirty-one distinct classes of oligosaccharides can be identified on CTLA4-Ig molecules comprised of monomers having sequences from SEQ ID NO:2, e.g., SEQ ID NO: 5, 6, 7, 8, 9, or 10.

[00409] High pH anion exchange chromatography (HPAEC) has been used extensively to profile oligosaccharides Released from glycoproteins without the need for derivitization. The high resolution of HPAEC and the fact that the separation is influenced by the type of sugar residue present, type of linkage and the size of the glycan are reasons for the widespread use of the technique. The dominating factor in separation is charge, highly charged oligosaccharides eluting later than less charged glycans. Chromatographic profiles are often divided into domains defined by the number of charged species, typically sialic acid residues, on the glycans (FIG. 17).

[00410] To obtain more information about the structure of the unknown oligosaccharides the HPAEC peaks can be collected, desalted and characterized by MS and/or NMR. One consideration to HPAEC-PAD profiling of oligosaccharide distributions is the variability inherent to the detection mode. Electrochemical cell aging and electrode surface fouling result in profile variability. It has also been reported that oligosaccharide structures and the degree of sialylation can cause variability among detection cells when using HPAEC with pulsed amperometric detection (HPAEC-PAD). This variability can affect quantitative and relatively quantitative results used to evaluate the effect of process changes or determine batch to batch consistency. Because of its speed and specificity, mass spectrometry (MS) has gained popularity as a technique for assessment of oligosaccharide profiling of glycoprotiens. Although MS profiles cannot be used to directly determine anomeric .configuration or branching patterns, MS data can be used to identify structural classes and detect qualitative changes in glycoform distributions.

[00411] The porous graphitized carbon (PGC) chromatographic profiling method for ' enzymatically released N-linked oligosaccharides uses both ultraviolet (UV) and mass spectroscopic (MS) detection to profile and characterize N-linked oligosaccharides, either directly from enzymatic digest mixtures or from isolated oligosaccharides. This method can be used to profile and characterize oligosaccharide released from CTLA4-Ig glycoproteins. The LC/MS PGC method can evaluate the consistency of the oligosaccharide distributions resulting from the production process, as well as any changes in the oligosaccharide distributions resulting from process modifications. In a chromatographic microanalysis ofN-linked oligosaccharides of CTLA4-Ig molecules, enzymatically released N-linked oligosaccharides can be readily separated by the PGC column in order of increasing sialylation and increasing size. The range of structures present and the relative amounts of each class of structure are determined through a combination of MS and UV analysis (Example 3).

[00412] To optimize the LC/MS PGC method, optimization of mass spectral conditions can be useful. Optimization can include a set of surface mapping experiments in order to evaluate the effects of solvent composition and MS ionization parameters on oligosaccharide detection. Solvent composition parameters for evaluation comprise percentage acetonitrile (by volume) and eluent additives (trifluoroacetic acid and ammonium hydroxide). MS ionization parameters evaluated for evaluation include the desolvation temperature, capillary voltage and cone voltage settings for the electrospray source.

[00413] The ionization parameters can play a significant role in signal response. The model resulting from the surface mapping determination was used to set ionization parameters during the chromatographic determination. Higher values for both desolvation temperature and cone voltage result in greater response. The capillary voltage optimum varies depending on the eluent additive, the TFA containing solvent system having a slightly higher optimal capillary voltage. The factor with the largest effect is the volume percentage of acetonitrile, higher acetonitrile content resulting in higher responses.

[00414] ׳ Porous Graphitized Carbon Chromatography

[00415] Porous graphitized carbon (PGC) has been used for solid phase extraction desalting of oligosaccharides. PGC has also been known as an effective chromatographic media for oligosaccharide separation under both acidic and basic elution conditions. Chromatography conditions for both acidic and basic profiling of enzymatically released oligosaccharides from CTLA4-Ig molecules having monomer sequences from SEQ ID NO:2 are developed. Each condition is compatible with both UV and MS detection. As was observed in the infusion experiments, the acidic elution conditions result in higher MS sensitivity than the basic conditions. The MS response for neutral oligosaccharides eluted under acidic conditions, detected as TFA adducts, are five to nine times the intensity of the corresponding peak eluted under basic conditions. The difference in signal response is less dramatic for the acidic oligosaccharides, averaging three times the signal response for monosialylated glycans and equal signal response for di-sialylated glycans. The increased number of peaks in the TFA eluted chromatogram (FIGS. 14A-B) compared to the NH4OH eluted chromatogram (FIG. 15A-B) is a result of separation of anomeric forms of oligosaccharides. Collection and concentration of individual peaks eluted from the TFA gradient result in splitting of the single peak into two peaks of identical mass upon reinjection. Basic elution of the oligosaccharides from the PGC column results in a simpler profile (FIG. 15A-B). The basic elution conditions do not result in complete anomeric separation, however significant peak broadening is observed. ^,The peaks resolution can be increased by increasing the column temperature, which will accelerate the interchange of anomeric forms (Itoh S., et al., J. Chromatogr. A. 2002 968(1 -2), 89-100). However, the sensitivity (ion count) for the detected oligosaccharides remains reduced compared to the acidic elution conditions. It has been reported that addition of salts such as ammonium acetate can increase sensitivity. (Churms SC, J. Chromatogr. A. 500 (1990) 555-583.)

[00416] Addition of ammonium acetate, ammonium trifluoroacetate or ammonium formate results in increased response but also results in asymmetric peak broadening. The resulting peak broadening and potential interference of the added salt with UV detection made salt addition an unattractive option. An alternative means of eliminating anomeric separation is to reduce the oligosaccharides to the corresponding alditols.

[00417] Higher sensitivity and chromatographic resolution make the acidic elution conditions useful for oligosaccharide profiling. A particular profiling system consists of a Luna Cl 8 column coupled through two dual-position six port valves to the Hypercarb 5 μΜ column (100 x 4.6 mm). The Hypercarb column is coupled to a UV detector (Waters 2996 PDA) in series with a Q-ToF Micro (Micromass) with a standard ESI probe. Through appropriate switch control, prepurified CTLA4-Ig samples can be profiled using the Hypercarb column alone, or digest mixtures can be profiled by direct injection of the digest mixture onto the Luna Cl 8 in series with the Hypercarb column. Typically, profiles are obtained from the N-linked oligosaccharides released from 10 to 20 nmoles of protein.

• [00418] In certain embodiments, the invention provides a population of CTLA4-Ig molecules that have a chromatogram according to any one or more of the chromatograms having representative peaks. Representative oligosaccharide profile chromatograms for CTLA4-Ig molecules having monomers with sequences from SEQ ID NO:2 are shown in

FIG. 13, FIGS. 14A-B, FIGS. 15A-B (PGC), FIG. 17 (HPAEC/PAD), and FIGS.18־־A-־B. ־ ־ Both of these'chromatographic profiles can.be broken down into four distinct domains containing oligosaccharide structures with increasing degrees of sialylation in the later eluting domains;. The PGC chromatographic system allows for direct interface, with a mass detector. The mass resolution and signal to noise ratios are acceptable even for oligosaccharides which are present in low percentages. Chromatographic resolution of individual oligosaccharide structures appears greater in the PGC chromatographic separation as compared to the HPAEC.

[00419] . . Collecting peaks from the HPAEC method requires desalting and the high pH employed introduces the possibility of peeling reactions that could interfere with accurate structural identification of peaks. Because the chromatographic conditions used with PGC chromatography are-free of salts, the eluted oligosaccharide peaks can be collected and concentrated with minimal manipulation. This allows for the collection and concentration of eluted peaks, followed by injection of the collected oligosaccharides onto the HPAEC system. Re-injection of the collected oligosaccharides onto a HPAEC-PAD system allows for structural assignment of some of the peaks present in the HPAEC profile (FIG. 17). Due to incomplete peak resolution on the anion exchange column, not all of the isolated peaks could be mapped to the HPAEC profile.

[00420] . The profiles resulting from direct injection of digest mixtures and those for isolated oligosaccharides from the same protein sample are not identical. The profiles resulting from direct injection (FIGS. 18A-B) have different anomer ratios suggesting that the concentration of collected oligosaccharides is resulting in increased anomerization. More importantly, the profile resulting from direct injection contains a peak, which corresponds to a tetra-sialylated structure. This structure is not identified in the profile of the collected and isolated oligosaccharides. In addition to shortened assay time, profiling directly from digest mixtures can result in a more accurate representation of the oligosaccharide distribution, by avoiding glycan degradation during collection and concentration.

[00421] Relative Quantitation

[00422] The surface mapping performed on infusion samples indicates that the volume percentage of acetonitrile has a significant effect on the ionization efficiency of eluting oligosaccharides. The dependence of signal intensity on acetonitrile content in.the mobile phase makes relative quantitation of oligosaccharides by MS dependent on the retention time of the eluting peak. Variations in column condition can effect elution times on PGC columns. For this reason, it would be difficult to obtain consistent relative quantitation from the ion chromatogram elution profile. The UV trace at 206 nm should not be affected by the solvent composition to the same extent as the ion trace. The relative quantitation was performed using the UV trace, the ion trace was used for characterization and qualitative comparisons only. Replicate injections for oligosaccharides isolated from a single glycoprotein lot resulted in percent relative standard deviations (%RSD) of less than 4% for each of the four oligosaccharide domains quantified.

[00423] O-linked Structures in CTLA4-Ig molecules comprising monomers of SEO ID NO:2

[00424] In addition to the N-linked carbohydrates, CTLA4-Ig molecules can contain Olinked carbohydrates. The O-linked oligosaccharide structures can be analyzed using a series of orthogonal mass spectrometry techniques. These techniques include several endopeptidase cleavages followed by LC/MS/MS analysis.

[00425] With respect to CTLA4-Ig molecules formed of monomers having a sequence from SEQ ID NO: 5, 6, 7, 8, 9, or 10, the two major sites of O-linked glycosylation were characterized using exact mass electrospray ionization and the major structures at each O-link site were determined. These data are summarized in FIG. 9. Data are consistent with there being three major O-linked structures: (GalNAc)! (Gal)! (NeuAc)!; (GalNAc)! (Gal)! (NeuAc)₂; (GalNAc)! (GlcNac)! (Gal)₂ (NeuAc)₂. Each structure is observed in differing amounts on each site. These amounts are relatively quantitative and represent data obtained from multiple analyses. The O-linked oligosaccharides contribute a substantial amount of sialic acid to CTLA4-Ig. There are two major O-linked oligosaccharide attachment points per chain. The primary site of occurrence for O-linked oligosaccharides is Ser¹⁶⁵, which is occupied in about 95% of the time. The secondary site of occurrence for O-linked oligosaccharides is Ser^15s/156 which is occupied = 25% of the time. The orthogonal data presented herein provides an overview of the predominant carbohydrate structures present on such CTLA4-Ig molecules and is summarized in FIG. 9.

[00426] In general, the O-linked carbohydrates have, far greater.heterogeneity of structure than are present in N-linked carbohydrates. In addition, there is no consensus sequence for Olink attachment. Thus, a series of orthogonal techniques were developed for use in the structural characterization of the. O-linked oligosaccharides: LC/MS intact analysis and

LC/MS glycopeptide analysis.

[00427] Based on Edman degradation and MALDI, an O-link site was reported to be Ser¹⁶⁵ (with respect to SEQ ID NO:2). To obtain direct data for the presence of Ser¹⁶⁵glycosylation, MS/MS sequencing using b' and y ion series on the T9 peptide (see Table 4 and Table 5) was performed. Table 4 lists the ion series for the T9 peptide in four different states of glycosylation. In all four states, the b' ion series, bl .. b6 ions are in agreement. However, the b' ion series, b7 .. b_max'vary by the different glycosylation states at b7 (Ser¹⁶⁵). As a confirmation, the corresponding y ion series is reported. In all four y ion series, the yl..yl9 ions are in complete agreement. However, the y ion series, y20 ..ymax vary by the different glycosylation states at y20 (Ser¹³⁹). The b' and y ion series

סד ו taken together support the implication of Edman sequencing that Ser is the primary site of Olinked glycosylation on the T9 peptide. T9 is a peptide that contains several serine and theronine residues.

[00428] Table 4 presents LC/MS/MS b’ and y” ions for the T9 peptide with and without the O-linked ladder of (GalNAc)! (Gal)! (NeuAc)!. The b’ ion series are identical for all spectra until b7. where the spectrum then differs by the.O-linked carbohydrate, structure listed above each series. The y’ ion series are identical for all spectra until y 19 where the spectrum then differs by the O-linked carbohydrate structure listed above each series.

Table 4: LC/MS/MS b’ and y” ions for the T9 peptide.

<td colspan="3"> T9-GalNAc-Gal-NeuAc</td><td colspan="3"> T9-GalNac-Gal</td><td colspan="3"> T9-GalNac</td><td colspan="3"> T9</td>

<td></td><td> b’</td><td> y”</td><td></td><td> b’</td><td> y”</td><td></td><td> b’</td><td> y”</td><td></td><td> b’</td><td> y”</td>

<td> 1 Thr 26</td><td> 102.1</td><td> -</td><td> 1 Thr 26</td><td> 102.1</td><td> -</td><td> 1 Thr 26</td><td> 102.1</td><td> -</td><td> 1 Thr 26</td><td> 102.1</td><td> -</td>

<td> 2 His 25</td><td> 239.1</td><td> 3243.6</td><td> 2 His 25</td><td> 239.1</td><td> 2952.5</td><td> 2 His 25</td><td> 239.1</td><td> 2790.5</td><td> 2 His 25</td><td> 239.1</td><td> 2587.4</td>

<td> 3 Thr 24</td><td> 340.2</td><td> 3106.6</td><td> 3 Thr 24</td><td> 340.2</td><td> 28152.5</td><td> 3 Thr 24</td><td> 340.2</td><td> 2653.4</td><td> 3 Thr 24</td><td> 340.2</td><td> 2450.3</td>

<td> 4 Ser 23 ’ '</td><td> 427.2</td><td> 3095.5</td><td> 4 Ser 23</td><td> 427.2</td><td> 2714.4</td><td> 4 Ser 23</td><td> 427.2</td><td> 2552.4</td><td> 4 Ser 23</td><td> 427.2</td><td> 2349.3</td>

<td> 5 Pro</td><td> 524.2</td><td> 2918.5</td><td> 5 Pro</td><td> 524.2.</td><td> 2627.4</td><td> 5 Pro</td><td> 524.2</td><td> 2465.3</td><td> 5 Pro</td><td> 524.2</td><td> 2262.3</td>

<td> 22</td><td></td><td></td><td> 22</td><td></td><td></td><td> 22</td><td></td><td> -</td><td> 22</td><td></td><td></td>

<td> 6 Pro 21</td><td> 621.3</td><td> 2821.4</td><td> 6 Pro 21</td><td> 621.3</td><td> 2530.3</td><td> 6 Pro 21</td><td> 621.3</td><td> 2368.3</td><td> 6 Pro 21</td><td> 621.3</td><td> 2165.2</td>

<td> 7 Oik 20</td><td> 1364.6</td><td> 2724.4</td><td> 7 Oln 20</td><td> 1073.52</td><td> 2433.3</td><td> 7 Oli 20</td><td> 911.4</td><td> 2271.2</td><td> 7 Ser 20.</td><td> 708.3</td><td> 2068.1</td>

<td> 8 Pro 19</td><td> 1461.6</td><td> 1981.1</td><td> 8 Pro 19</td><td> 1170.5</td><td> 1981.1</td><td> 8 Pro 19</td><td> 1008.5</td><td> 1981.1</td><td> 8 Pro 19</td><td> 605.4</td><td> 1981.1</td>

<td> 9 Ala 18</td><td> 1532.6</td><td> 1884.1</td><td> 9 Ala 18</td><td> 1241.6</td><td> 1884.1</td><td> 9 Ala 18</td><td> 1079.5</td><td> 1884.1</td><td> 9 Ala 18</td><td> 876.4</td><td> 1884.1</td>

<td> 10 Pro 17</td><td> 1629.7</td><td> 1813.0</td><td> 10 Pro 17</td><td> 1338.6</td><td> 1813.0</td><td> 10 Pro 17</td><td> 1176.6</td><td> 1813.0</td><td> 10 Pro 17</td><td> 973.5</td><td> 1813.0</td>

<td> 11 Glu 16</td><td> 1758.7</td><td> 1716.0</td><td> 11 Glu 16</td><td> 1467.6</td><td> 1716.0</td><td> 11 Glu 16</td><td> 1305.6</td><td> 1716.0</td><td> 11 Glu 16</td><td> 1102.5</td><td> 1716.0</td>

<td> 12 Leu 15</td><td> 1871.8</td><td> 1586.9</td><td> 12 Leu 15</td><td> 1580.7</td><td> 1586.9</td><td> 12 Leu 15</td><td> 1418.7</td><td> 15S6.9</td><td> 12 Leu 15</td><td> 1215.6</td><td> 1586.9</td>

<td> 13 Leu 14</td><td> 1984.9</td><td> 1473.8</td><td> 13 Leu 14</td><td> 1693.8</td><td> 1473.8</td><td> 13 Leu 14</td><td> 1531.8</td><td> 1473.8</td><td> 13 Leu 14</td><td> 1328.7</td><td> 1473.8</td>

<td> 14 Gly 13</td><td> 2041.9</td><td> 1360.8</td><td> 14 Gly 13 </td><td> 1750.8</td><td> 1360.8</td><td> 14 Gly 13</td><td> 1588.8</td><td> 1360.8</td><td> 14 Gly 13</td><td> 1385.7</td><td> 1360.8</td>

<td> 15 Gly 12</td><td> 2099.0</td><td> 1303.7</td><td> 15 Gly 12</td><td> 18070.9</td><td> 1303.7</td><td> 15 Gly 12</td><td> 1645.8</td><td> 1303.7</td><td> 15 Gly 12</td><td> 1442.7</td><td> 1303.7</td>

<td> 16 Ser 11</td><td> 2186.0</td><td> 1246.7</td><td> 16 Ser 11</td><td> 1894.9</td><td> 1246.7</td><td> 16 Ser 11</td><td> 1732.8</td><td> 1246.7</td><td> 16 Ser 11</td><td> 1529.8</td><td> 1246.7</td>

<td> 17 Ser 10</td><td> 2273.0</td><td> 1159.7</td><td> 17 Ser 10</td><td> 1981.9</td><td> 1159.7</td><td> 17 Ser 10</td><td> 1819.9</td><td> 1159.7</td><td> 17 Ser 10</td><td> 1616.8</td><td> 1159.7</td>

Table 5: O-linked glycopeptide fragments with the corresponding sequence numbers, amino acid sequences and theoretical masses

<td> Enzyme</td><td> Enzyme Fragment</td><td> Sequence Fragment</td><td> Amino Acid Sequence (SEQ ID NO:2)</td><td> Unmodified Mass</td>

<td> Trypsin</td><td> T9</td><td> 159-184</td><td> THTSPPSPAPELLGGSSVFLFPPKPK</td><td> 2688.44</td>

<td> AspN</td><td> D8</td><td> 150-156</td><td> DQEPKSS</td><td> 790.36</td>

<td> Tryp/ chrmo</td><td> N/A</td><td> 159-171</td><td> THTSPPSPAPELL</td><td> 1345.7</td>

[00429] The O-linked carbohydrate structures at Ser¹⁶⁵ represent a heterogeneous population of three major species. In FIG. 19, the T9 glycopeptide is observed in the ־ deconvoluted spectrum. There is a base peak at 2689.2 amu which is in agreement with the theoretical mass for this peptide of 2689.11 amu. The spectrum illustrates three major O-linked structures. The spectrum illustrates the base peptide with a sugar ladder consistent with the O-linked structure (GalNAc)! (Gal)! (NeuAc)!. The magnified bold portion of the spectrum has been enhanced 10-fold and identifies two additional O-linked structures consistent with (GalNAc)! (Gal)! (NeuAc)? and (GalNAc)! (GlcNAc)! (Gal)2 (NeuAc)!.

[00430] Mass spectrometry was used to assess the relative abundance of each O-linked species. In FIG. 19, the (GalNAc)! (Gal)! (NeuAc)! glycan is observed in a 10:1 ratio with the (GalNAc)! (Gal)! (NeuAc)2 glycan and in a 30:1 ratio with the (GalNAc)! (GlcNAc)! (Gal)2 (NeuAc)₂ glycan. In one embodiment therefore, the invention provides a population ־comprising CTLA4-Ig molecules that have a 10:1 ratio of (GalNAc)! (Gal)! (NeuAc)! glycan to (GalNAc)! (Gal)! (NeuAc)₂ glycan. In another embodiment, the invention provides a population comprising CTLA4-Ig molecules that have a 30:1 ratio of (GalNAc)! (Gal)! (NeuAc)! glycan to (GalNAc)! (GlcNAc)! (Gal)₂ (NeuAc)₂ glycan. The (GalNAc)! (Gal)! (NeuAc)2 glycan is observed in a ratio of 20:1 with the (HexNAc)2 (Gal)2 (NeuAc)2 glycan.

In another embodiment, the invention provides a population comprising CTLA4-Ig molecules that have a 20:1 ratio of the (GalNAc)! (Gal)! (NeuAc)2 glycan to (HexNAc)2 (Gal)2 (NeuAc)2 glycan. In another embodiment, the invention provides a population of CTLA4-Ig molecules that comprise all of the said ratios in this paragraph. In addition, a negative ion electrospray spectrum confirms these three predominant structures; the relative abundance of each is shown in FIG. 9.

[00431] With respect to CTLA4-Ig molecules comprising monomers of SEQ ID NO:2, in addition to the Ser¹⁶⁵ site, a second O-link site is observed at Ser¹⁵⁵ or Ser¹⁵⁶. This site is referred to as Ser^155/I56. The D8 peptide containing Ser^155/156 was generated from an AspN digestion and corresponds to amino acids 150-156 of SEQ ID NO:2. The peptide is separated and detected by LC/MS. Hie spectrum (not shown herein) for the D8 O-linked glycopeptide shows a base peak of 790.2 amu that is in agreement with the theoretical mass of 790.8 amu. The spectrum illustrates the peptide ion and a series of ions which are consistent with the structure, (GalNAc)! (Gal)1 (NeuAc)!. The peptide is predominantly non-glycosylated; the glycosylated (GalNAc)! (Gal)! (NeuAc)! species constitutes approximately 22% peak area.

[00432] The O-linked oligosaccharide structures of the CTLA4-Ig single chain were characterized using a series of orthogonal mass spectrometry techniques. These techniques include endopeptidase cleavages with LC/MS analysis of the two predominant sites of O-linked glycosylation with the use of electrospray ionization to determine the predominant structures at each O-link site. These data are summarized in FIG. 20. There can be four predominant O-linked structures: (GalNAc) !(Gal)!(NeuAc)1; (GalNAc)!(Gal)!

(NeuAc)2; (HexNAc)2(Gal)2(NeuAc)2; (HexNAc)₂(Gal)₂,(NeuAc)3. These structures are detected in differing amounts on each site. Greater than 95% ofthe CTLA4-Ig single chain has at least (HexNAc)2 (Gal)₂ (NeuAc)₂.

[00433] Another assay was developed to confirm the O-linked carbohydrates and look for less prevalent structures. This technique utilized trypsin and chymotrypsin codigestion to produce a peptide confirmed by MS/MS to be THTSPPSPAPELL (amino acids 159-171־ of SEQ ID NO:2). This peptide allowed for the identification of one monosialylated, two di-sialylated and one tri-sialylated O-linked species. A definitive structure has not been elucidated for the tri-sialylated species, however two possibilities are proposed: a peptide containing a core 2 structure with 3 sialic acids or two core 1 structures present on two different amino acid residues.

[00434] A complementary technique, intact analysis by MS, was used to confirm the presence of heterogeneous O-link glycosylations of CTLA4-Ig molecules. CTLA4-Ig dimers and׳ CTLA4-Ig single chain were treated with PNGase F to remove the N-linked oligosaccharides. The molecule was then detected by the mass spectrometer and the corresponding ions were deconvolved into the spectrum. In the single chain material, the predominant glycan composition is (HexNAc)2(Hex)2(NeuAc)2, while the reference is predominantly (HexNAc)l(Hex)l(NeuAc)l. The glycosylation compositions are in agreement with those observed during the LC/MS peptide analysis. In addition to a change in the Olinked glycosylati on pattern, a second major modification was observed. A mass shift of 113 ± 4 u is observed between the single chain non-reduced species and the reduced CTLA4Ig standard. The mass shift of 113 ± 4 u disappeared upon reduction with DTT. In the dimer material, the resulting ion envelope was deconvolved into a spectrum (not shown herein) with a major peak at 79944 amu, which corresponds to the presence of two (GalNAc)! (Gal)! (NeuAc)! structures. The next largest peak, at 80600 amu, corresponds to three O-link. structures or a combination of at most one branched O-link structure. The third largest peak corresponds to either four O-linked structures or a combination containing at most two branched O-link structures.

[00435] Determination of sialic acid content

[00436] Another aspect of glycoprotein characterization is determination of sialic acid. Sialic acid content Can be a signature characteristic of glycoprotein. Sialic acid content of a glycoprotein of the present invention can be assessed by conventional methods. For example, sialic acid can be separately determined by a direct colorimetric method (Yao et al., 1989, Anal. Biochem. 179:332-335), using at least triplicate samples. Another method of sialic acid determination involves the use of thiobarbaturic acid (TBA), as described by Warren et al., 1959, J. Biol. Chem., 234:1971 -1975. Yet another method involves high performance chromatography, such as described by Η. K. Ogawa et al., 1993, J. Chromatography, 612:145-149.

[00437] In one embodiment, a method to determine the amount of N-Acetyl Neuraminic Acid (NANA) and N-Glycolyl Neuraminic Acid (NGNA) is through acid hydrolysis treatment of the glycoprotein of interest (for example, see Example 3). In this method, ־ NANA and NGNA are cleaved from the protein by acid hydrolysis. In one embodiment the glycoprotein is substantially purifed by methods suitable for its purification. The released NANA and NGNA are separated by HPLC on a Rezex Monosaccharide RHM column and detected by UV absorbance (206 nm). NANA and NGNA are quantitated based on the response factors of concurrently run NANA and NGNA standards. The results can be reported as molar ratios (MR) of NANA and NGNA respectively, to protein.

[00438] The purpose of the acid hydrolysis method of measuring sialic acid content is to measure the amount of total sialic acid (NANA and NGNA) to protein in CTLA4-Ig or CTLA4^A29YLI04E-Ig samples (molar ratios). It is important to note, however, that these sialic acid molar ratios include both bound and free NANA and NGNA. Molar ratio results are obtained based on the peak area comparison of NANA and NGNA from hydrolyzed CTLA4Ig or CTLA4^A29YL104E-Ig samples versus non-hydrolyzed NANA and NGNA standards. Hydrolyzed standards of NANA and NGNA can also be used.

[00439] For example, molar ratios were obtained for CTLA4-Ig molecules having SEQ ID NO:2 amino acid sequences. Without hydrolysis, the peak of interest in chromatograms of NANA and NGNA standards appears as a single peak. When the NANA standard and

CTLA4-Ig samples are hydrolyzed, the resulting chromatograms show NANA as a major peak followed closely by a small shoulder peak (&lt;10% of the major peak area; referred to as degraded NANA); the same concentration of NANA standards with-and without hydrolysis resulted in very close peak areas, including the degradant. No peak is clearly seen in the chromatograms for a degraded NGNA species, although the area counts of the NGNA peak in a hydrolyzed NGNA standard were seen to decrease approximately 8 - 9%. Mass spectrometry (MS) experiments demonstrated that the NANA degradant in both the hydrolyzed NANA standard and the hydrolyzed CTLA4-Ig samples results from loss of 18 Daltons (water) from NANA. Therefore, the method appropriately includes the small shoulder peak in the integration of NANA peak in hydrolyzed CTLA4-Ig. It was also demonstrated by MS experiments that NGNA degraded upon hydrolysis with a loss of 18 Daltons. The NGNA degradant eluted between NANA and NANA degradant so that UV did not detect it. In CTLA4-Ig material, NGNA content is roughly 5% of NANA content and, as a result, co-elution of the NGNA degradant causes less than 0.5% change of the NANA peak area, which is within the variability range of the NANA peak area. The method cannot include the area of degraded NGNA in the NGNA result; therefore the NGNA result can be low by &lt; 10%, also within the variability of the method.

[00440] Because NGNA is thought to be more immunogenic than NANA, there is a clinical preference for a recombinant thereapeutic that contains a low NGNA molar ratio. In one embodiment of the invention, the preponderance of sialic acid in a population of CTLA4Ig molecules is NANA and not NGNA, wherein in this population the molar ratio of moles sialic acid per mole CTLA4-Ig molecules or dimer is from about 5 to about 18. In another embodiment, the preponderance of sialic acid in a population of CTLA4^A29YLI04E-Ig molecules is NANA and not NGNA, wherein in this population the molar ratio of moles sialic acid per mole CTLA4^A29YL104E-Ig molecules or dimer is from about 5.5 to about 8.5.

[00441] CTLA4-Ig and CTLA4^A29YL104E־Ig Expression Cassettes

[00442] The invention provides for a nucleic acid encoding a CTLA4-Ig molecule, which is an expression cassette in one embodiment. The invention also provides for a nucleic acid encoding a CTLA4^A29YL104E-Ig molecule. In one embodiment, the nucleic acid encoding CTLA4-Ig molecule is contained within an expression cassette. In another embodiment, the nucleic acid encoding the CTLA4-Ig molecule is contained within an expression cassette derived from a plasmid having the nucleotide sequence of SEQ ID NO:17. In further embodiments, the nucleic .acid.encoding the CTLA4^A29YLI04E-Ig molecule is contained within an expression cassette. In certain embodiments, the nucleic acid encoding the

CTLA4^A29YL104E-Ig molecule is contained within an expression cassette derived from a plasmid deposited as ATCC Accession No. PTA-2104.

[00443] The nucleic acids of the invention can be a cDNA, cDNA-like, DNA or RNA nucleic acid molecule of interest in an expressible format, such as an expression cassette, which can be expressed from the natural promoter or a derivative thereof or an entirely heterologous promoter. Alternatively, the nucleic acid of interest can encode an anti-sense RNA. The nucleic acid of interest can encode a protein (for example a glycoprotein, such as CTLA4-Ig or CTLA4^A29YL104E-Ig glycoprotein), and may or may not include introns.

[00444] In one embodiment, the nucleic acid encoding a peptide having CTLA4 activity can be obtained from T cell genomic DNA or from mRNA present in activated T lymphocytes. In another embodiment, the nucleic acid encoding a CTLA4^A29YL104E-Ig also can be obtained from T cell genomic DNA or from mRNA present in activated T lymphocytes. In another embodiment of the invention, the gene encoding a protein of interest, for example CTLA4 or CTLA4^A29YL104E-Ig, can be cloned from either a genomic library or a cDNA according to standard protocols that one skilled in the art practices. A cDNA, for example encoding CTLA4 or CTLA4^A29YLI04E-Ig, can be obtained by isolating total mRNA from a suitable cell line. Using methods known in the art, double stranded cDNAs can be prepared from the total mRNA and subsequently can be inserted into a suitable bacteriophage vector or plasmid. Genes can also be cloned using PCR techniques well established in the art. In one embodiment, a gene that encodes CTLA4 or CTLA4^A29YL104E-Ig can be cloned via PCR in accordance with the nucleotide sequence information provided by this invention.

[00445] In another embodiment, a DNA vector containing a CTLA4 or CTLA4^A29YL104E-Ig cDNA can act as a template in PCR reactions wherein oligonucleotide primers designed to amplify a region of interest can be used as to obtain an isolated DNA fragment encompassing that region. In a particular embodiment of the invention, the region of interest targeted in CTLA4 cDNA can be the extracellular domain of CTLA4, including the extracellular domain of human CTLA4. In certain embodiments, the region of interest targeted in a CTLA4^A29YLI04E-Ig cDNA can be the extracellular domain of CTLA4 with amino acid changes at amino acid positions 55 and 130 of SEQ ID NO:2, (for example, see SEQ ID NO:

18) including the extracellular domain of human CTLA4 harboring the amino acid changes described above.

[00446] To express a fusion protein in the context of this invention, the chimeric gene fusion in one embodiment (for example a gene encoding a CTLA4-immunoglobulin (CTLA4-Ig) fusion protein or CTLA4^A29YL104E-Ig fusion protein) includes a nucleotide sequence, which encodes a signal sequence whereby upon transcription and translation of the chimeric gene, directs the newly synthesized fusion protein for secretion. In one embodiment, a native CTLA4 signal sequence (e.g., the human CTLA4 signal sequence described in Harper, K., et al. (1991, J. Immunol. 147,1037-1044) can be used. In an alternative embodiment of the invention, a heterologous signal sequence can be used to direct CTLA4-Ig or CTLA4^A29YL104E-Ig secretion (for example, the oncostatin-M signal sequence (Malik N., et al., 1989, Mol Cell Biol 9(7), 2847-2853) or an immunoglobulin signal sequence). One skilled in the art understands that the nucleotide sequence corresponding to the signal sequence can be inserted into the chimeric gene fusion by standard recombinant DNA techniques, such as by performing an in-frame ligation of the signal sequence at the 5' end of a nucleic acid sequence encoding CTLA4.

[00447] Under the provisions of the Budapest Treaty, DNA encoding the amino acid sequence corresponding to a CTLA4-Ig fusion protein has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA, 20110, on May 31,1991. It has been assigned ATCC Accession No. 68629. Additonally, an expression plasmid comprising a nucleic acid sequence encoding the amino acid sequence corresponding to a CTLA4^A29YL104E-Ig was deposited under the provisions of the Budapest Treaty on June 19, 2000 with theATCC. The deposited plasmid has been assigned ATCC Accession No. PTA-2104. The deposited plasmid is also referred to as pD16 LEA29Y and pD16 L104EA29Y. CTLA4^A29YL104E-Ig s are further described in U.S. Patent No. 7,094,874 and co-pending U.S. Patent Application Nos. 09/579,927, 60/287,576, and 60/214,065, and in International Patent Publication No. WO 01/923337 A2, all of which are incorporated by reference in this application in their entireties.

[00448] An expression vector of the invention can be used to transfect cells, either eukaryotic (for example, yeast, mammalian, or insect cells) or prokaryotic in order to produce proteins (for example, fusion proteins such as CTLA4-Ig, CTLA4^A29YL104E-Ig molecules, and the like) encoded by nucleotide sequences of the vector. One skilled in the art understands that expression of desired protein products in prokaryotes is most often carried out in E. coli with vectors that contain constitutive or inducible promoters. Some E. coli expression vectors (also known in the art as fusion-vectors) are designed to add a number of amino acid residues, usually to the N-terminus of the expressed recombinant protein. Said fusion vectors can serve three functions: 1) to increase the solubility of the desired recombinant protein; 2) to increase expression of the recombinant protein of interest; and 3) to aid in recombinant protein purification by acting as a ligand in affinity purification. Some examples of fusion expression vectors include, but are not limited to: a) pGEX (Amrad Corp., Melbourne, Australia) which fuse glutathione S-tranferase to desired protein; b) pcDNA™3.1/V5-His A B &amp; C (Invitrogen Corp, Carlsbad, CA) which fuse 6x-His to the recombinant proteins of interest; and c) pMAL (New England Biolabs, Beverly, Mass.) which fuse maltose E binding protein to the target recombinant protein.

[00449] The cells suitable for culturing according to the processes and methods of the present invention can harbor introduced expression vectors (constructs), such as plasmids and the like. The expression vector constructs can be introduced via transfection, lipofection, transformation, injection, electroporation, or infection. The expression vectors can contain coding sequences, or portions thereof, encoding the proteins for expression and production in the culturing process. Such expression vectors can include the required components for the transcription and translation of the inserted coding sequence. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination which are described in J. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley &amp; Sons, New York, N.Y.

[00450] A selectable marker can be used in a recombinant expression vector (for example, a plasmid), wherein the vector is stably integrated into the genome of the cell, to confer resistance to the cells harboring the vector. This allows for their selection in an appropriate selection medium. A number of selection systems can be used, including but not limited to, the hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the Herpes Simplex Virus thymidine kinase (HSV TK), (Wigler et al., 1977, Cell, 11:223), (Szybalska and Szybalski,

1992, Proc. Natl. Acad. Sci. USA, 48:202), and adenine phosphoribosyltransferase (AP-RT), (Lowy etal.,. 1980, Cell, 22:817) genes, which can be employed in hgprt-, tk-, or apr/-cells, respectively.

[00451] The following non-limiting examples of marker genes, which can be contained within an expression vector, can also be used as the basis of selection for anti-metabolite resistance: gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA, 78:2072); dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA, 77:3-57; and O'Hare et al., 1981, Proc. Natl. Acad. Sci. 78:1527); hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene, 30:147); and neo, which confers resistance to the aminoglycoside G418 (Clinical Pharmacy, 12:488-505; Wu and Wu, 1991, Biotherapy, 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol., 32:573-596; Mulligan, 1993, Science, 260:926-932; Anderson, 1993, Ann. Rev. Biochem., 62:191-21; May, 1993. TIB Tech, 11 (5):155-215). Recombinant DNA techniques commonly known in the art can be routinely applied to elect the desired recombinant cell clones. Such techniques are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY (1993); Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley &amp; Sons, NY (1994); Colberre-Garapin et al., 1981. J. Mol. Biol., 150:1, which are incorporated by reference herein in their entireties.

[00452] The expression levels of an expressed protein molecule can be increased via amplification of the expression vector (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3, Academic Press, New York, 1987). An increase in the level of an inhibitor present in the culture medium of a host cell will increase the number of copies of the marker gene when a marker in the expression vector system expressing a protein of interest is amplifiable. Since the amplified region is associated with the protein-encoding gene, protein production will concomitantly increase (Crouse et al., 1983, Mol. Cell. Biol3:257 ״). Vectors that harbor the nucleic acid sequences that encode for the selectable markers glutamine synthase (GS) or dihydrofolate reductase (DHFR) can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of such vectors is the availability of cell lines, for example the murine myeloma cell line, NSO and the

Chinese Hamster Ovary, CHO, cell line DG44, which are glutamine synthase negative and־ dihydrofolate reductase negative, respectively.

[00453[ In one embodiment of the present invention, a nucleic acid sequence encoding a soluble CTLA4 or CTLA4^A29YL104E-Ig fusion protein molecule can be inserted into an expression vector designed for expressing foreign sequences in a eukaryotic host. The regulatory components of the vector can vary according to the eukaryotic host chosen for use. Vectors used to express soluble CTLA4 or CTLA4^A29YL104E-Ig in eukaryotic host cells can include enhancer sequences for optimization of protein expression.

[00454] Mammalian cells (such as BHK cells, VERO cells, CHO cells and the like) can harbor an expression vector (for example, one that contains a gene encoding the CTLA4-Ig fusion protein or the CTLA4^A29YLI04E-Ig fusion protein) via introducing the expression vector into an appropriate host cell. Accordingly, the invention encompasses expression vectors containing a nucleic acid sequence that encodes a CTLA4-Ig or CTLA4^A29YLI04E-Ig fusion protein and encompasses host cells into which such expression vectors can be introduced via methods known in the art. As described herein, an expression vector of the invention can include nucleotide sequences that encode a CTLA4-Ig or CTLA4^A29YLI04E-Ig fusion protein linked to at least one regulatory sequence in a manner that allows expression of the nucleotide sequence in a host cell. To those skilled in the art, regulatory sequences are well known and can be selected to direct the expression of a protein of interest in an appropriate host cell as described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences can comprise the following: enhancers, promoters, polyadenylation signals, and other expression control elements. Practitioners in the art understand that designing an expression vector can depend on factors, such as the choice of host cell to be transfected and/or the type and/or amount of desired protein to be expressed.

[00455] Cloning and expression plasmids (for example, pcSD and piLN) are constructed as described in Examples 11. In one embodiment of this invention, an isolated DNA fragment from plasmid pSV2d7z/r- is ligated to the pcDNA3 vector backbone generating the expression vector pcSD. Vector pcSD is comprised of the following features: a cytomegalovirus (CMV) promoter followed by a multiple cloning site (MCS); a bovine growth hormone (BGH) polyadenylation signal and transcriptional termination sequence; a mouse dhfr cDNA sequence for selection and amplification; an ampicillin resistance gene;

and a pUC origin of replication for selection and maintenance in Escherichia coli. Vector piLN is constructed containing cDNAs encoding portions of the amino acid sequence corresponding to a fragment of the extracellular domain of the human CTLA4 receptor Example 11, where the cDNA encoding a first amino acid sequence is joined to DNA encoding a second amino acid sequence, which corresponds to an IgC region that permits the expression of the CTLA4 receptor gene by altering the solubility of the expressed CTLA4 protein (see FIG. 1 and brief description for FIG. 1 for residues corresponding to CTLA4 extracellular portion and IgGl constant region). In one embodiment, an oncostatin M signal peptide sequence can be fused to the amino acid sequence corresponding to the extracellular domain of CTLA4 which subsequently is fused to a second amino acid sequence corresponding to an Ig domain (for example, the human IgC_y! domain) as previouslydescribed in FIG. 1. The oncostatin M signal sequence allows for soluble forms of the CTLA4 gene (for example CTLA4-Ig) protein product to be generated.

[00456] To construct a pcSD expression vector containing a gene encoding the CTLA4immunoglobulin fusion protein, methods known in the art (for example, restriction site subcloning) can be used. The starting material for one embodiment of the invention can be a digested and excised DNA fragment from the cloning vector piLN described in Example 11. In another embodiment, the excised DNA fragment from said vector contains the amino acid sequence of the oncostatin M signal sequence and-CTL4Ig fusion protein, wherein said DNA fragment is ligated to the digested pcSD vector. The oncostatin M-CTLA4-Ig DNA fragment can be inserted between the CMV promoter and a cassette containing the BGH polyadenylation signal and transcriptional termination sequence. This would place a CTLA4Ig gene product under the control of the CMV promoter in the plasmid designated pcSDhuCTLA4-Ig (FIG. 21; SEQ ID NO: 17).

[00457] Additionally, cloning and expression plasmids (for example, pD16LEA29Y) can be derived from the Invitrogen plasmid pcDNA3. Vector pD16LEA29Y (FIG. 22) comprises the following features: the neomycin resistance gene from pcDNA3 was replaced with the murine dihydrofolate reductase (DHFR) gene under control of the enhancerless (weakened) SV40 promoter; the gene encoding a CTLA4^A29YLI04E-Ig is expressed from the CMV promoter, and the poly adenylation signal is from the bovine growth hormone gene; the expression cassette for the gene of interest is flanked by transcription termination sequences, i.e., 5׳ to the promoter and 3' to the poly A site; the vectors contain two distinct restriction site polylinkers, one 3' to the promoter for cloning the gene of interest, and one 5' to the promoter for vector linearization prior to transfection; the vector contains an ampicillin resistance gene and the ColEl origin of replication for plasmid propagation in E. coli., the CTLA4^A29YL104E-Ig sequence (SEQ ID NO:3) is preceded by the Oncostatin M signal peptide and assembled in the expression vector known as vector pD16LEA29Y.

[00458] The vector is constructed containing cDNAs encoding portions of the amino acid sequence corresponding to a fragment of the extracellular domain of the human CTLA4 receptor (SEQ ID N-O:2), wherein the amino acid Ala at position 55 is replaced by the amino acid Tyr and the amino acid Leu at position 130 is replaced by the amino acid Glu (FIG. 3). These amino acid changes are depicted in the CTLA4^A29YLI04E-Ig amino acid sequence having SEQ ID NO: 4. The cDNA encoding a first amino acid sequence (for example, the sequence that encodes a CTLA4^A29YL104E-Ig) is joined to DNA encoding a second amino acid sequence, which corresponds to an IgC region that permits the expression of the CTLA4^A29YU04E-Ig receptor gene by altering the solubility of the expressed CTLA4^a29yl104eIg protein (see FIG. 3 and the brief description for FIG. 3 for residues corresponding to the modified CTLA4 extracellular portion and IgGl constant region) having SEQ ID NO: 3.

[00459] In one embodiment, an oncostatin M signal peptide sequence can be fused to the amino acid sequence corresponding to the extracellular domain of CTLA4 which subsequently is fused. to .a second amino acid sequence corresponding to an Ig domain (for example, the human IgC_y! domain) as previously described in FIG. 3. The oncostatin M signal sequence allows for soluble forms of the CTLA4 gene (for example a CTLA4^a29yl104eIg) protein product to be generated.

[00460] Stable Transfection to Generate Cell Line

[00461] Vectors that contain DNA encoding a protein of interest (for example, fusion constructs, glycoproteins, and the like) can be transformed into suitable host cells (for example bacterial cells) in order to produce large quantities of cloned DNA. Some nonlimiting examples of bacterial cells for transformation include the bacterial cell line E. coli strains DH5a or MCI 061/p3 (Invitrogen Corp., San Diego, Calif.), which can be transformed using standard procedures practiced in the art, and colonies can then be screened for the appropriate plasmid expression.

[00462] Expression vectors for eukaryotic cells, such as mammalian cells, can include promoters and control sequences compatible with mammalian cells. In one embodiment of the invention, these regulatory elements can be, for example, a CMV promoter found in the pcSD or pD16LEA29Y vector, or the avian sarcoma virus (ASV) located in the piLN vector. Other commonly used early and late promoters include, but are not limited to, those from Simian Virus 40 (SV 40) (Fiers, et al., 1973, Nature 273:113), or other viral promoters such as those derived from bovine papilloma, polyoma, and Adenovirus 2 virus. The regulatable promoter, hMTII (Karin, et al., 1982, Nature 299:797-802) can also be used, in addition to others known in the art. For recombinant protein expression in cultured insect cells (for example, SF 9 cells), some baculovirus vectors available include the pVL series (Lucklow, V. A., and Summers, M. D., 1989, Virology 170:31-39) and the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165). A practitioner skilled in the art also understands that enhancer regions (those sequences found upstream or downstream of the promoter region in noncoding DNA regions) are also important in optimizing expression. Origins of replication can be employed, if needed, from viral sources, for example if utilizing a prokaryotic host for introduction of plasmid DNA. However, chromosome integration is a common mechanism for DNA replication in eukaryotic organisms.

[00463] Although in an embodiment of this invention mammalian host cells (such as CHO cells) are employed for expression of desired protein (for example, fusion proteins, glycoproteins, and the like), other eukaryotic organisms also may be used as hosts.

Laboratory strains of the budding yeast Saccharomyces cerevisiae (also known as Baker's yeast or Brewer’s yeast) can be used as well other yeast strains, such as the fission yeast Schizosaccharomyces pombe. Yeast vectors harboring DNA encoding a protein of interest (for example fusion constructs, glycoproteins, and the like such as CTLA4-Ig or CTLA4^A29YL104E-Ig), can utilize the 2μ origin of replication of Broach, Meth. Enz. 101:307 (1983), or other origins of replications compatible with yeast (for example, Stinchcomb et al., 1979, Nature 282:39; Tschempe et al., 1980, Gene 10:157; and Clarke et al., 1983, Meth, Enz. 101:300). A regulatory element contained within yeast vectors can be a promoter for the synthesis of glycolytic enzymes (Hess et al., 1968, J. Adv. Enzyme Reg. 7:149; Holland et al., 1978, Biochemistry 17:4900).

[00464] One skilled in the art can also utilize other promoters wherein growth conditions can regulate transcription of said regulatable gene, and can include the following non-limiting ־־examples: isocytochrome C, alcohol dehydrogenase 2, enzymes responsible for maltose and _ galactose utilization, acid phosphatase, and degradative enzymes associated with nitrogen metabolism. Similar to mammalian expression systems, terminator sequences in yeast expression vectors are also desirable at the 3'.end of the.coding sequences, and are found in the 3' untranslated region following the open reading frame in yeast-derived genes. Some non-limiting examples of yeast vectors suitable for recombinant protein expression in yeast (for example, in 5. cerevisiae) include pMFa (Kurjan and Herskowitz, (1982) Cell 30:933943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYepSecl (Baldari, et al., 1987, Embo J. 6:229-234), pYES2 (Invitrogen Corporation, San Diego, Calif.), as well as those belonging to the pRS family of yeast vectors.

[00465] . Clones, for example bacterial clones,, which contain DNA encoding a protein of interest (for example, fusion constructs, glycoproteins, and the like), obtained as described above may then transfected into suitable host cells, such as mammalian cells, for expression of the desired product. Transfection techniques are carried out using standard techniques established in the art appropriate to said host cells, wherein the transfection technique depends on the host cell used. For example, mammalian cell transfection can be accomplished using lipofection, protoplast fusion, DEAE-dextran mediated transfection, CaPO4 co-precipitation, electroporation, direct microinjection, as well as other methods known in the art which can comprise: scraping, direct uptake, osmotic or sucrose shock, lysozyme fusion or erythrocyte fusion, indirect microinjection such as via erythrocytemediated techniques, and/or by subjecting host cells to electric currents. As other techniques for introducing genetic information into host cells will be developed, the above-mentioned list of transfection methods is not considered to be exhaustive.

[00466] Expression of DNA encoding a protein of interest (for example, fusion constructs, glycoproteins, and the like) in eukaryotic host cells derived from multicellular organisms (for example, mammalian in origin) is particularly utilized in the context of this invention (Tissue Cultures, Academic Press, Cruz and Patterson, Eds. (1973)). Host .cells derived from . .

multicellular organisms have the ability to splice out introns and thus can be used directly to express genomic DNA fragments. As stated earlier, useful host cell lines include, but are not limited to, Chinese hamster ovary (CHO), BHK cells, monkey kidney (COS), VERO and HeLa cells. In the present invention, cell lines stably expressing the protein of interest (for example, fusion constructs, glycoproteins, and the like) are used. In one embodiment, a mammalian cell line, (such as a׳CHO cell line) is transfected (for example by electroporation) with an expression vector (for.example, pcSDhuCTLA4-Ig, pD16LEA29Y, and the like) containing a DNA sequence encoding a glycoprotein of interest. In one embodiment, the glycoprotein of interest can be a CTLA4-Ig protein, including the CTLA4-Ig protein(s) having an amino acid sequence contained in SEQ ID NO:2, encoded by a portion of the nucleotide sequence in SEQ ID NO: 1. In another embodiment, the glycoprotein of interest can be a CTLA4^A29YL104E-Ig, including the CTLA4^A29YL104E-Ig having an amino acid sequence contained in SEQ ID NO:4, encoded by a portion of the nucleotide sequence in SEQIDNO:23.

[00467] A recombinant protein, such as CTLA4-Ig or CTLA4^A29YL104E-Ig, can be expressed in eukaryotic host cells, such as mammalian cells (for example, CHO, BHK, VERO, or NS0 cells), insect cells (for example, using a baculovirus vector), or yeast cells. Those skilled in the art can use other suitable host cells, such as those described earlier, in the context of this invention. In one embodiment, eukaryotic, rather than prokaryotic, expression of a recombinant fusion protein, (such as CTLA4-Ig or CTLA4^A29YL104E-Ig) is employed.

Expression of eukaryotic recombinant proteins, such as human CTLA4-Ig or CTLA4^A29YL104E-Ig, in eukaryotic cells, such as CHO cells, can lead to partial and/or complete glycosylation, as well as the formation of intra- or inter- chain disulfide bonds. For transient amplification and expression of a desired protein, a vector harboring DNA encoding a protein of interest (for example fusion constructs, glycoproteins, and the like such as CTLA4-Ig or CTLA4^A29YL104E-Ig), is delivered into eukaryotic cells by a transfection method known in the art but not integrated into the cell’s genome. Expression of transfected genes can be measured within 16-96 hours. Mammalian cells (such as COS cells) can be used in conjunction with vectors such as pCDM8 to transiently express a desired protein (Gluzman, Y., 1981. Cell 23:175-182: Seed. B. 1987. Nature 329:8401.

[00468] It is understood in the art that for stable transfection of mammalian cells, a small fraction of cells can integrate DNA into their genomes and successful integration can depend on the expression vector and transfection method utilized. For stable amplification and expression of a desired protein, a vector harboring DNA encoding a protein of interest (for example fusion constructs, glycoproteins, and the like such as CTLA4-Ig or CTLA4^a29yli04eIg) is stably integrated into the genome of eukaryotic cells (such as mammalian cells), resulting in the stable expression of transfected genes. In order to identify and select clones.

stably expressing a gene that encodes a protein of interest, a gene that encodes a selectable marker (for example, resistance to antibiotics) can be introduced into the host cells along with the gene of interest. Selectable markers used by one skilled in the art can be those that confer resistance to drugs, such as G4.18 and hygromycin. The gene encoding a selectable marker can be introduced into a host cell on a separate plasmid or can be introduced on the same plasmid as the gene of interest. Cells containing the gene of interest can be identified by drug selection wherein cells that have incorporated the selectable marker gene will survive in the presence of said drug, while cells that have not incorporated the selectable marker gene die. Surviving cells can then be screened for. the production of the desired protein (for example, a־ CTLA4-Ig protein or CTLA4^A29YLIQ4e=Ig). .

(00469] As described earlier, CHO cells deficient in expression of the dihydrofolate reductase (dhfr) gene can survive only with the addition of nucleosides. When said cells are stably transfected with a DNA vector harboring the dhfr gene, cells are then capable of producing the necessary nucleosides. By using dhfr as the selectable marker, one skilled in the art understands that in the presence of the anti-metabolite, methotrexate, gene amplification of dhfr as well as the transfected gene of interest (for example, CTLA4-Ig or CTLA4^A29YL104E-Ig) readily occurs. In one embodiment of this invention, mammalian cells, such as CHO dhfr- cells, are transfected with an expression vector, such as pcSDhuCTLA41g (Examples 11-13) or pD16LEA29Y, to generate a population of cells that can be stably amplified and that can stably express a desired protein product, (such as CTLA4-Ig or beta p CTLA4^A29YLI04E-Ig olypeptide, respectively). In another embodiment, the dhfr- negative cell line DG44 (Invitrogen Corp. Carlsbad, CA) can be employed for stable transfection. In another embodiment of this invention, transfection can occur via electroporation.

[00470] As is readily practiced in the art, transfected mammalian cells (for example dhfrnegative CHO cells) are maintained in non-selective medium containing serum for 1-2 days post-transfection. Cells then are treated with trypsin and re-plated in serum-containing medium, in the presence of a selective pressure (for example, a drug such as methotrexate). Cells are cultured in selective serum-containing medium for 2-3 weeks, with frequent changes of selective medium in order to eliminate debris and dead cells,.until distinct colonies can be visualized. Individual colonies can then be trypsinized and placed into multiwell plates for further propagation and amplification in the presence of selective medium in order to identify producers that express a high level of the desired protein (for example, fusion constructs, glycoproteins, and the like) via methods established in the art such as

ELIS As or immunoprecipitation. In one embodiment of this invention, the method described above was carried out for transfecting dhfr- negative CHO cells (for example DG44 cells) in order to establish a stable cell line expressing a recombinant protein of interest (for example, a־CTLA4-Ig protein) (see for example, Examples 12-13). In another embodiment, a stable • cell line expressing a CTLA4^A29YL104E-Ig was established (see EXAMPLE 23).

[00471] A stable CHO line of the invention stably expresses CTLA4-Ig protein molecules as CTLA4-Ig monomers having the sequence (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2. This cell line can secrete a population of CTLA4-Ig molecules that can exist as multimeric forms (such as dimers, tetramers, and the like), wherein the multimeric form can have different monomer sequences of SEQ ID NO:2. The expression cassette integrated into this cell line comprises SEQ ID NO:1, and is contained within pcSDhuCTLA4-Ig. ־

[00472] The invention also provides a stable CHO line, which stably expresses a CTLA4^A29YLI04E-Ig. In one embodiment, the cell line expresses CTLA4^A29YL104E-Ig monomers having the sequence (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4. In another embodiment, the cell line can secrete a population of CTLA4^A29YL104E-Ig molecules that can exist as multimeric forms (such as dimers, tetramers, and the like), wherein the multimeric form can have different monomer sequences of SEQ ID NO:4. The expression cassette integrated into this cell line comprises SEQ ID NO:3, and is contained within pD16LEA29Y.

[00473] Subcloning To Generate a Clonal Population of Cells

[00474] Cells identified as being producers of a desired protein (for example, fusion constructs, glycoproteins, and the like) are isolated from cell culture and subsequently ־ ' amplified under production-equivalent conditions wherein culture medium can contain ' serum. Subcloning methods known in the art, such as, but not limited to, soft-agaf cloning, can be employed. The stable recombinant cell clones obtained can then be further multiplied under serum- and animal product-free conditions. According to the present invention, a ’ stable cell clone expressing the desired protein product (for example CTLA4-Ig, a

CTLA4^A29־^YLI04E-Ig,-and the like) is achieved via obtaining a recombinant cell clone from a cell culture that is obtained after culturing a recombinant-original cell clone in serumcontaining medium and re-adapting the cells to serum- and animal product- free medium. In one embodiment, the cell clones expressing CTLA4-Ig can be continued to be cultured in serum- and animal product- free medium through at least 50 generations. In another embodiment of the invention, the cell clones can be continued to be cultured as described .above through at least 75 generations. According to the invention, cell clones can also be continued to be cultured in serum- and animal product- free medium through at least 100 generations.

[00475] In a further embodiment, the cell clones expressing a CTLA4^A29YL104E-Ig can be continued to be cultured in serum- and animal product- free medium through at least 27 generations. In another embodiment, the cell clones can continue to be cultured as described above through at least 75 generations. Additionally, cell clones can be cultured in serum- and animal product- free medium through at least 100 generations.

[00476] In one embodiment, the invention provides a cell line that produces CTLA4-Ig molecules comprising SEQ ID NO :2 monomers, wherein the cell line is stable for over 100 generations, and wherein cell line stability comprises: (1) doubling time at generation 100 is less than about 24.5 ± 2.6 hours; (2) cell viability at generation 100 is greater than 95%, (3) production titer for CTLA4-Ig in 5-L bioreactors is greater than 1.69 mg/mL at generation 100; (4) sialic acid molar ratio to protein is about 9.3 to about 11.0 at generation 105.

[00477] The stable recombinant cell clone of this invention is present in isolated form wherein isolation can occur according to methods practiced in the art (for example, soft-agar cloning or limited dilution cloning or the like). In this invention, the stable recombinant cell clone is derived from a recombinant mammalian cell (for example, a CHO cell) that contains DNA sequences encoding a recombinant protein of interest (for example, fusion constructs, glycoproteins, and the like, such as CTLA4-Ig or CTLA4^A29YLI04E-Ig), which can grow in suspension or adherently. A recombinant protein expressed by the cell line of this invention can be a therapeutic glycoprotein, such as CTLA4-Ig or CTLA4^A29YL104E-Ig. According to the present invention, stable recombinant cell clones derived from eukaryotic cells (such as mammalian CHO cells, DG44 cells, or dhfr- negative CHO cells), which contain a DNA sequence encoding a recombinant glycoprotein, such as CTLA4-Ig or CTLA4^A29YLI04E-Ig, and which are capable of stably expressing the recombinant glycoprotein over several generations is useful.

[00478] In one embodiment of the invention, a population of mammalian host cells stably expressing a protein of interest (for example, fusion constructs, glycoproteins, and the like, such as CTLA4-Ig or CTLA4^A29YLI04E-Ig) is obtained under serum- and animal product-free conditions via amplifying the stably transfected cells. According to the invention, a recombinant cell clone can then be characterized in that it is stable in serum-free and animal product-free culturing medium through at least 105 generations, for example.

[00479] In one embodiment of the invention, the clonal population of cells produces CTLA4-Ig molecules. Some of the specific characteristics of this population of CTLA4-Ig molecules are listed below in Table 6. A population of CTLA4-Ig molecules can at least includes CTLA4-Ig dimer molecules that comprise two monomer molecules that each can׳ have one of the following sequences: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2. Thus, the population of CTLA4-Ig molecules can include predominantly homodimers or heterodimers. The population can include both homodimers and heterodimers. In one embodiment, the invention provides for a population of CTLA4-Ig molecules having the characteristics shown in Table 6 or a phannaceutical equivalent thereof. As used herein, a pharmaceutical equivalent is where a population of molecules has a safety and efficacy profile equivalent to the original population (standard population) for treating a patient, as would be understood by a governmental agency, such as the FDA. For example, the CTLA4-Ig population of this invention can have the characteristics shown in Table 6. In another embodiment, the population of CTLA4-Ig molecules of the invention can have the characteristics shown in Table 6 or equivalents thereof singly or in any combination or permutaion thereof.

[00480] In another embodiment, the clone of interest can also be characterized according to the recombinant product expressed and its biochemical characteristics (for example, CTLA4-Ig having a particular extinction coefficient value). An extinction coefficient value (also referred to as an absorptivity value (a_s)) can be derived theoretically or experimentally. At 280 nm, the absorptivity value (a_s) of CTLA4-Ig was determined to be 1.01 mL mg’¹ cm’¹ using the method of Mach, et al. (Analytical Biochemistry, Vol. 200, pp. 74-80,1992) as detailed below.

Equation 1 was used to determine the molar absorptivity (ε).

Equation 1: ε = [(Number of disulfide bonds x 134) + (Number of Tryptophan residues x 5,540) + (Number of Tyrosine residues x 1,480)]

CTLA4-Ig has 9 disulfide bonds, 8 tryptophan residues and 32 tyrosine residues to give a molar absorptivity (ε) of 92,886 M^cm’¹ as shown in Equation 2.

Equation 2: ε = (9 x 134) + (8 x 5,540) + (32 x 1,480)] = 92,886 M^cm’¹

The absorptivity constant (a_s) was calculated by dividing the molar absorptivity (ε) by the molecular weight where the molecular weight was determined by MALDI-TOF as shown in Equation 3:

Equation 3: a_s =c/M01ecular Weight = 92,886 M^cm'¹ / 92,278 Da = 1.01 mL mg’¹ cm’¹

[00481] A comparison of the theoretically derived absorptivity value to the experimentally determined absorptivity values on two lots of CTLA4-Ig (comprising SEQ ID NO:2) material was carried out using amino acid analysis. The average experimentally determined absorptivity constant is 1.00 + 0.05 mL mg’¹ cm’¹. The experimental value confirms the theoretical value of 1.01 mL mg’¹ cm¹ within the error of the experimental determination. Thus, in one embodiment, the invention provides a cell line that produces CTLA4-Ig molecules that have an absorptivity value or extinction coefficient of about 1.00 ± 0.05 mL mg‘¹ cm‘¹.

[00482] According to this invention, the recombinant clone of interest can also be characterized according to the number of sites a DNA sequence that encodes a protein of interest (for example, fusion constructs, glycoproteins, and the like, such as CTLA4-Ig) is integrated into the host cell genome. One skilled in the art understands that standard Southern hybridization techniques will allow for such an analysis. In one embodiment of the invention, a single hybridizing fragment of approximately 1.2 kb was detected in each of the .EcoRI, and Xbal restriction digests of genomic DNA prepared from the recombinant cell clone of the invention, consistent with the expected size of the CTLA4-Ig gene (Southern hybrid; FIG. 23). The figure depiction is consistent with a single integration site of the plasmid as well as there being no insertions or deletions in the CTLA4-Ig gene being detectable by Southern hybridization analysis.

[00483] In one embodiment, the invention provides CHO cell populations capable of producing CTLA4-Ig molecules, wherein each cell of the population comprises at least 30 copies of a nucleic acid that codes for a CTLA4-Ig protein, wherein the 30 or more copies are integrated in tandem at a single site in the genome of the cell, and wherein the population of cells are clonal. In other embodiments, the CHO cell populations capable of producing CTLA4-Ig molecules comprise a population wherein at least 75%, 80%, 85%, 90%, 95%, or 99% of the cells in the populations comprises at least 30 copies of a nucleic acid that codes for a CTLA4-Ig protein.

[00484] In another embodiments, the invention provides a cell line that when cultured in conditions according to FIG. 10 or Example 14, produces CTLA4-Ig molecules comprised of SEQ ID NO:2 in an amount that is least 1.0, 1.3, 1.6, 1.8, 2.0, or 2.5 grams of CTLA4-Ig molecules per liter cell culture at the production stage.

[00485] According to the invention, a mammalian cell line (for example a dhfr negative CHO cell line) is generated which expresses a desired protein (for example a CTLA4-Ig protein) that when grown in a suspended culture can produce a population of molecules that is secreted into the culture supernatant. This population of molecules can have, for example, one or more or all of the following characteristics listed in TABLE 6.

Table 6. Illustrative CTLA4-Ig Characteristics

<td></td><td> Characteristic</td><td></td>

<td> 1</td><td> N-terminal Sequence</td><td> aa 26 (Ala) of SEQ ID NO:2 aa 27 (Met) of SEQ ID NO :2</td>

<td> 2</td><td> C-terminal Sequence</td><td> aa 382 (Gly) of SEQ ID NO:2 aa 383 (Lys) of SEQ ID NO:2</td>

<td> 3</td><td> B7 Binding</td><td> 70-130%</td>

<td> 4</td><td> pi</td><td> 4.3-5.6</td>

<td> 5</td><td> Sialic Acid Ratio NANA NGNA</td><td> &gt;8.0 moles per mole CTLA4-Ig molecules 8.0 — 12.0 moles per mole CTLA4-Ig molecules &lt;1.5 moles per mole CTLA4-Ig molecules</td>

<td> 6</td><td> dimer</td><td> &gt;95%...</td>

<td> 7</td><td> HMW Species (e.g. tetramer)</td><td> &lt;4.0%</td>

<td> 8</td><td> Low Molecular Weight Species (e.g. monomer)</td><td> &gt;0.5%</td>

<td> 9</td><td> Exctinction Coefficient</td><td> 1.0±0.05 ml/mg-cm</td>

<td> 10</td><td> Free Sulfhydryl Groups</td><td> &lt;0.24 free thiols per molecule</td>

<td> 11</td><td> Amino Monosaccharide Composition: GlcNAc</td><td> 15-35 moles per mole CTLA4-Ig molecules</td>

<td> 12</td><td> Amino Monosaccharide Composition: GalNAc</td><td> 1.7-8.3 moles per mole CTLA4-Ig molecules</td>

<td> 13</td><td> Neutral Monosaccharide Composition: Galactose</td><td> 8-17 moles per mole CTLA4-Ig molecules</td>

<td> 14</td><td> Neutral Monosaccharide Composition: Fucose</td><td> 3.5- 8.3 moles per mole CTLA4-Ig molecules</td>

<td> 15</td><td> Neutral Monosaccharide Composition: Mannose</td><td> 7.7-22 moles per mole CTLA4-Ig molecules</td>

[00486] According to Table 6, the percent of CTLA4-Ig dimer, percent of HMW species (for example CTLA4-Ig multimers such as a tetramer), and percent of LMW species (for example CTLA4-Ig monomer) are with respect to a population of CTLA4-Ig molecules. The moles of sugars and the moles of sialic acid described in Table 6 are with respect to mole of CTLA4-Ig molecules or dimer. The percent of B7 binding found in Table 6 is in reference to CTLA4-Ig binding experiments performed by surface plasmon resonance (SPR) on a BIAcore instrument described earlier wherein the percentage is a comparison to B7 binding to a CTLA4-Ig control.

[00487] In one embodiment, a mammalian cell line (such as, a dhfr negative CHO cell line) generates a population of CTLA4-Ig molecules displaying characteristic attributes numbers 1-5 from Table 6. In another embodiment of the invention, a mammalian cell line generates a population of CTLA4-Ig molecules having characteristic attributes numbers 1-10 from Table 6. In other embodiments, a mammalian cell line generates a population of CTLA4-Ig molecules displaying characteristic attributes numbers 1-15 from Table 6. In a further embodiment, the amount of free sulfhydryl groups on CTLA4-Ig is about &lt;0.20 free thiols per molecule.

[00488] Upon purification of the cell culture supernatant that contains the desired protein (for example a CTLA4-Ig protein) secreted by a population of transfected mammalian cells (for example a dhfr negative CHO cell), the population of molecules can have further characteristics. In addition to those characteristics listed in Table 6, this population of molecules can have, for example, one or more or all of the following characteristics: a pH range from about 7.0 — 8.0; ability to inhibit human cell IL-2 activity by 50 - 150%;

Monocyte Chemotactic Protein (MCP-1) present in the final purified product at &lt;5 ng/mg

CTLA4-Ig. dimer or CTLA4-Ig molecules; concentration of DNA present in the final purified product at &lt;2.5 pg/mg CTLA4-Ig dimer; CHO host cell protein present in the final purified product at &lt;50 ng/mg CTLA4-Ig dimer; concentration of Triton X-100 in the final purified product at &lt;1.0 ppm; amount of Protein A at &lt;5 ng/mg CTLA4-Ig dimer; amount of bacterial endotoxins in the final purified product at &lt;0.3 EU/mg CTLA4-Ig dimer; amount of Bioburden in the final purified product at &lt;3.0 CFU/lOml.

[00489] In one embodiment, Monocyte Chemotactic Protein (MCP-1) is present in the final purified product at &lt; 3 ng/mg CTLA4-Ig dimer or CTLA4-Ig molecules; the concentration of DNA present in the final purified product at &lt;1.0 pg/mg CTLA4-Ig dimer; CHO host cell protein present in the final purified product at &lt; 10 ng/mg CTLA4-Ig dimer; amount of Protein'A at &lt; Ing/mg CTLA4-Ig dimer; amount of bacterial endotoxins in the final purified product at &lt; 0.15 EU/mg CTLA4-Ig dimer; and amount of Bioburden in the final purified product at &lt;1.0 CFU/lOml; a pH range from about 7.2 - 7.8. In a particular embodiment, Monocyte Chemotactic Protein (MCP-1) is present in the final purified product at &lt;1 ng/mg CTLA4-Ig dimer or CTLA4-Ig molecules. In a further embodiment, CTLA4-Ig molecules inhibit human cell IL-2 activity by 60 — 140%.

[00490] In another embodiment of the invention, the clonal population of cells produces CTLA4^A29YL104E-Ig molecules. Some of the specific characteristics of this population of CTLA4^A29YL104E-Ig molecules are listed in Table 7. A population of CTLA4^A29YL104E-Ig molecules can at least includes CTLA4^A29YL,04E-Ig dimer molecules that comprise two monomer molecules that each can have one of the following sequences: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4. Thus, the population of CTLA4^A29YL104E-Ig molecules can include predominantly homodimers or heterodimers, or any mixture thereof. In one embodiment, the invention provides for a population of CTLA4^A29YL104E-Ig molecules having the characteristics shown in Table 7 or a pharmaceutical equivalent thereof. As used herein, a pharmaceutical equivalent is where a population of molecules has a safety and efficacy profile equivalent to the original population (standard population) for treating a patient, as would be understood by a governmental agency, such as the FDA. For example, the CTLA4^A29YLl04E-Ig population of this invention can have the characteristics shown in Table 7. In another embodiment, the population of

CTLA4^A29YL104E-Ig molecules of the invention can have the characteristics shown in Table 7 or equivalents thereof singly or in any combination or permutation thereof.

[00491] In one embodiment, the invention provides CHO cell populations capable of producing CTLA4^A29YL104E-Ig molecules, wherein each cell of the population comprises at least 30 copies of a nucleic acid that codes for a CTLA4^A29YL104E-Ig protein, wherein the 30 or more copies are integrated in tandem at a single site in the genome of the cell, and wherein the population of cells are clonal. In other embodiments, the CHO cell populations capable of producing CTLA4^A29YL,04E-Ig molecules comprise a population wherein at least 75%, 80%, 85%, 90%, 95%, or 99% of the cells in the populations comprises at least 30 copies of a nucleic acid that codes for a CTLA4^A29YL104E-Ig.

[00492] In another embodiments, the invention provides a cell line that when cultured in conditions according to FIG. 24 or Examples 19-20, produces CTLA4^A29YLI04E-Ig molecules comprised of SEQ ID NO:4 in an amount that is least 22, 22.5, 23, 27.5, or 28 grams of CTLA4^A29YE104E-Ig molecules per liter cell culture at the production stage.

[00493] According to the invention, a mammalian cell line (for example a dhfr negative CHO cell line) is generated which expresses a desired protein (for example a CTLA4^A29YL104E-Ig) that when grown in a suspended culture can produce a population of molecules that is secreted into the culture supernatant. This population of molecules can have, for example, one or more or all of the following characteristics listed in TABLE 7.

[00494] Table 7. Illustrative Characteristics of a CTLA4^A29YLI04E-Ig

<td></td><td> Characteristic</td><td></td>

<td> 1</td><td> N-terminal Sequence</td><td> aa 26 (Ala) of SEQ ID NO:4 aa 27 (Met) of SEQ ID NO:4</td>

<td> 2</td><td> • C-terminal Sequence</td><td> aa 382 (Gly) of SEQ ID NO:4 aa 383 (Lys) of SEQ ID NO:4</td>

<td> 3</td><td> B7 Binding</td><td> 70-130%</td>

<td> 4</td><td> Pl</td><td> 4.5-5.5</td>

<td> 5</td><td> Sialic Acid Ratio</td><td> &gt;5.0 moles per mole Total CTLA4-Ig protein</td>

<td> 5</td><td> Dimer</td><td> &gt;95%</td>

<td> 7</td><td> HMW Species (e.g., tetramer)</td><td> &lt;4%</td>

<td> 8</td><td> LMW species (e.g., monomer)</td><td> &lt;1%</td>

<td> 9</td><td> Amino Monosaccharide Composition: GlcNAc</td><td> 24-28 moles per mole Total CTLA4-Ig protein</td>

<td> 10</td><td> Amino Monosaccharide Composition: GalNAc</td><td> 2.7-3.6 moles per mole Total CTLA4-Ig protein</td>

<td> 11</td><td> Neutral Monosaccharide Composition: Galactose</td><td> 11-13 moles per mole Total CTLA4-Ig protein</td>

<td> 12</td><td> Neutral Monosaccharide Composition: Fucose</td><td> 6.4-7.0 moles per mole Total CTLA4-Ig protein</td>

<td> 13</td><td> Neutral Monosaccharide Composition: Mannose</td><td> 14-16 moles per mole Total CTLA4-Ig protein</td>

[00495] According to Table 7; the percent of CTLA4^A29YL104E-Ig dimer, percent of HMW species (for example CTLA4^A29YL104E-Ig multimers such as a tetramer), and percent of LMW species (for example CTLA4^A29YLI04E-Ig monomer) are with respect to a population of CTLA4^A29YLI04E-Ig molecules. The moles of sugars and the moles of sialic acid described in Table 7 are with respect to mole of CTLA4^A29YLI04E-Ig molecules or dimer. The percent of B7 binding found in Table 7is in reference to CTLA4^A29YL104E-Ig binding experiments performed by surface plasmon resonance (SPR) on a BIAcore instrument described earlier wherein the percentage is a comparison to B7 binding to a CTLA4^A29YLI04E-Ig control.

[00496] In one embodiment, a mammalian cell line (such as, a dhfr negative CHO cell line) generates a population of CTLA4^A29YL104E-Ig molecules displaying characteristic attributes numbers 1-5 from Table 7. In another embodiment of the invention, a mammalian cell line generates a population of CTLA4^A29YL104E-Ig molecules having characteristic attributes numbers 1-10 from Table 7. In other embodiments, a mammalian cell line generates a population of CTLA4^A29YLI04E-Ig molecules displaying characteristic attributes numbers 1-13 from Table 7.

[00497] Upon purification of the cell culture supernatant that contains the desired protein (for example a CTLA4^A29YL104E-Ig) secreted by a population of transfected mammalian cells (for example a dhfr negative CHO cell), the population of molecules can have further characteristics. In addition to those characteristics listed in Table 7, this population of molecules can have, for example, one or more or all of the following characteristics: Monocyte Chemotactic Protein (MCP-1) present in the final purified product at &lt;5 ng/mg CTLA4^A29YL104E-Ig dimer; concentration of DNA present in the final purified product at &lt;2.5 pg/mg CTLA4^A29YL104E-Ig dimer; and CHO host cell protein present in the final purified product at .&lt;50 ng/mg CTL-A4^A29YL104E-Ig dimer.

[00498] General Culturing of CelLLines.

[00499] According to this invention, mammalian cells are cultured to produce a desired protein, including a glycoprotein, as conventionally known by one skilled in the art. The mammalian cells expressing a glycoprotein of interest should express or be manipulated to express the appropriate enzymes such that under satisfactory conditions, post-translational modifications most pertinent to glycosylation occur in vivo. The enzymes include those necessary for the addition and completion of N- and O-linked carbohydrates, such as those described in Hubbard and Ivatt, Ann, Rev. Biochem., 50:555-583(1981) for N-linked oligosaccharides. The enzymes optionally include oligosaccharyltransferase, alphaglucosidase I, alpha-glucosidase II, ER alpha(l,2)mannosidase, Golgi alpha-mannodase I, Nacetylyglucosaminyltransferase I, Golgi alpha-mannodase II, Nacetylyglucosaminyltransferase II, alpha(l,6)fucosyltransferase, beta (l,4)galactosyltransferase, and an appropriate sialyltransferase.

[00500] A delay in apoptosis (programmed cell death) can have an effect of increasing cell viability during a cell culturing processes. A decrease in apoptosis, and in turn, an increase in the lifetime of a particular cell can increase protein production from a cell culture. Apoptotic events can be inhibited in a cell by introducing into a cell (such as a mammalian cell, an insect cell, or a yeast cell) one or more anti-apoptotic proteins, which inhibit apoptosis in cells at precise points along the apoptotic pathway. Another method to inhibit apoptosis is to inhibit release of pro-apoptotic molecules from the mitochondria in the cell. Variants of proapoptotic proteins known in the art, such as a dominant-negative form of caspase-9, can be used as an inhibitor of apoptosis in a cell. Such a variant protein can be introduced into a cell in order to delay programmed cell death. Inhibition of apoptosis of a cell, in turn, prolongs the time during which a particular cell produces protein, resulting in an overall increase in the production of a desired protein by a particular cell. Several genes that encode caspase inhibitors (such as X-linked inhibitor of apoptosis (XIAP) or variants therof) or anti-apoptotic genes (for example, Bcl-2 and Bcl-x_Lor variants thereof), can be transfected into genetically engineered mammalian cells (such as, CHO cells, VERO cells, BHK cells, and the like) (Sauerwald, T. et al., 2003, Biotechnol Bioeng. 81:329-340; Sauerwald, T. et al., 2002, Biotechnol Bioeng. 77:704-716; Mastrangelo, A., et al,, 2000, Biotechnol Bioeng. 67:544WO 2007/076032

564; Kim, N., et al., 2002, J Biotechnol. 95:237-248; Figueroa, B., et al., 2001, Biotechnol

- Bioeng. 73:211-222).

[00501] In one embodiment, mammalian cells, which produce a recombinant protein, can be transfected with a vector containing an anti-apoptotic gene (such as bcl-2). In another embodiment, recombinant mammalian cells can be transfected with a plasmid that contains a gene encoding for a caspase inhibitor, or a gene that encodes a variant of a pro-apoptotic molecule as described above, a gene that encodes a protein that is known to an individual skilled in the art to possess anti-apoptotic activity, or any combination thereof.

[00502] In another embodiment, the overall product quality (for example enhanced glycosylation) of a desired recombinant protein (such as a therapeutic protein) can be enhanced. To increase glycosylation of a recombinant protein, mammalian cells (for example CHO cells, VERO cells, BHK cells, and the like) can be transfected with nucleic acids encoding one or more enzymes that are involved in glycosylation (such as a2,3sialyltransferase, p1,4-galactosyltransferase, and the like) of proteins (Weikert et al., 1999, Nature Biotechnol 17:1116-21). In one embodiment, a plasmid that encodes β1,4galactosyl transferase can be introduced into mammalian cells expressing a protein of interest. In another embodiment, a plasmid that encodes a2,3-sialyltransferase can be introduced into mammalian cells expressing a protein of interest.

[00503] Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art (Cleveland et al., J. Immunol. Methods, 56: 221-234 (1983)) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)), and vary according to the particular host cell selected.

[00504] Without limitation, cell culture medium (such as inoculum medium, feed medium, basal medium, and the like) can refer to a nutrient solution used for growing and or maintaining cells, especially mammalian cells. These solutions ordinarily provide at least one component from one or more ofthe following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range. The nutrient solution can be supplemented electively with one or more components from any of the following categories: (1) hormones and other growth factors such as, serum, insulin, transferrin, and epidermal growth factor; (2) salts, for example, magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) protein and tissue .hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (6) antibiotics, such as gentamycin; (7) cell protective agents, for example pluronic polyol; and (8) galactose. An example of basal medium can be Cell Growth Basal Medium. An example of incoculum medium can be Inoculum Cell Growth Basal Medium. An example of feed medium can be Production Bioreactor Feed Medium.

[00505] Commercially available media can be utilized and include, for example, Minimal Essential Medium (MEM, Sigma, St. Louis, Mo.); Dulbecco's Modified Eagles Medium (DMEM, Sigma); Ham's F10 Medium (Sigma); HyClone cell culture medium (HyClone, Logan, Utah); RPMI-1640 Medium (Sigma); and chemically-defined (CD) media, which are formulated for particular cell types, e.g., CD-CHO Medium (Invitrogen, Carlsbad, Calif.). Any of these media can be supplemented as necessary with the previously defined supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessary or desired. The mammalian cell culture that can be used with the present invention is prepared in a medium suitable for the particular cell being cultured. In one embodiment, the cell culture medium can be one of the aforementioned that is generally free of serum from any mammalian source (for example, fetal bovine serum (FBS)). In another embodiment of this invention, the mammalian cell culture can be grown in the commercially available chemically defined (CD)-CHO Medium, supplemented with additional components specified in Table 15. In a further embodiment, the mammalian cell culture can be grown in CD-CHO Medium, supplemented with additional components specified in Table 20 or 21.

[00506] The methods of the present invention include the culturing of numerous cell types.

In one embodiment of the invention, the cells are animal or mammalian. In another embodiment, the cells can express and secrete large quantities of a desired protein. In another embodiment of the invention, cells can express and secrete large quantities of a glycoprotein of interest into the culture medium. The animal or mammalian cells can also be molecularly modified to express and secrete a protein of interest The protein produced by the host cell can be endogenous or homologous to the host cell. The protein also can be heterologous (for example, foreign), to the host cell whereby genetic information coding for the protein of interest is introduced into the host cell via methods standard in the art (for example by electroporation, transfection, and the like). In one embodiment, a mammalian glycoprotein can be produced and secreted by a Chinese hamster ovary (CHO) host cell into the culture medium.

[00507] In some embodiments, the invention provides populations of CTLA4-Ig molecules produced by the methods of production discussed herein, including the method of massproduction that is described in Example 14 and shown in FIG. 10. The process can result in the production of CTLA4-Ig molecules of high molecular weight (HMW) (for example, see Examples 14 and 15). In another embodiment, populations of CTLA4^A29YLI04E-Ig molecules are provided that are produced by the production methods discussed herein, such as the method of mass-production that is described in EXAMPLES 19 and 20, and shown in FIG. 24. The process can result in the production of CTLA4^A29YL104E-Ig molecules of high molecular weight (HMW) (for example, see EXAMPLES 19 and 20). In some embodiments, the HMW species can be about 15-25% of the molecules or dimer produced by a method for production, including a chemically defined (CD)-CHOl fermentation process. In other embodiments, the present invention provides methods for isolation, purification and characterization of CTLA4-Ig or CTLA4^A29YLI04E-Ig HMW components produced by a CD-CHO1 fermentation process. CTLA4-Ig or CTLA4^A29YLI04E-Ig HMW components, are multimers (i.e, tetramers, hexamers, etc.), which have a higher molecular weight than CTLA4-Ig or CTLA4^A29YLI04E-Ig dimers.

[00508] Animal or mammalian host cells capable of harboring, expressing, and secreting large quantities of a glycoprotein of interest into the culture medium for subsequent isolation and/or purification include, but are not limited to, Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec, Genet, 12:555556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909; and WO 01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/dhfr-, Urlaub and Chasin, 1980, Proc, Natl. Acad, Sci. USA, 77:4216), and dpl2.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey .kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK,־ATCC CCL-34); human lung cells (W138; ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells. In one aspect of this invention, CHO cells are utilized, particularly, CEOIdhfr- and CHO DG44 cells.

[00509] Examples of mammalian glycoproteins that can be produced by the methods of this invention include, without limitation, cytokines, cytokine receptors, growth factors (e.g., EGF, HER-2, FGF-a, FGF-β, TGF- a, TGF-β, PDGF, IGF-1, IGF-2, NGF, NGF- β); growth factor receptors, including fusion or chimeric proteins. Other examples include, but are not limited to growth hormones (e.g., human growth hormone, bovine growth hormone); insulin (e.g., insulin A chain and insulin B chain), proinsulin; erythropoietin (EPO); colony stimulating factors (e.g., G-CSF, GM-CSF, M-CSF); interleukins (e.g., ILrl through IL-12); vascular endothelial growth factor (VEGF) and its receptor (V.EGF--R); interferons (e.g., IFNa, β, or γ); tumor necrosis factor (e.g., TNF-a and TNF-β) and their receptors, TNFR-1 and TNFR-2; thrombopoietin (TPO); thrombin; brain natriuretic peptide (BNP); clotting factors (e.g., Factor VIII, Factor IX, von Willebrands factor, and the like); anti-clotting factors; tissue plasminogen activator (TPA), e.g., urokinase or human urine or tissue type TPA; follicle stimulating hormone (FSH); luteinizing hormone (LH); calcitonin; CD proteins (e.g., CD3, CD4, CD8, CD28, CD19, etc.); CTLA proteins (e.g., CTLA4); T-cell and B-cell receptor proteins; bone morphogenic proteins (BNPs, e.g., BMP-1, BMP-2, BMP-3, etc.);

neurotrophic factors, e.g., bone derived neurotrophic factor (BDNF); neurotrophins, e.g., 3-6;

• renin; rheumatoid factor; RANTES; albumin; relaxin; macrophage inhibitory protein (e.g., MIP-1, MIP-2); viral proteins or antigens; surface membrane proteins; ion channel proteins; enzymes; regulatory proteins; antibodies; immunomodulatory proteins, (e.g.,HLA, MHC, the B7 family); homing receptors; transport proteins; superoxide dismutase (SOD); G-protein coupled receptor proteins (GPCRs); neuromodulatory proteins; Alzheimer's Disease associated proteins and peptides, (e.g., A-beta), as well as others known in the art. Suitable proteins, polypeptides, and peptides that can be produced by the methods of the present invention include, but are not limited to, fusion proteins, polypeptides, chimeric proteins, as well as fragments or portions, or mutants, variants, or analogs of any of the aforementioned .

proteins and polypeptides.

[00510] The methods of the invention can also be used to produce CTLA4-Ig molecules which are variants of SEQ ID NO: 5, 6, 7, 8, 9, or 10. In one embodiment, a CTLA4-Ig molecule can comprise a monomer having one or more changes in residues 55 (A55Y) and 130 (L130E) (residues referred to are from SEQ ID NO:2). See the descriptions of variants and mutants of CTLA4-Ig described in U.S. Publication No. US 2002/0182211 Al, which is hereby incorporated by reference in its entirety. In another embodiment, a CTLA4-Ig variant can comprise a CTLA4-Ig molecule having a mutation within the CTLA-4 region or a mutation in the Ig region, or any combination thereof. In one embodiment, a CTLA4-Ig variant molecule comprises a CTLA4-Ig molecule having an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOS: 5, 6, 9 ,8 ,ר, or 10. In one embodiment, the CTLA4-Ig variant molecule is capable of binding to CD80 or CD86. In another embodiment, the variant is able to form a dimer. In a further embodiment, the variant exhibits a carbohydrate profile similar to that exhibited by a non-mutated CTLA4-Ig molecule population. In one embodiment, the CTLA4-Ig variant molecules have the same potential N-linked and O-linked glycosylation sites present in SEQ ID NO:2. In another embodiment, a CTLA4-Ig variant molecule has the same N-linked and O-linked glycosylation sites present in SEQ ID NO:2, and has additional glycosylation sites. The mutations can include, but are not limited to, nucleotide deletions, insertions, additions; amino acid deletions, substations, additions; nucleic acid frameshifts; the substitutions can be either non-conservative (e.g., a glycine substituted with a tryptophan) or conservative substitutions (e.g., a leucine substituted for an isoleucine).

[00511] CTLA4-Ig variant molecules include, but are not limited to, CTLA4LI 04EA29YIg (using the residue numbering system according to SEQ ID NO:2, CTLA4L104EA29YIg herein is referred to as CTLA4-L130EA55YIg), as well as those CTLA4-Ig variant molecules described in U.S. patent application Ser. Nos. 09/865,321 (U.S. Pub. No. US2002/0182211), 60/214,065 and 60/287,576; in WO 01/92337 A2; in U.S. Pat. Nos. 6,090,914, 5,844,095 and 5,773,253; and as described in R. J. Peach et al., 1994, J Exp Med, .180:2049-2058. In one embodiment, CTLA4-Ig variant molecules produced in the present methods can be secreted from a cell that comprises an expression vector coding for a

CTLA4-Ig variant protein.

[00512] A CTLA4-Ig variant, L130EA55Yig, is a genetically engineered fusion protein similar in structure to CTAL4-Ig molecule. L130EA55Y-Ig has the functional extracellular binding domain of modified human CTLA-4 and the Fc domain of human immunoglobulin of the IgGl class. Two amino acid modifications, leucine to glutamic acid at position 104 (L104E) of an L104EA29Y variant, which corresponds to position 130 of SEQ ID NO:2, and alanine to tyrosine at position 29 (A29Y) of an L104EA29Y variant, which corresponds to position 55 of SEQ ID NO:2, were made in the B7 binding region of the CTLA-4 domain to generate L130EA55Y. L130EA55Y-Ig can comprise two homologous glycosylated polypeptide chains of approximately 45,700 Daltons each, which are held together by one inter-chain disulfide bond and non-covalent interactions. DNA encoding L.130EA55Y.-Ig was deposited as DNA encoding L104EA29Y-Ig on Jun. 20, 2000, with the American Type Culture Collection (ATCC) under the provisions of the Budapest Treaty. It has been accorded ATCC accession number PTA-2104. LI 04EA29Y-Ig (corresponding to L130EA55Y-Ig in this application) is further described in co-pending U.S. patent application Ser. Nos. 09/579,927, 60/287,576 and 60/214,065, and 09/865,321 and in WO/Ol/923337 A2, all of which are incorporated by reference in this application in their entireties.

[00513] Since the recombinant protein L13OEA55Y-Ig is different at only 2 amino acids (Tyr at amino acid position 55 and Glu at amino acid position 130) compared to CTLA4-Ig monomers having an Ala at amino acid position 55 and Leu at amino acid position 130 of SEQ ID NO:2, and because these 2 mutations do not affect N- or O- linked glycosylation, CTLA4-Ig variant molecule populations comprising L130EA55Y-Ig may have the same profile or a very similar glycosylation profile as do populations comprising wild type CTLA4-Ig. Further, because the recombinant protein L130EA55Y-Ig is different at only 2 amino acids (Tyr at amino acid position 55 and Glu at amino acid position 130) compared to CTLA4-Ig monomers having an Ala at amino acid position 55 and Leu at amino acid position 130 of SEQ ID NO:2, the present methods of this invention should be able to produce L130EA55Y-Ig with similar characteristic attributes as described in Table 6.

[00514] The methods of the invention can also be used to produce CTLA4^A29YL104E-Ig molecules, which are variants of SEQ ID NOS: 11, 12, 13,14, 15, or 16. In one embodiment, a CTLA4^A29YL104E-Ig can comprise a monomer having one or more changes in SEQ ID NO:3.

For example, descriptions of other CTLA4^A29YL,04F-Ig molecules are described in U.S. Patent

Application Publication Nos. U.S. 2002/0039577, U.S. 2003/0007968, U.S. 2004/0022787,

U.S. 2005/0019859, and U.S. 2005/0084933, and U.S. Patent No. 7,094,8874, which are hereby incorporated by reference in their entirety.

[00515] In one embodiment, CTLA4^A29YL104E-Ig comprises one or more mutations within the CTLA-4 region (SEQ ID NO: 18), or a mutation in the Ig region, or any combination thereof. In other embodiments, a CTLA4^A29YL104E-Ig molecule comprises a CTLA4^a29yl104eIg having an amino acid sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOS: 11, 12, 13, 14, 15, or

16. In a further embodiment, a CTLA4^A29YL104E-Ig molecule as described above is capable of binding to CD80 or CD86. In another embodiment, the CTLA4^A29YL,04E-Ig is able to form a dimer. In a further embodiment, the CTLA4^A29YL104E-Ig exhibits a carbohydrate profile similar to that exhibited by a non-mutated CTLA4^A29YL104E-Ig molecule population. In yet other embodiments, the CTLA4^A29YL104E-Ig molecules have the same potential N-linked and O-linked glycosylation sites present in SEQ ID NO:4. In another embodiment, a CTLA4^A29YL104E-Ig has the same N-linked and O-linked glycosylation sites present in SEQ . ID NO:4, and has additional glycosylation sites. The mutations can include, but are not limited to, nucleotide deletions, insertions, additions; amino acid deletions, substitutions, additions; nucleic acid frameshifts; the substitutions can be either non-conservative (e.g., a glycine substituted with a tryptophan) or conservative substitutions (e.g., a leucine substituted for an isoleucine).

[00516] In one embodiment of the invention, a population of CTLA4-Ig variant molecules can be produced by mammalian cells (for example dhfr negative CHO cells), which express a gene encoding the desired protein (for example a L130EA55Y-Ig protein or the like), grown in suspension according to the mass-production method of this invention. According to this invention, a recombinant CTLA4-Ig variant protein produced by mammalian cells can be recovered according to the harvesting parameters described herein. In other embodiments, a recombinant CTLA4-Ig variant protein produced by mammalian cells can be purified according to the purification scheme described in this invention (Example 15).

[00517] In one embodiment of the invention, a population of CTLA4^A29YLI04E-Ig molecules can be produced by mammalian cells (for example dhfr negative CHO cells), which express a gene encoding the desired protein (for example a CTLA4^A29YL104E-Ig), grown in suspension according to the mass-production method of this invention. According to this invention, a recombinant CTLA4^A29YLI04E-Ig produced by mammalian cells can be recovered according to the. harvesting parameters described herein. In other embodiments, a recombinant CTLA4^A29YLI04E-Ig produced by mammalian cells can be purified according to the purification scheme described in this invention (EXAMPLES 19-20).

• [00518] Types of Cell Cultures and General Culturing Processes

[00519] A protein of interest, for example a glycoprotein, a fusion protein and the like, can be produced by growing cells expressing the desired protein product under a variety of cell culture conditions. A practitioner skilled in the art understands that cell cultures and culturing runs for protein production can include, but are not limited to, three general types: continuous culture, batch culture, and fed-batch culture. In a continuous culture process, a fresh culture medium supplement (for example, feeding medium) is supplied to cells during the culturing period while old culture medium is removed. The product produced during a continuous culture can also be harvested, for example, on a daily basis or continuously. As long as the cells remain alive, and the environmental and culturing conditions are maintained, cells can remain in culture as long as is desired in a continuous culturing process.

[00520] In a batch culture process, cells are initially cultured in medium and this culturing medium is neither replaced, nor removed, nor supplemented. The cells are not “fed” with new medium during or before the end of the culturing run thus culturing continues until nutrients are exhausted. The protein product is harvested at the end of the culturing run.

[00521] For fed-batch culture processes, the culturing run time can be increased by supplementing the culture medium one or more times daily (or continuously) with fresh medium during the run. In this process, the cells are supplied with fresh medium, a “feeding medium”, during the culturing period. Fed-batch cultures can include the-various feeding schedules described previously, for example, daily, every two days, every other day, etc.; more than once per day, or less than once per day, and so on. Fed-batch cultures also can be fed continuously with feeding medium. At the end of the culturing/production run, the protein product of interest is then harvested.

[00522] Cell culture systems for the small- or large-scale production of proteins, including glycoproteins, produced by mammalian host cells are useful within the context of this invention. Those having skill in the art understand that tissue culture dishes, spinner flasks, and T-flasks are typically used for culturing methods on a laboratory scale. The processes that can be used for culturing on a larger scale (e.g., 500 L, 5000 L, 10,000 L, 20,000 L and the like) include, but are not limited to, a hollow fiber bioreactor, a fluidized bed bioreactor, a stirred tank bioreactor system, or a roller bottle culture. The later two processes can be utilized with or without microcarriers.

[00523] The systems can be operated in a batch, fed-batch, or continuous mode. For production-scale culturing, the stirred-tank bioreactor is the system of choice because of its flexibility. These reactors can maintain cells in suspension by agitation through mechanical stirring with gas bubble sparging .or an impeller. The stirred-tank bioreactors can be scaled up to large production-scale volumes (for example, 20,000 liters) and can be operated in different feed modes; These systems provide a large surface area for cell growth and the efficient. transfer of metabolic wastes, oxygen, and nutrients, as well as maintain a homogenous environment throughout the reactor by preventing cells from settling to the bottom via continuous stirring or mixing of the components within the reactors. For the production of a desired glycoprotein, the present invention embodies large-scale, fed-batch cell cultures maintained in a stirred-tank bioreactor, fed daily with feeding medium containing D-galactose. In another embodiment, fed-batch cell cultures can also be maintained in a stirred-tank bioreactor, fed daily with feeding medium that contains suitable concentrations of the limiting cell culture nutrients important for protein glycosylation, such as glucose and glutamine (Chee et al., 2005, Biotechnol. Bioeng. 89:164-177).

[00524] The cells of the culture producing a protein of interest can be propagated according to any scheme br routine that is most suitable for the particular mammalian host cell and the particular production plan contemplated. Cell culture conditions can be . . developed to enhance expansion or growth of a population of mammalian host cells in the growth phase of the cell culture for a period of time that is maximized for such expansion and growth. The growth phase of the cell culture comprises the period of exponential cell growth (for example, the log phase) where cells are primarily dividing rapidly. During this phase, the rate of increase in the density of viable cells is higher than at any other time point.

[00525] Also, cell culture conditions can be developed to enhance protein production during the production phase of the cell culture for a period of time. The production phase of the cell culture comprises the period of time during which cell growth is stationary or is maintained at a near constant level. The density of viable cells remains approximately constant over a given period of time. Logarithmic cell growth has terminated and protein production is the primary activity during the production phase. The medium at this time is generally supplemented to support continued protein production and to achieve the desired glycoprotein product.

[00526] Culture conditions, such as temperature, pH, dissolved oxygen (DO2), and the like, are those used in culturing mammalian host cells that are understood by the individual skilled in the art. An appropriate temperature range for culturing mammalian host cells, such as CHO cells, is between 30 to 40^qC, and in one embodiment about 37°C. The pH generally is adjusted to a level between about 6.5 and 7.5 using either an acid or base. A suitable DO₂is between 5-90% of air saturation. ^,These culture conditions can be used to facilitate the culturing of mammalian cells that produce a desired protein or glycoprotein product.

[00527] A mammalian host cell population can be expanded and grown in a growth phase culture wherein cells, possibly removed from storage, are inoculated into a culturing medium acceptable for promoting growth and high viability. The cells can then be maintained in a production phase for a suitable period of time by the addition of fresh culturing medium to the host cell culture. During the production phase, cells can be subjected to various shifts in temperature to enhance protein production. Multiple temperature shift culturing processes are described in patent applications U.S. Ser. No. 10/742,564, filed Dec. 18, 2003, and U.S. Ser. No. 10/740,645, filed on Dec. 18, 2003. The contents from these applications are incorporated by reference herein in their entirety. In this invention, the two or more temperature shifts comprising the cell culture processes can result in an increased number of viable cells that survive in culture until the end of the process or production run. During the production phase of the culture, the greater the number of cells that survive can result in a greater amount of protein or glycoprotein product produced, increasing the amount of protein product at the end of the process.

[00528] A particular aspect of this invention embodies a fed-batch, large-scale (for example 500 L, 5000 L, 10000 L, and the like), mammalian cell culture, that is fed daily or with feeding medium described in Tables 14, 15, comprising D-galactose in order for cells to produce a glycoprotein of interest. To increase the quality of the protein produced in this embodiment, two or more temperature shifts can be employed during the culture period to extend the protein production phase beyond that which occurs when no temperature shift is used, or when only one temperature shift is used. In another embodiment, the invention entails a fed-batch, large-scale (for example 500 L, 5000 L, 10000 L, and the like), mammalian cell culture, that is fed 1 or more times daily with feeding medium described in Table 22, which comprise D-galactose in order for cells to produce a glycoprotein of interest (for example, CTLA4^A29YL104E-Ig). One or more temperature shifts also can be employed during the culture period to extend the protein production phase beyond that which occurs when no temperature shift is used in order to increase the quality of the glycoprotein. Alternatively, dextran sulfate can be added to the culture with a concomitant temperature shift.

[00529] Mass-Production of Recombinant Protein in Bioreactors

[00530] The present invention provides methods for conventional stirred tank bioreactor cultivation of eukaryotic cells (for example, a 20000 L cell culture volume), particularly to produce large-scale or industrial amounts of desired protein products that are expressed by such cells. The cultivation process is a fed-batch culturing process of eukaryotic cells grown in suspension, with harvesting of culture supernatant, wherein eukaryotic cells, for example mammalian cells, expressing a protein of interest, secrete desired protein product into the culture medium.

[00531] Methods for large-scale cultivation of mammalian cells, particularly to produce large amounts of desired protein products that are expressed by such cells, are embodied in the present invention. The methods can be carried out by steps comprising:

(i) inoculating cells into a seed culture vessel (for example, a T-175 flask) containing serum-free culture medium and propagating the seed culture (for example, a starter culture that used to inoculate a larger volume) at least until the cells reach a minimal cross-seeding density whereby the density is a pre-determined value needed for sufficient propagation of cells in the subsequent culturing volume;

(ii) transferring the propagated seed culture to a larger culture vessel (for example, roller bottles or cell bags) containing culture medium lacking animal-derived components in order to expand the culture;

(iii) transferring the expanded seed culture to a large-scale culture vessel containing serum-free culture medium to further propagate to the cell culture; and (iv) maintaining the large-scale culture in medium lacking animal-derived components, at least until said cells reach a target density or display a specific biochemical characteristic.

[00532] In some embodiments, the method can comprise the step .of (iv) harvesting of the culture medium and replacing that medium with fresh-medium.

[00533] _k In other embodiments, methods for large-scale cultivation of mammalian cells can be carried out by steps comprising:

(i) inoculating cells into a seed culture vessel (for example, a T-175 flask) containing serum-free culture medium (for example, inoculoum medium) and propagating the seed culture (for example, a starter culture that used to inoculate a larger volume) at least until the cells reach a minimal cross-seeding density whereby the density is a pre-determined value needed for sufficient propagation of cells in the subsequent culturing volume;

(ii) transferring the propagated seed culture to a larger culture vessel (for example, roller bottles or cell bags) containing culture medium lacking animal-derived components (for example, inoculum medium) in order to expand the culture;

(iii) transferring the expanded seed culture to a large-scale culture vessel (such as 1000-L bioreactors) containing serum-free culture medium (for example, basal medium) to further propagate to the cell culture; and (iv) maintaining the large-scale culture in medium lacking animal-derived components (for example, feed medium), at least until said cells reach a target density or display a specific biochemical characteristic.

[00534] In some embodiments of the invention, the method can comprise the step of:

(v) harvesting of the culture medium and replacing the spent medium with fresh medium.

[00535] The present invention is applicable to any cell type in any formulation of medium lacking animal-derived components in order to produce large-scale quantities of desired protein products, and can utilize either of the following two processes, or variations thereof: a) microcarrier processes, or b) suspension cell processes. Culturing of cells, for example mammalian cells, can utilize either process, operated in two distinct phases, a growth phase and a production phase. In another embodiment of the invention, any formulation of medium which contains animal-derived components (some non-limiting examples being BovineSerum Albumin (BSA) or FBS) can be employed as well for the production of large-scale protein quantities as described above.

[00536] One skilled in the art understands that a microcarrier process, not limited to a standard microcarrier-process or a perfusion microcarrier process, can b.e used for .cell, culturing wherein cells are attached to and/or immobilized in a macroporous carrier. In a standard microcarrier-process, cells are inoculated into a seed culture vessel containing serum-free culture medium and propagated until the cells reach a minimum seeding density. Subsequently, the propagated seed culture is transferred to a large-scale culture vessel containing serum-free culture medium and microcarriers. In this growth phase, the cells are grown on microcarriers until the carriers are fully colonized, for example by cells migrating into the carriers in the case of a process using macroporous carriers.

[00537] Medium exchange can occur when microcarriers settle to the bottom of the culture vessel, after which a predetermined percentage of the tank volume is removed and a corresponding percentage tank volume of fresh medium is added to the vessel. Microcarriers are then re-suspended in the culturing medium. A skilled artisan understands that the process of medium removal and replacement can be repeated at a predetermined interval, for example every 24 hours whereby the amount of replaced medium is dependent on cell density and can typically be from 25% to 80% of the tank volume. 60-95% of the tank medium in the tank can be changed every 24 hours when the cell density reaches a pre-determined value suitable for protein expression. Those having skill in the art often use the aforementioned medium . exchange % value throughout the production phase as well.

[00538] During the production phase, culture medium can be exchanged by allowing the microcarriers to settle to the bottom of the tank, after which the selected % of the tank volume is removed and a corresponding % tank volume, for example 60-95% as described earlier, of fresh culturing medium is added to the vessel. Microcarriers are then re-suspended in the culturing medium and the medium removal and replacement process can be repeated . daily.

[00539] The microcarrier perfusion process resembles the standard microcarrier process and also is operated in the growth/expansion and production phases. The main difference between the two processes is the method employed to change the culture medium. A defined amount of the tank volume, for example 60-95% of the total , tank volume, is changed all at once in the standard microcarrier process, whereas in the perfusion process the medium is added continuously. Essentially, a % tank volume medium is changed gradually over a predetermined length of time while the microcarriers are kept in the vessel by using a separation device (or perfusion device) that allows the medium to leave the vessel but retains the microcarriers within the tank. The growth phase in this process is as described for a standard microcarrier process except for the gradual medium exchange.

[00540] Two non-limiting options for a suspension cell process include a suspension cell perfusion process and a suspension cell batch process. In the perfusion process, cells in a culturing medium are freely suspended without being immobilized in carriers and, and like the microcarrier processes, can be operated in־ two distinct phases (for example, a growth phase and a production phase). During the growth phase of a suspension cell-perfusion process, cells are inoculated into a seed culture vessel containing serum-free culture medium and propagated until cells reach a target cross-seeding density. The propagated seed culture can then be transferred to a large-scale culture vessel, which contains culturing medium lacking animal-derived components, and propagated until a pre-determined cell density value suitable for protein expression is reached. A continuous perfusion of the culture vessel with fresh culture medium is performed to execute the medium exchange process.

[00541] In the suspension cell batch process, cell culturing can be carried out via the following non-limiting formats: a) simple batch process or b) fed-batch process. Cells are inoculated into a seed culture vessel containing culture medium lacking animal-derived components in a simple batch process and propagated until the cells reach a pre-determined cross-seeding density. Subsequently, the propagated seed culture is transferred to a largescale culture vessel containing serum-free culture medium and the culturing vessel is operated until the nutrients in the culture medium have been exhausted. In a fed-batch process, feeding a concentrated solution of nutrients (for example a feed medium) to the tank can extend the nutrient supply in the medium of this culturing process, thus extending the process time and ultimately leading to an increase in the production of the desired protein within the culture vessel. The method of adding the feed medium can vary. It can be added either as a single pulse bolus (once, twice, three times etc., a day) or can be fed gradually throughout a 24rhour period. This feed allows cells to be propagated in a large-scale culture vessel and the medium, which can contains the secreted protein product of interest, to be harvested at the end of the run before any of the nutrients become exhausted. Instead of removing all of the contents from the vessel, one skilled in the art would remove only a portion of the tank volume (can be about 80%).

[00542] An optional aspect of the fed-batch-process is the use of temperature shifts. In this process, temperatures employed as the operating temperatures during the production phase are lower than the temperature used during the growth phase. Said temperature ranges for a fed batch process, for example the process used in this invention, could consist of an initial growth phase at a temperature suitable for growth of the particular cell line in use followed by a decrease in the operating temperature at a pre-determined cell density.

[00543] In one embodiment, a process for a large-scale fed-batch culture process comprises the following: (i) inoculating cells into a seed culture vessel (for example, a T-175 flask) containing serum-free culture medium and propagating the seed culture at least until the cells reach a pre-determined cross-seeding density at a temperature suitable for growth; (ii) transferring the propagated seed culture to a larger culture vessel (for example, roller bottles or cell bags) containing culture medium lacking animal-derived components in order to expand the culture at a suitable temperature suitable; (iii) transferring the expanded seed culture to a large-scale culture vessel containing serum-free culture medium to further propagate to the cell culture at a suitable temperature; and (iv) maintaining the large-scale culture at a decreased temperature suitable for protein expression, in medium lacking animalderived components, with daily replacements by fresh feed medium, at least until said cells reach a target density or critical length of time.

[00544] The step of replacement with fresh feed medium in (iv) can entail removing a predetermined volume, for example 80%, of the tank volume and replacing it with the same volume of fresh feed medium.

[00545] In a further embodiment, a process for a large-scale fed-batch culture process comprises the following: (i) inoculating cells into a seed culture vessel (for example, a T-175 flask) containing serum-free culture medium and propagating the seed culture at least until the cells reach a pre-determined cross-seeding density at a temperature suitable for growth; (ii) transferring the propagated seed culture to a larger culture vessel (for example, roller bottles or cell bags) containing culture medium lacking animal-derived components in order to expand the culture at a suitable temperature suitable; (iii) transferring the expanded seed culture to a large-scale culture vessel (for example, a 1000-L bioreactor) containing serumfree culture medium to further propagate to the cell culture at a suitable temperature; and (iv) maintaining the large-scale culture at a decreased temperature suitable for protein expression, in medium lacking animal-derived components, with-daily replacements by fresh feed medium, at least until said cells reach a target density or critical length of time.

[00546] The step of replacement with fresh feed medium in (iv) can entail removing a predetermined volume, for example about 80% of the tank volume, and replacing it with the same volume of fresh feed medium.־

[00547] In one embodiment of this invention, the cells cultured in a fed-batch process are mammalian cells, for example CHO cells, which express a desired protein product. Mammalian cells are inoculated into a seed culture vessel (for example, a T-l 75 flask) containing serum-free culture medium, for example CD-CHO medium (Example 13), and propagated at a temperature suitable for growth, for example at about 35-39°C, for 3-4 days until the cells reach a pre-determined cross-seeding density (for example, having >6.0x10⁶viable cells, or wherein the final culture viability >80%). The propagated seed culture is then transferred to a large culture vessel (for example, roller bottles) containing culture medium lacking animal-derived components for expansion at a suitable temperature (for example at about 35-39°C) for approximately 3-4 days. The cell culture is further expanded in a larger culture vessel (for example, a 20 L cell bag, a 100L cell bag, and the like) containing serum free medium, for example CD-CHO medium, at a temperature suitable for growth, for example at about 35-39°C, for 3-4 days until the cells reach a target seeding density (for example, having >1-2 xlO⁶ viable cells/ml, or wherein the final culture viability >80%). In one embodiment, the inoculum expansion involves a minimum of 4 passages. In another embodiment of the invention, inoculum expansion entails no more than 20 passages.

[00548] The expanded seed culture can then used to inoculate a large-scale culturing tank (for example, a 1000L, a 4000L bioreactor and the like), containing serum-free culture medium (for example CD-CHO medium) to further propagate the cell culture at a suitable temperature, for example at about 35-39°C, for 3-6 days, until the cells reach a target seeding density (for example, having >1-2 xlO⁶ viable cells/ml, or wherein the final cell culture viability >80%). A large-scale culture (for example a 10,000L, 15,000L, 20,000L culture in a bioreactor and the like) is subsequently maintained in serum-free culture medium, wherein the medium is a feed medium (for example eRDF medium, Example 14), at a temperature lower than the growth temperature (for example at or about 33-35°C for 3-4 days, and at or about 31-33 C for 6-8 days), suitable for protein expression and production of the secreted protein product. Πιε feed medium is replaced daily with fresh feed medium, whereby the tank’s replacement with fresh feed medium entails removal of a predetermined volume, for example 80% of the tank volume, and replacing the tank with the same volume of fresh feed medium. The commercial scale culture is maintained until said cells reach a target value of production parameters that can be, but are not limited to, a length of time, a target cell density, or biochemical protein characteristic (such as a NANA molar ratio as previously described) wherein the viable cell density can be 3.0-8.0 x 10⁶ cells/ml; a NANA molar ratio can be >6.0; a final cell culture viability can be >30%; and a final protein product titer can be >0.5 g/L).

[00549] In a particular embodiment of this invention, the cells cultured in a fed-batch process are mammalian cells, for example CHO cells, which express a desired protein product (for example, a CTLA4-Ig molecule). CHO cells are inoculated into a seed culture vessel (for example, a T-175 flask) containing serum-free culture medium, for example CDCHO medium, and propagated at a temperature suitable for growth, for example at about 37 C,. for •3-4 days until the cells reach a pre-determined cross-seeding density (for example, having >10.0x10⁶ viable cells, or wherein the final culture viability >84%). The propagated seed culture is then transferred to a large culture vessel (for example, roller bottles) containing culture medium lacking animal-derived components for expansion at a suitable temperature (approximately 37 C) for about 4 days. The cell culture is further expanded in a larger culture vessel (for example, a 20 L cell bag, a 100L cell bag, and the like) containing serum-free medium, for example CD-CHO medium, for 4 days at a temperature suitable for growth (for example at about 37°C) until the cells reach a target seeding density (for example, having >1-2 xlO⁶ viable cells/ml, or wherein the final culture viability >91%). The inoculum expansion can involve a minimum of 7 passages.

[00550] The expanded seed culture is then used to inoculate a large-scale culturing tank (for example, a 4000L bioreactor and the like), containing serum-free culture medium (for example CD-CHO medium) to further propagate the cell culture at a suitable temperature, for example at about 37 C, for 5-6 days, until the cells reach a target seeding density (for example, having >1-2 xlO⁶ viable cells/ml, or wherein the final cell culture viability >86%). A commercial-scale culture (for example a 20,000 L culture in a bioreactor) is subsequently maintained in serum-free culture medium, wherein the medium is a feed medium (for example eRDF medium), at a temperature lower than the growth temperature, which is suitable for protein expression and production of the secreted protein product (for example,

CTLA4-Ig). The commercial-scale culture is first lowered from.about 37°C to about 34°C for 4 days, and then subjected to a second temperature shift by lowering the temperature from about 34°C to about 32°C for 8 days. The feed medium is replaced daily with fresh feed medium, whereby replacing the feed medium in the bioreactor tank entails removal of a predetermined volume, for example 80% of the tank volume, and replacing it with the same volume of fresh feed medium. The commercial scale is maintained until said CHO cells and/or secreted protein product reach a target value of the following non-limiting production parameters: a viable cell density of 4.0-7.0 x 10⁶ cells/ml; a NANA molar ratio 28.0; a final cell culture viability >38%; and a final protein product titer of >0.6 g/L.

[00551] In another embodiment of this invention, the cells cultured in a fed-batch process are mammalian cells, for example CHO cells, which express a desired protein product. Mammalian cells are inoculated into a seed culture vessel (for example, a T-l 75 flask) containing serum-free culture medium, for example CD-CHO medium (EXAMPLE 19), and propagated at a temperature suitable for growth, for example from about 35°C to about 39°C, for about 3-4 days; or from about 36’C to about 38°C, for about up to about 4 days until the cells reach a pre-determined cross-seeding density (for example, having a cell density of greater than or equal tol .5 x 10⁶, or wherein the final culture viability is greater than or equal to about 80%). The propagated seed culture is then transferred to a large culture vessel (for example, roller bottles) containing culture medium lacking animal-derived components for expansion at a suitable temperature (for example, from about 35°C to about 39’C, or from about 36°C to about 38“C) for about 3-4 days or up to about 4 days. The cell culture is further expanded in a larger culture vessel (for example, a 20 L cell bag, a 100L cell bag, and the like) containing serum free medium, for example CD-CHO medium, at a temperature suitable for growth, for example from about 35°C to about 39°C, or from about 36°C to about 38 C, for about 3-4 days or up to about 4 days until the cells reach a target seeding density (for example, having at least about 1.5 x 10⁶ viable cells/ml, or wherein the final culture viability is greater than or equal to 80%). In one embodiment, the inoculum expansion involves a minimum of 4 passages. In another embodiment of the invention, inoculum expansion entails no more than 20 passages. In some embodiments, the CD-CHO medium is a CD-CHO inoculum medium.

[00552] The expanded seed culture can then be used to inoculate a large-scale culturing tank (for example, a 1000L, a 4000L bioreactor, and the like), containing serum-free culture medium (for example CD-CHO medium, such as CD-CHO inoculum medium and/or CDCHO basal medium) to further propagate the cell culture at a suitable temperature, for example from about 35°C to about 39°C, or from about 36°C to about 38°C for from about 3 to about 6 days, or for from about 4 to about 5 days, or for about 4.7 days, or for less than or equal to about 113 hours, until the cells reach a target seeding density (for example, having about 2.3 xlO⁶ viable cells/ml, or wherein the final cell culture viability is at least about 88%).

[00553] A commercial-scale culture (for example a 10,000L, 15,000L, 20,000L, 30,000L culture in a bioreactor and the like) is subsequently maintained in serum-free culture medium, wherein the medium is a feed medium (for example, eRDF medium, EXAMPLE 19), at a temperature of from about 35°C to about 39°C for from about 3 to about 6 days, or from about 4 to about 5 days, suitable for protein expression and production of the secreted protein product. Alternatively, a polyanionic compound (for example, such as dextran sulfate) can be added to a culture maintained in serum-free culture medium, wherein the medium is a feed medium (for example, eRDF medium, EXAMPLE 19) as described below and in U.S. Pat. App. Publication No. 2005/0019859, which is hereby incorporated by reference in its entirety. The culture can be concomitantly subjected to a single step temperature lowering (for example, at or about 32°C to at or about 36° C for from about 3 to about 14 days, or for from about 10 to about 13 days, or for from about 234 to about 304 hours.

[00554] In one embodiment of the invention, the culture can also be concomitantly subjected to a multi-step temperature lowering (for example, at or about 33°C to at or about 35°C for about 3-6 days, and at or about 31 °C to at or about 33°C for about 6-8 days). The above described processed are suitable for protein expression and production of the secreted protein product.

[00555] The feed medium in the instances described above can be replaced daily (1, 2, 3, etc. times daily) or every few days with fresh feed medium. The tank’s replacement with fresh feed medium entails removal of a predetermined volume, for example 80% of the tank volume, and replacing the tank with the same volume of fresh feed medium. The commercial scale culture is maintained until said cells reach a target value of production parameters that can be, but are not limited to, a length Of time, a target cell density, or biochemical protein characteristic (such as a NANA molar ratio as previously described) wherein the viable cell density can be 3.0-8.0 x 10⁶ cells/ml; a NANA molar ratio can be >5.0, or about 6, or from about 5.2 to about 7.6; a final cell culture viability can be greater than or equal to about 30% or greater than or equal to about 37%; and a final protein product titer can be from. about 0.46 to about 0.71 g/L; greater than or equal to 0.5 g/L, or greater than or equal to 20 g/L.

(00556] In accordance with the present invention, a cell culture process involving the delayed addition of polyanionic compound is provided. The process comprises adding polyanionic compound to a cell culture at a time after inoculation (for example, during the growth phase or during the production phase of the culturing process). The delayed addition of pblyanionic compound achieves increased cell viability. In one embodiment, the invention is directed to a cell culturing process that comprises culturing host cells, which express a protein of interest, and adding polyanionic compound to the cell culture at a time after inoculation.

[00557] Polyanionic compounds include, but are not limited to, dextran sulfate (available from Sigma-Aldrich, St. Louis, Mo.), heparin (available from Sigma-Aldrich), heparan sulfate (available from Sigma-Aldrich), mannan sulfate, chondroitin sulfate (available from Sigma-Aldrich), dermatan sulfate (available from Sigma-Aldrich), keratan sulfate (available from Sigma-Aldrich), hyaluronate (available from Sigma-Aldrich), poly(vinyl sulfate) (available from Sigma-Aldrich), kappa-carrageenan (available from Sigma-Aldrich), and suramin (available from Sigma-Aldrich). The compounds are readily available from the listed sources, or readily obtainable through means known to one of skill in the art. These compounds are frequently available in the form of a salt, including but not limited to sodium salt, but may also be used in non-salt forms. A polyanionic compound includes all forms thereof, including but not limited to salt forms, such as sodium salts.

[00558] Particularly useful, non-limiting examples of polyanionic compounds of the invention include poysulfated compounds: dextran sulfate, heparin, heparan sulfate, mannan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, poly(vinyl sulfate), kappacarrageenan, and suramin. In one embodiment, the polyanionic compound is dextran sulfate. Dextran sulfate may have an average molecular weight of 5,000 to 500,000 Da. In another embodiment of the invention, dextran sulfate having a molecular weight of 5,000 Da is used.

[00559] According to methods of the invention, polyanionic compound may be added to the cell culture one time, two times, three times, or any number of times during the specified cell culture period (for example, at a time after inoculation, such as during the growth phase or the production phase). One or more polyanionic compounds may be used in conjunction. For example, any given single addition of a polyanionic compound may include the addition of one or more other polyanionic compounds. Similarly, if there is more than one addition of a polyanionic compound, different polyanionic compounds may be added at the different additions. ׳ Additional compounds and substances, including polyanionic compounds, may be added to the culture before, with, or after the addition of polyanionic compound, either during or not during the specified time period. In a particular embodiment, there is a single, for example one time, addition of polyanionic compound. In another embodiment, one polyanionic compound is added.

[00560] Polyanionic compound may be added to the cell culture by any means. Means of adding polyanionic compound include, but are not limited to, dissolved in water, dissolved in culture medium, dissolved in feed medium, dissolved in a suitable medium, and in the form in which it is obtained. In particular, polyanionic compound is added dissolved in water. In accordance with the invention, polyanionic compound is added to bring the concentration in the culture to an appropriate level. As non-limiting examples, polyanionic compound is added to a concentration of 1-1000 mg/L, 1-200 mg/L, 1-100 mg/L, or 25-75 mg/L. Particularly useful concentrations of polyanionic compound added to the cell culture include, but are not limited to, about 25-200 mg/L; about 25-100 mg/L; and about 50-100 mg/L. In one embodiment of the invention, the concentration of polyanionic compound added to the culture is about 50 mg/L. In another embodiment, the concentration of polyanionic compound added to the culture is about 100 mg/L.

[00561] Methods of the invention provide that the culture may be run for any length of time after addition of polyanionic compound. The culture run time may be determined by one of skill in the art, based on relevant factors such as the quantity and quality of recoverable protein, and the level of contaminating cellular species (e.g. proteins and DNA) in the supernatant resulting from cell lysis, which will complicate recovery of the protein of interest. In some embodiments of the cell culturing process, polyanionic compound is added at a time after inoculation (for example, during the growth phase of the cell culture process or during the production phase of the cell culture process). Polyanionic compound is added at a time after inoculation that is during on or about the end of the growth phase. In particular, polyanionic compound is added at a time after inoculation that is during the production phase, for example, at the onset of the production phase.

[00562] In a particular embodiment of this invention, the cells cultured in a fed-batch ~ process are mammalian cells, for example CHO cells, which express a desired protein product (for example, a CTLA4^A29YLI04E-Ig molecule). CHO cells are inoculated into a seed culture vessel (for example, a T-175 flask) containing serum-free culture medium, for example CD-CHO medium (such as CD-CHO inoculum medium), and propagated at a temperature suitable for growth, for example at about 37°C, for about 3-4 days until the cells reach a pre-determined cross-seeding density (for example, having >1.5xl0⁶ viable cells, or wherein the final culture viability >80%). The propagated seed culture is then transferred to a large culture vessel (for example, roller bottles) containing culture medium lacking animalderived components for expansion at a suitable temperature (at about 37°C) for about 4 days. The cell culture is further expanded in a larger culture vessel (for example, a 20 L cell bag, a 100L cell bag, and the like) containing serum-free medium, for example CD-CHO medium (such as CD-CHO inoculum medium), for about 4 days at a temperature suitable for growth (for example, at about 3 7 °C) until the cells reach a target seeding density (for example, having >1.5x10⁶ viable cells, or wherein the final culture viability >80%). The inoculum . expansion can involve a minimum of 7 passages.

[00563] The expanded seed culture is then used to inoculate a large-scale culturing tank (for example, a 1000-L, a 4000-L bioreactor, and the like), containing serum-free culture medium (for example CD-CHO medium, such as CD-CHO basal medium) to further propagate the cell culture at a suitable temperature, for example at about 37°C, for about 5-6 days, until the cells reach a target seeding density (for example, having about 2.3 xlO⁶ viable cells/ml, or wherein the final cell culture viability >88%). A commercial-scale culture (for example a 10,000L, 15,0000L, or 20,000 L culture and the like in a bioreactor) is subsequently maintained in serum-free culture medium, wherein the medium is a feed medium (for example eRDF medium), at a temperature lower than the growth temperature, which is suitable for protein expression and production of the secreted protein product (for example, a CTLA4^A29YL104E-Ig). . ..

[00564] ׳The commercial-scale culture is lowered, for example, from about 37°C to about 34°C for about 4 days. Polyanionic compound may be added concomitantly to the culture when the temperature is lowered. Alternatively, the commercial-scale culture is lowered from about 35°C-37°C to about 32°C - 36°C for about 12 days and polyanionic compound is concomitantly added to the culture as the temperature is lowered.

[00565] The feed medium in the instances described above can be replaced daily (1,2, 3, etc. times daily) or every few days with fresh feed medium. The tank’s replacement with ־ fresh feed medium entails removal of a predetermined volume, for example 80% of the tank volume, and replacing the tank with the same volume of fresh feed medium. In one embodiment, the feed medium is added daily for about 2 to 3 days until, for example, the glucose concentration falls to 1 g/L. In another embodiment, the feed medium is added every 8 hours, for example, once the glucose concentration has reached Ig/L. The commercial scale is maintained until said CHO cells and/or secreted protein product reach a target value of the following non-limiting production parameters: a NANA molar ratio of about 6.0, or from about 5.2 to about 7.6; a final cell culture viability >37%; and a final protein product titer of from about 0.46 to about 0.71 g/L.

[00566] In an embodiment of the present invention, the cells being cultivated can be mammalian cells, or an established mammalian cell line, including, without limitation, CHO (e.g., ATCC CCL 61), HEK293 (e.g., ATCC CRL 1573; Graham et al., J. Gen, Virol. 36:5972,1977), COS-1 (e.g., ATCC CRL 1650), DG44 (CHO cell line) (Cell, 33: 405, 1983, and Somatic Cell and Molecular Genetics 12: 555, 1986), and baby hamster kidney (BHK) cell lines. Other useful non-limiting examples are myelomas, 3T3 cells, Namalwa cells, and fusions of myelomas with other cells. In some embodiments, the cells can be mutant or recombinant cells, such as, for example, cells that express a different spectrum of enzymes that catalyze post-translational modification of proteins (e.g., processing enzymes such as propeptides or glycosylation enzymes such as glycosyl transferases and/or glycosidases) than the cell type from which they were derived. In one particular aspect of this invention, CHO/dhfr- cells particularly are utilized.

[00567] The culturing vessels used for expanding the cell culture can be, but are not limited to, Erlenmyer flasks, T-175 flasks, roller bottles, and cell bags. The large-scale culture vessels can be, for example airlift reactors where agitation is obtained by means of introducing air from the bottom of the vessel or conventional stirred tank reactors (CSTR), where agitation is obtained by means of conventional impeller types. Among the parameters׳ . controlled within specified limits are temperature, pH, and dissolved oxygen tension (DOT). The temperature-control medium in this system is water, and can be heated or cooled as necessary. The water can be passed through a piping coil immersed in the cell culture medium or through a jacket surrounding the vessel. The pH, for example, can be regulated by addition of base to the cell culture medium when required or by varying the CO₂ concentration in the head-space gas. DOT -can be maintained by sparging with pure oxygen or air or mixtures thereof.

[00568] The invention therefore provides a method for producing a recombinant protein, the method comprising at least two steps: (a) expanding mammalian cells that secrete a recombinant protein (i.e., a protein that the mammalian cells do not normally express or overexpress, where the recombinant protein is expressed in the cells via an expression vector or construct that has been transfected into the cells or the parents of the cells) from a seed culture to a liquid culture of at least 10,000L, and (b) isolating the recombinant protein from the at least 10,000 L liquid culture. Tn one embodiment, this method can be used such that the recombinant protein is produced at a concentration of at least 0.5 grams per liter of liquid culture prior to purification of the protein from the liquid culture. In another embodiment, the method according to the invention can be used to produce a recombinant protein at a concentration of at least from about 0.46 to about 0.71 grams per liter of liquid culture prior to purification of the protein from the liquid culture.

[00569] In one embodiment, the expansion step can involve (i) culturing the cells in a serum-free medium with at least four passages so as to obtain a cell density of at least about 1.0 x 10⁵ viable cells per mL, and (ii) maintaining the cells in culture for a time sufficient to produce at least about 0.5 of the recombinant protein. In one embodiment, the number of passages does not exceed 36 passages. In another embodiment, the number of passages can exceed 36 passages where the cells are stable over generations with respect to copy number of the nucleic acid coding for the recombinant protein, cell viability, and doubling time.

[00570] The time sufficient to produce at least about 0.5 to about 1.3 g/L of the recombinant protein can be any amount of time as long as the cell viability does not fall below 5%, 10%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, 95%, 98% and/or as long as the number of cell generations does not exceed 50, 75, 100, 105, or 125 generations. The maintaining step can also comprise temperature shift steps, such as lowering the temperature of the culture first from 37 ± 2°C to 34 ± 2°C and at a later time from 34 ± 2°C to 32 ± 2°C. The temperature of 32 ±2°C can be maintained for at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14,15, 16, 17, 18, 20, 30, 50, or 100 days. The temperature of 32 ± 2°C can be maintained for at least 20, 50, 75, or 100 cell generations. The temperature of 32 ± 2°C can be maintained until the cell density of the culture is from about 30 to about 100 x 10^s cells per mL of liquid culture.

[00571] In other embodiments, the invention provides methods for producing a recombinant protein, the method comprising at least the steps of: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000L so that the recombinant protein concentration is at least 0.5 grams/L of liquid culture; and (b) isolating the recombinant protein from the at least 10,000 L liquid culture when the liquid culture: (i) contains greater than or equal to about 6.0 moles of NANA per mole of protein (glycoprotein in this case); (ii) has a cell density of from about 33 to about 79 x 10⁵ cells per mL; (iii) cell viability in the liquid culture is not less than about 38% or is greater than or equal to about 38%; (iv) endotoxin is less than or equal to about 76.8 EU per mL of liquid culture; and/or (v) bioburden is less than 1 colony forming unit per mL of liquid culture.

[00572[ In a further embodiment, the expansion step can involve (i) culturing the cells in a serum-free medium with at least four passages so as to obtain a cell density of at least about 1.0 x 10⁶ viable cells per mL, and (ii) maintaining the cells in culture for a time sufficient to produce at least from about 0.46 to about 0.71 grams of the recombinant protein per literof liquid culture. In one embodiment, the number of passages does not exceed 36 passages. In another embodiment, the number of passages can exceed 36 passages where the cells are stable over generations with respect to copy number of the nucleic acid coding for the recombinant protein, cell viability, and doubling time.

[00573] The time sufficient to produce at from about 0.46 to about 0.71 g/L of the recombinant protein can be any amount of time as long as the cell viability does not fall below 5%, 10%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, 95%, 98% and/or as long as the number of cell generations does not exceed 27, 50, 75, 100, 105, or 125 generations. The maintaining step can also comprise temperature shift steps, such as lowering the temperature of the culture first from 37 ± 2°C to 34 ± 2°C. The temperature of 34 ± 2°C can be maintained for at least 5, 6, 7, 8, 9,10, 11,12, 13, 14, 15, 16, 17, 18, 20, 30, 50, or 100 days. Alternatively, the maintaining step can a temperature shift step, such as lowering the temperature of the culture from 37 ± 2°C to 34 ± 2°C.

[00574] Polyanionic compound can be added to the cultures as temperature lowering

--. . -commences. The concentration of polyanionic compound added to the culture can be about 1 mg/L, 5 mg/L, 10 mg/L, 12.5 mg/L, 15 mg/L, 25 mg/L, 50 mg/L, 75 mg/L, 100 mg/L, 200 mg/L, 250 mg/L, 500 mg/L, 750 mg/L, or 1000 mg/L. The temperature of 32 ± 2°C can be maintained for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 30, 50, or 100 days. The temperature of 34 ± 2°C can be maintained for at least 20, 27, 50, 75, or 100 cell generations.

[00575] In further embodiments, the invention provides methods for producing a recombinant protein, the method comprising at least the steps of: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000L so that the recombinant protein concentration is at least from about 0.46 to about 0.71 grams per liter of liquid culture; and (b) isolating the recombinant protein from the culture of at least 10,000 L liquid culture when the liquid culture: (i) contains about 6 moles . of NANA per mole of protein (glycoprotein in this case); (ii) cell .viability in the liquid culture is not less than about 37%; (iii) endotoxin is less than or equal to about 4.8 EU per mL of liquid culture; and/or (iv) bioburden is less than 1 colony forming unit per mL of -liquid culture.

[00576] The recombinant protein produced by these methods of the invention .can be a secreted protein, a glycoprotein, a cytokine, a hormone, a CTLA4-Ig protein, or a CTLA4^A29YL104E-Ig protein. In one embodiment, the mammalian cells are progeny or subclones of cells provided by the invention. In another embodiment, the mammalian cells are progeny or subclones of cells derived from the cell line of the invention. In a further embodiment, the mammalian cells are a clonal population from cells transfected with an expression cassette comprising SEQ ID NO:1. In a particular embodiment, the mammalian cells are a clonal population from cells transfected with an expression cassette comprising SEQ ID NO: 3.

[00577] General Techniques for the Purification of Recombinant Protein from Culture

[00578] Following the protein production phase of the cell culture process, the protein of interest, for example a glycoprotein, is recovered from the cell culture medium using techniques understood by one skilled in the art. In particular, the protein of interest is recovered from the culture medium as a secreted polypeptide, although it also can be recovered from host cell lysates. The culture medium or lysate is initially centrifuged to remove cellular debris and particulates. The desired protein subsequently is purified from contaminant DNA, soluble proteins, and polypeptides, with the following non-limiting purification procedures well-established in the art: SDS-PAGE; ammonium sulfate precipitation; ethanol precipitation; fractionation on immunoaffinity or ion-exchange columns; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as QAE or DEAE; chromatofocusing; gel filtration using, for example, Sephadex G-75™ column; and protein A Sepharose™ columns to remove contaminants such as IgG. Addition of a protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF), or a protease inhibitor cocktail mix also can be useful to inhibit proteolytic degradation during purification. A person skilled in the art will recognize that purification methods suitable for a protein of interest, for example a glycoprotein, can require alterations to account for changes in the character of the protein upon expression in recombinant cell culture.

[00579] Purification techniques and methods that select for the carbohydrate groups of the glycoprotein are also of utility within the context of the present' invention. For example, such techniques include, HPLC or ion-exchange chromatography using cation- or anion-exchange resins, wherein the more basic or more acidic fraction is collected, depending on which carbohydrate is being selected for. Use of such techniques also can result in the concomitant removal of contaminants.

[00580] Tn the present invention, CHO cells capable of producing CTLA4-Ig or CTLA4^A29YLl04E-Ig fusion proteins are grown as a suspension in a CHO specific medium to a predetermined cell density. CHO cells grown in suspension in the serum-free expression medium subsequently produce CTLA4-Ig or CTLA4^A29YL104E-lg molecules, which are secreted by the CHO cells into the culture medium. The cell suspension can be cleared via centrifugation and CTLA4-Ig molecules can then be separated from the cleared culture supernatant by standard purification techniques. Non-limiting examples of suitable purification procedures for obtaining greater purity and homogeneity of CTLA4-Ig or CTLA4^A29YL104E-Ig, either individually or in combination, are: affinity chromatography on sepharose; fractionation on anion-exchange columns (AEC); and hydrophobic interaction chromatography (HIC).

[00581] In some embodiments, isolating CTLA4-Ig molecules or other proteins (including glycoproteins) from the methods of production described herein can at least include the following steps: (i) obtaining a cell culture supernatant; (ii) subjecting the supernatant to anion exchange chromatography to obtain an eluted protein product; (iii) subjecting the eluted protein product of step (ii) to hydrophobic interaction chromatography so as to obtain an enriched protein product; (iv) subjecting the enriched protein product to affinity chromatography to obtain an eluted and enriched protein product; and (v) subjecting the eluted and enriched protein product of (iv) to anion exchange chromatography. The enriched protein product obtained in step (iii) can be characterized, for example, in that its percentage of any HMW protein or contaminant is less than 5, 10, 15 or 25%. The anion exchange chromatography of step (ii) can be carried out, for example, by using a wash buffer comprising about 25-100 mM HEPES and about 300-900 mM NaCI and having a pH of about 7.0-8.0. The hydrophobic interaction chromatography of step (iii) can be carried out, for example, by using a single wash buffer having a pH of about 7.0 and comprising about 25 mM HEPES and about 850 mM NaCI; or a wash buffer having a pH of about 8.0 and comprising about 25 mM Tris and about 250 mM NaCI. The affinity chromatography of step (iv) can be carried out, for example, by using an elution buffer having a pH of about 3.5 and comprising about 100 mM glycine. The affinity chromatography of step (v) can be carried out,-for example, by using a wash buffer having a.pH of about 8.0 and comprising about 25 mM HEPES and from about 120 mM NaCI to about 130 mM NaCI, or a wash buffer having a pH of about 8.0 and comprising about 25 mM HEPES and about 200 mM NaCI. The anion exchange .chromatography of step (ii) can be carried out using a column having an anion exchange resin having a primary, secondary, tertiary, or quartenary amine functional group. The hydrophobic interaction column of step (iii) can be carried out using a hydrophobic interaction resin having a phenyl, an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group.

[00582] In other embodiments, isolating CTLA4^A29YLI04E-Ig molecules or other proteins (including glycoproteins) from the methods of production described herein can at least include the following steps: (i) obtaining a cell culture supernatant; (ii) subjecting the supernatant to affinity chromatography to obtain an eluted protein product; (iii) subjecting the eluted protein product of step (ii) to anion exchange chromatography so as to obtain an enriched protein product; and (iv) subjecting the enriched protein product to hydrophobic interaction chromatography to obtain an eluted and enriched protein product with reduced high molecular weight (HMW) protein complexes. The enriched protein product obtained in step (iv) can be characterized, for example, in that its percentage of any HMW protein or contaminant is less than 5,10,15 or 25%. The affinity chromatography of step (ii) can be carried out, for example, by using an elution buffer having a pH of about 3.0 and comprising about 250 mM glycine. The affinity chromatography of step (ii) can be carried out, for example, by using a wash buffer having a pH of about 7.5 and comprising about 25 mM NaH2PO4 and about 150 mM NaCl. The anion exchange chromatography of step (iii) can be carried out, for example, by using a wash buffer comprising about 50 mM HEPES and about 135 mM NaCl and having a pH of about 7.0. The anion exchange chromatography of step (iii) can be carried out, for example, by using an elution buffer comprising about 50 mM HEPES and about 200 mM NaCl and having a pH of about 7.0. The hydrophobic interaction chromatography of step (iv) can be carried out, for example, by using a wash buffer having a pH of about 7.0 and comprising about 50 mM HEPES and about 1.2 M (NH4)2SO₄. The anion exchange chromatography of step (iii) can be carried out using a column having an anion exchange resin having a primary, secondary, tertiary, or quartenary amine functional group. The hydrophobic interaction column of step (iv) can be carried out using a hydrophobic interaction resin having a phenyl, an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group.

[00583] In one embodiment, the invention provides a method for purifying CTLA4-Ig molecules from a liquid cell culture so that the purified CTLA4-Ig is substantially free of Monocyte Chemotactic Protein-1 (MCP-1). In one embodiment, the invention provides for a pharmaceutically acceptable composition of CTLA4-Ig molecules, wherein the composition comprises no more than 0.5 ppm MCP-1, 1 ppm MCP-1, 2 ppm MCP-1, 3 ppm MCP-1, 4 ppm MCP-1, 5 ppm MCP-1, 6 ppm MCP-1, 7 ppm MCP-1, 8 ppm MCP-1, 9 ppm MCP-1 or 10 ppm MCP-1. In another embodiment, in the composition, the amount of MCP-1 cannot exceed 1 %, 0.5%, or 0.1 % of the weight of purified CTLA4-Ig. In another embodiment, the composition of CTLA4-Ig molecules is substantially free of MCP-1 where there is less than 50, 45, 40, 38, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/mL of MCP-1 in the QFF eluate liquid. In another embodiment, the invention provides a method for purifying CTLA4Ig molecules from a liquid cell culture so that the purified CTLA4-Ig is substantially free of MCP-1 and comprises less than 2.5% of CTLA4-Ig tetramer.

[00584]. . The amount of Monocyte chemotactic protein 1 (MCP-1) in the composition can be quantified using an ELISA method. The coating antibody is a goat anti-mouse MCP-1 IgG antibody. The secondary antibody is a rabbit anti-rat MCP-1 IgG antibody. Detection is accomplished using horseradish peroxidase conjugated goat anti-rabbit IgG antibody and the substrate TMB. The horseradish peroxidase reagent produces a colorimetric reaction that develops in proportion to the amount of protein captured. The ELISA quantifies the MCP-1 level relative to a material standard curve. In one embodiment, MCP-1 was quantified in the composition and the MCP-1 levels were in ranges from 0-0.097 ng/mg and 0.014 0.154 ng/mg.

[00585] In another embodiment, the invention provides a method for purifying CTLA4^A29YL104E-Ig molecules from a liquid cell culture so that the purified CTLA4^a29yl104eIg is substantially free of Monocyte Chemotactic Protein-1 (MCP-1). In one embodiment, the amount of MCP-1 cannot exceed 1%, 0.5%, or 0.1% of the weight of purified CTLA4^A29YL104E-Ig. In another embodiment, CTLA4^A29YL104E-Ig is substantially free of MCP-1 where there is less than 50, 45, 40, 38, 35, or 30 ng/mL of MCP-1 in the HIC eluate liquid. In a further embodiment, the invention provides a method for purifying CTLA4^A29YLI04E-Ig molecules from a liquid cell culture so that the purified CTLA4^a29yl104eIg is substantially free of MCP-1 and comprises less than 2.5% of CTLA4^A29YLI04E-Ig tetramer.

[00586] Glycoprotein Recovery from the Cell Culture and Purification

[00587] The present invention describes a series of steps for the separation of a glycoprotein (for example, CTLA4-Ig or CTLA4^A29YLI04E-Ig) from an impure, cell-culture supernatant, protein pool that contains the glycoprotein of interest (such as CTLA4-Ig or CTLA4^A29YLI04E-Ig) and undesirable contaminants. The impure, cell-culture supernatant can be used as the starting material for the purification of the CTLA4-Ig or CTLA4^A29YL104E-Ig glycoprotein.

[00588] In one embodiment of the present invention, the impure, cell-culture supernatant that contains CTLA4-Ig glycoprotein and undesirable contaminants is applied to an anionexchange medium. The CTLA4-Ig glycoprotein present in the impure, cell-culture supernatant binds to the anion-exchange medium. The anion-exchange medium is then washed to remove any unbound material .from .the anion-exchange medium. CTLA4-Ig glycoprotein is.eluted after the unbound material is removed, and the eluate is collected.

[00589] In one embodiment of the present invention, the impure, cell-culture supernatant that contains CTLA4^A29YLI04E-Ig glycoprotein and undesirable contaminants is applied to an affinity chromatography medium. The CTLA4^A29YU04E-Ig glycoprotein present in the impure, cell-culture supernatant binds to the affinity chromatography medium. The affinity chromatography medium is then washed to remove any unbound material from the anionexchange medium. CTLA4^A29YL104E-Ig glycoprotein is eluted after the unbound material is removed, and the eluate is collected.

[00590] In a particular embodiment of this invention, Q-Sepharose Anion-Exchange

Chromatography (AEC), for example using a Q-Sepharose XL column (GE Healthcare), is employed to separate CTLA4-Ig glycoprotein from the harvest material, as well as for decreasing bulk contaminants. This column can be used as an early step in the purification of CTLA4-Ig glycoprotein from a mammalian cell culture, for fractionation of the harvested cell culture medium. In another embodiment, Q-Sepharose Anion-Exchange Chromatography, for example Q-Sepharose Fast Flow (GE Healthcare), can be used after an affinity chromatography purification step. The very high flow property of the anion exchange columns allows the large volume of CTLA4-Ig glycoprotein or harvested cell culture medium to be readily concentrated before subsequent chromatography steps, such as SP-Sepharose or HIC, by adjusting conditions so that the CTLA4-Ig glycoprotein binds the column. For a wash buffer of pH from about pH 5 to 9, in particular about 8, 75mM HEPES and 360mM NaCl concentrations are useful. Typically, for an elution buffer of pH from about pH 5 to 8, in particular about 7, 25mM HEPES and 325mM NaCl concentrations are useful.

[00591] Suitable resins for separating CTLA4-Ig glycoprotein from the harvested culture medium were those having immobilized amine functional groups. Most useful are the quarternary amine functional group resins, for example those on Q-Sepharose Fast Flow resins from GE Healthcare, where a quarternary ammonium ligand is bound to high-porosity, cross-linked agarose. Also useful are the primary, secondary and tertiary amine functional group resins, for example those on DEAE Sepharose Fast Flow resins from GE Healthcare, where a tertiary di ethyl aminoethyl ligand is bound to high-porosity, cross-linked agarose.

(00592] In another embodiment of the present invention, the CTLA4-Ig glycoproteincontaining eluate from the anion-exchange medium is collected and then contacted with a hydrophobic interaction resin. As described below, the CTLA4-Ig glycoprotein-containing volume passes through the HIC column and the collected pool, which can be further purified, is then bound to an anion-exchange resin.

[00593] HIC is useful for the separation of desired CTLA4-Ig glycoprotein dimers from high molecular weight material and other protein impurities from the mammalian cell culture. For example, CTLA4-Ig-expressing-CHO cell culture contains high molecular weight aggregates of CTLA4-Ig glycoprotein. Also found in the mammalian cell culture medium are CHO cell protein impurities. These undesirable products could generate an unwanted antigenic response in a patient and contribute to poor product quality or activity. HIC effectively separates hydrophobic variants, CTLA4-Ig glycoprotein dimers from CTLA4-Ig glycoprotein HMW complexes and CHO protein impurities via the latter products binding to the HIC resin and the CTLA4-Ig glycoprotein dimers passing through the column. Thus, a CTLA4-Ig glycoprotein pool could be obtained that is substantially free of these species, and that is particularly suited for another chromatographic step, such as anion-exchange chromatography. A source of CTLA4-Ig glycoprotein mixtures for use with HIC is mammalian cell culture, for example a CHO cell culture. In particular, the culture can be subjected to at least one prior purification step as discussed previously.

[00594] In another embodiment of this invention, the HIC method can be modified to collect a pool of other glycoproteins (for example, CTLA4-Ig HMW complexes). HMW aggregates can bind to the HIC resin (for example, comprising CTLA4-Ig tetramer and the like). These HMW complexes have a higher avidity and bind more efficaciously in vivo than CTLA4-Ig dimer alone. Thus, one skilled in the art can obtain a pool of CTLA4-Ig HMW aggregates by eluting the CTLA4-Ig pool off of the HIC.

[00595] The most useful HIC resins for separating CTLA4-Ig glycoprotein forms are those having immobilized phenyl functional groups. Of the phenyl-HIC resins, Phenyl Sepharose Fast Flow High Sub (high substitution) by GE Healthcare is most useful. Phenyl Toyopearl media by TosoHaas and TSK Phenyl 5PW are non-limiting examples of other phenyl-HIC resins that can be used. Other HIC functional groups include, but are not limited to, the propyl, octyl, alkoxyl, butyl, and isoamyl moieties.

[00596] ־For example, a Phenyl Sepharose 4 Fast Flow column chromatography,

Hydrophobic Interaction Chromatography (HIC), process can be used to reduce the amount of CTLA4-Ig or CTLA4^A29YL104E-Ig high molecular weight species eluted in a HIC purification step (see Example 15 and EXAMPLE 20). Therefore, the cleaning peak from the HIC column is enriched in CTLA4-Ig or CTLA4^A29YU04E-Ig HMW species.

[00597] The unbound fraction containing CTLA4-Ig glycoprotein from the HIC purification step can be subjected to an additional purification method, such as affinity chromatography, and the resulting eluate can then be applied to an anion-exchange medium. The CTLA4-Ig glycoprotein binds to the anion-exchange resin, which can be subsequently washed to remove unbound proteins. After the unbound proteins are removed, CTLA4-Ig glycoprotein is eluted from the second anion-exchange resin. The eluate is collected and can be further concentrated.

[00598] In another embodiment of this invention, affinity chromatography, for example rProtein A Sepharose Fast Flow (GE Healthcare), is employed to further enrich CTLA4-Ig glycoprotein, which can be further followed by an anion-exchange chromatography step, for example Q-Sepharose Fast Flow (GE Healthcare). The affinity chromatography step can also reduce the levels of CHO proteins and Monocyte Chemotactic Protein (MCP-1, a chemokine) impurities. Affinity chromatography involves adsorptive separation, where a molecule of interest to be purified, for example CTLA4-Ig glycoprotein, binds specifically and reversibly to a ligand immobilized on some matrix or resin. Some non-limiting examples of affinity purification columns include lectin; affinity tag (for example, a GST column or 6X-His column), Streptavidin; heparin; or antibody (for example, a Protein A column or a Protein G column). In particular, this invention utilizes a protein A resin for binding the CTLA4-Ig glycoprotein. For a wash buffer of pH from about 5 to 9, more effective at about 8, 25mM Tris and 250mM NaCl concentrations are useful. For an elution buffer of pH from about 2 to 5, more effective at about 3.5, lOOmM glycine concentration is useful. The affinity chromatography eluate can then be neutralized and loaded onto an anion exchange chromatography column, Q-Sepharose Fast Flow being most useful.

[00599] . To further reduce the levels of protein A, DNA, and non-desired CTLA4-Ig glycoprotein species in the product after the foregoing recovery/initial purification steps, another ion-exchange step can be incorporated into the purification procedure. This invention can employ commercially available ion-exchange columns, such as a Q-Sepharose

Fast Flow column from GE Healthcare, or a DEAE Sepharose Fast Flow column, also from GE Healthcare. As.determined herein, the most suitable resins for separating CTLA4-Ig glycoprotein from the harvested culture medium were those having immobilized amine functional groups. Other useful groups are the quarternary amine functional group resins, for example those in a Q-Sepharose Fast Flow column from GE Healthcare, where a quarternary ammonium ligand is bound to high-porosity, cross-linked agarose. Also useful are the primary, secondary and tertiary amine functional group resins, for example those in a DEAE Sepharose Fast Flow column from GE Healthcare, where a tertiary diethylaminoethyl ligand is bound to high-porosity,’ cross-linked agarose. In a particular embodiment of the invention, a column that utilizes a strong anion exchanger, such as a Q-Sepharose Fast Flow column, is utilized.

[00600] In one embodiment of the invention, a CTLA4-Ig glycoprotein eluate is loaded onto an anion exchange column, for example Q-Sepharose Fast Flow. The column is washed and CTLA4-Ig glycoprotein is subsequently eluted from the anion exchange column. For a wash buffer of pH from about 5 to 9, in one embodiment, pH 8, 25mM HEPES and 100140mM NaCl concentrations are useful. For.an.elution buffer .of pH from about 5 to 9, or in another embodiment, pH 8, 25mM HEPES and 200mM NaCl concentrations are useful.

CTLA4-Ig glycoprotein eluted from the anion-exchange medium is recovered, concentrated and washed, by diafiltration or other suitable method known to one skilled in the art, to provide a final purified CTLA4-Ig glycoprotein product. The CTLA4-Ig glycoprotein product prepared in accordance with the process of the present invention is of high purity, for example containing >95% of the CTLA4-Ig dimer, containing <5% of the CTLA4-Ig HMW product, and containing <1 % of CTLA4-Ig monomer.

[00601] The purification method can further comprise additional steps that inactivate and/or remove viruses and/or retroviruses that might potentially be present in the cell culture medium of mammalian cell lines. A significant number of viral clearance steps are available, including but not limited to, treating with chaotropes such as urea or guanidine, detergents, additional ultrafiltration/diafiltration steps, conventional separation, such as ion-exchange or size exclusion chromatography, pH extremes, heat, proteases, organic solvents or any combination thereof.

[00602] In another embodiment of this invention, affinity chromatography, for example MabSelect Protein A Sepharose resin (GE Healthcare), is employed to capture

CTLA4^A29YLI04E-Ig glycoprotein, which can be further followed by an anion-exchange chromatography step, for example Q-Sepharose Fast Flow (GE Healthcare). The affinity chromatography step can also reduce the levels of CHO proteins and Monocyte Chemotactic Protein (MCP-1, a chemokine) impurities. Affinity .chromatography involves adsorptive separation, where a molecule of interest to be purified, for example CTLA4^a29yl1 °^4E-Ig glycoprotein, binds specifically and reversibly to a ligand immobilized on some matrix or resin. Some non-limiting examples of affinity purification columns include lectin; affinity tag (for example, a GST column or 6X-His column); Streptavidin; heparin; or antibody (for example, a Protein A column or a Protein G column). In particular, this invention utilizes a protein A resin for binding the CTLA4^A29YL104E-Ig glycoprotein. For a wash buffer of pH from about 5 to 9, more effective at about 7.5, 25mM Tris, 25mM NaH₂PO₄, and 250mM NaCI concentrations are useful. For an elution buffer of pH from about 2 to 5, more effective at about 3.5, 100-300 mM glycine concentration is useful. The affinity chromatography eluate can then be neutralized and loaded onto an anion exchange chromatography column, Q-Sepharose Fast Flow being most useful.

[00603] To further reduce the levels of protein A, DNA, and non-desired CTLA4^a29yl104eIg glycoprotein species in the product after the foregoing recovery/initial purification steps, an ion-exchange step can be incorporated into the purification procedure. ^,This invention can employ commercially available ion-exchange columns, such as a Q-Sepharose Fast Flow column from GE Healthcare, Q-Sepharose XL column (GE Healthcare), or a DEAE Sepharose Fast Flow column, also from GE Healthcare. The most suitable resins for separating CTLA4^A29YLI04E-Ig glycoprotein are those having immobilized amine functional groups. Other useful groups are the quarternary amine functional group resins, for example those in a Q-Sepharose Fast Flow column from GE Healthcare, where a quarternary ammonium ligand is bound to high-porosity, cross-linked agarose. Also useful are the primary, secondary and tertiary amine functional group resins, for example those in a DEAE Sepharose Fast Flow column from GE Healthcare, where a tertiary diethylaminoethyl ligand is bound to high-porosity, cross-linked agarose. In a particular embodiment of the invention, a column that utilizes a strong anion exchanger, such as a Q-Sepharose Fast Flow column, is utilized.

[00604] In one embodiment of the invention, a CTLA4^A29YL104E-Ig glycoprotein eluate is loaded onto an anion exchange column, for example Q-Sepharose Fast Flow. The column is washed and CTLA4^A29YLI04E-Ig glycoprotein is subsequently eluted from the anion exchange column. For a wash buffer of pH from about 5 to 9, in one embodiment, pH 7, 25-55 mM

HEPES and 100-140mM NaCI concentrations are useful. For an elution buffer of pH from about 5 to 9, or in another embodiment, pH 7, 25-50 mM HEPES and 200 mM NaCI concentrations are useful. ..

[00605] In another embodiment of the present invention, the CTLA4^A29YLI04E-Ig glycoprotein-containing eluate from the anion-exchange medium is collected and then contacted with a hydrophobic interaction resin. HIC is useful for the separation of desired CTLA4^A29YLI04E-Ig glycoprotein dimers from high molecular weight material and other protein impurities from the mammalian cell culture. For example, CTLA4^A29YLI04E-Ig expressing-CHO cell culture contains high molecular weight aggregates of CTLA4^a29yli04eIg glycoprotein. Also found in the mammalian cell culture medium are CHO cell protein impurities. These undesirable products could generate an unwanted antigenic response in a patient and contribute to poor product quality or activity.

[00606] HIC effectively separates hydrophobic variants, CTLA4^A29Y*^104,־^E-Ig glycoprotein dimers from.CTLA4^A29YLI04E-Ig glycoprotein HMW complexes and CHO protein impurities via the latter products binding to the HIC resin and the CTLA4^A29YLI04E-Ig glycoprotein dimers passing through the column. Thus, a CTLA4^A29YLI04E-Ig glycoprotein pool could be obtained that is substantially free of these species. A source of CTLA4^A29YLI04E-Ig glycoprotein mixtures for use with HIC is mammalian cell culture, for example a CHO cell culture. In particular, the culture can be subjected to at least one prior purification step as discussed previously.

[00607] In another embodiment of this invention, the HIC method can be modified to collect apool of other glycoproteins (for example, CTLA4^A29YLI04E-Ig HMW complexes). HMW aggregates can bind to the HIC resin (for example, comprising CTLA4^A29YL104E-Ig tetramer and the like). These HMW complexes have a higher avidity and bind more efficaciously in vivo than CTLA4^A29YLI04E-Ig dimer alone. Thus, one skilled in the art can obtain a pool of CTLA4^A29YL104E-Ig HMW aggregates by eluting the CTLA4^A29YL104E-Igpool off of the HIC.

[00608] CTLA4^A29YLI04E-Ig glycoprotein eluted from the HIC medium is recovered, concentrated and washed, by diafiltration or other suitable method known to one skilled in the art, to provide a final purified CTLA4^A29YLI04E-Ig glycoprotein product. The

CTLA4^A29YL104E-Ig glycoprotein product prepared in accordance with the process of the present invention is of high purity, for example containing >95% of the CTLA4^A29YL104E-Ig dimer, containing <5% of the CTLA4^A29YL104E-Ig HMW product, and containing <1% of

CTLA4^A29YL104E-Ig monomer.

[00609] The most useful HIC resins for separating CTLA4^A29YLI04E-Ig glycoprotein forms are those having immobilized phenyl functional groups. Of the phenyl-HIC resins, Phenyl Sepharose Fast Flow High Sub (high substitution) by GE Healthcare is most useful. Phenyl Toyopearl media by TosoHaas and TSK Phenyl 5PW are non-limiting examples of other phenyl-HIC resins that can be used. Other HIC functional groups include, but are not limited to, the propyl, octyl, alkoxyl, butyl, and isoamyl moieties.

[00610] The purification method can further comprise additional steps that inactivate and/or remove viruses and/or retroviruses that might potentially be present in the cell culture medium of mammalian cell lines. A significant number of viral clearance steps are available, including but not limited to, treating with chaotropes such as urea or guanidine, detergents, additional ultrafiltration/diafiltration steps, conventional separation, such as ion-exchange or size exclusion chromatography, pH extremes, heat, proteases, organic solvents or any combination thereof.

[00611] In one aspect, purified CTLA4-Ig molecules which have been concentrated and subjected to diafiltration step can be filled into 2-L Biotainer® bottles, 50-L bioprocess bag or any other suitable vessel. CTLA4-Ig molecules in such vessels can be stored for about 60 'days' at 2° to 8°C prior to freezing. Extended storage of purified CTLA4-Ig at 2° to 8°C may lead to an increase in the proportion of CLTA4-Ig tetramer. Therefore, for long-term storage, CTLA4-Ig molecules can be frozen at about —70°C prior to storage and stored at a temperate of about -40°C. ׳The freezing temperature can vary from about -50°C to about -90°C. The freezing time can vary and largely depends on the volume of the vessel that contains CTLA4Ig molecules, and the number of vessels that are loaded in the freezer. For example, in one embodiment, CTLA4-Ig molecules are in 2-L Biotainer® bottles. Loading of less than four 2-L Biotainer® bottles in the freezer may require from about 14 to at least 18 hours of freezing time. Loading of at least four bottles may require from about 18 to at least 24 hours of freezing time. Vessels with frozen CTLA4-Ig molecules are stored at a temperature from about -35°G to about -55°C.

[00612] The storage time at a temperature of about -35°C to about -55°C can vary and can be as short as 18hours. The frozen CTLA4-Ig molecules can be thawed in a control manner. Thawing of frozen CTLA4-Ig molecules is controlled and can be done in an incubator at a temperature from about 20°C to about 24°C. The duration of the thawing steps depends on the loading of the incubator wherein loading of les's than four 2-L Biotainer® bottles may require less than about 24 hours of thawing time. Loading of four 2-L Biotainer® bottles may require about 18 hours. Thawed solution comprising CTLA4-Ig molecules can be mixed to avoid potential concentration gradients. Therefore, thawing can be done in a controlledtemperature incubator, which also allows for shaking of the vessels, which contain CTLA4Ig. The speed of shaking can be from about 40 to about 80rpm. Thawed CTLA4-Ig molecules can be further mixed for additional 5-1 Omin at a rotational rate of about 3rpm.

Thawed CTLA4-Ig molecules can be stored at 2° to 8°C, alequated and lyophilized during the production of pharmaceutical compositions comprising CTLA4-Ig.

[00613] The present invention can be further applied to the purification of other, non!uniting examples, of therapeutic glycoproteins produced in large scale. The process of this invention can be applicable to the production of other glycoproteins having more than one glycosylated variant in mammalian'cell’cultures. One skilled in the art will understand the ’ modifications that might become necessary in the course of the adaptation of the exemplified method to the production of different glycoproteins.

[00614] Formulations &amp; Kits

[00615] The invention also provides any of the described CTLA4-Ig molecules as a lyophilized mixture. Formulations comprising CTLA4-Ig to be lyophilized can further comprise three basic components: (1) an additional active ingredient(s) including other recombinant proteins or. small molecules (such as immunosuppressants), (2) an excipient(s) and (3) a solvent(s). Excipients include pharmaceutically acceptable reagents to provide good lyophilized cake properties (bulking agents) as well as to provide lyoprotection and/or cryoprotection of proteins (“stabilizer”), maintenance of pH (buffering agents), and proper conformation of the protein during storage so that substantial retention of biological activity (including active ingredient stability, such as protein stability) is maintained. With respect to excipients, an example of a formulation can include one or more of a buffering agent(s), a bulking agent(s), a protein stabilizer(s) and an antimicrobial(s). Sugars or polyols can be used as nonspecific protein stabilizers in solution and during freeze-thawing and freezedrying. Polymers can be used to stabilize proteins in solution and during freeze-thawing and freeze-drying. One popular polymer is serum albumin, which has been used both as a cryoprotectant and lyoprotectant. In one embodiment, the invention provides formulations that are albumin free. Various salts can be used as bulking agents. Illustrative salt bulking agents include, for example, NaCl, MgC12 and CaC12. Certain amino acids can be used as cryoprotectants and/or lyoprotectants and/or bulking agents. Amino acids that can be used include, but are not limited to, glycine, proline, 4-hydroxyproline, L-serine, sodium glutamate, alanine, arginine and lysine hydrochloride. Many buffering agents covering a wide pH range are available for selection in formulations. Buffering agents include, for example, acetate, citrate, glycine, histidine, phosphate (sodium or potassium), diethanolamine and Tris. Buffering agents encompasses those agents which maintain the solution pH in an acceptable range prior to lyophilization. Formulations have previously been described in. ״ U.S. Patent Application No. 60/752,150, filed December .20, 2005, which is hereby incorporated by reference in its entirety.

[00616] In one embodiment, the invention provides a lyophilized CTLA4-Ig mixture comprising at least 90%, 95%, 99%, or 99.5% CTLA4-Ig dimer. In one embodiment, the invention provides a lyophilized CTLA4-Ig mixture comprising at least 90%, 95%, 99%, or 99.5% CTLA4-lg dimer and not more than 5%, 4%, 3%, 2%, or 1% CTLA4-Ig tetramer. In another embodiment, the invention provides a lyophilized CTLA4-Ig mixture comprising at least 90%, 95%, 99%, or 99.5% CTLA4-Ig dimer, and not more than 5%, 4%, 3%, 2%, or 1% CTLA4-Ig tetramer, and not more than 2%, 1.5%, 1.0%, 0.8%, 0.5%, or 0.3% CTLA4-Ig monomer. In a further embodiment, the invention provides a lyophilized CTLA4-Ig mixture comprising at least 8.0 moles of sialic acid per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule. In another embodiment, the invention provides a lyophilized CTLA4-Ig mixture comprising: from about 15 to about 35 moles of GlcNac per mole of CTLAIg molecules or dimer; from about 1 to about 5 moles of GalNac per mole of CTLA4-Ig dimer or to CTLA4Ig molecule; from about 5 moles to about 20 moles of galactose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule; from about 2 to about 10 moles of fucose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule; and/or from about 5-15 moles of mannose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule

[00617] A CTLA4^A29YL104E-Ig drug substance is available as an aqueous solution at approximately 25 mg/mL (22,5-27.5 mg/mL) concentration in 25 mM sodium phosphate and 10 mM sodium chloride buffer at pH ~ 7.5. CTLA4^A29YLI04E-Ig has a tendency to form high molecular weight species in aqueous solution. Therefore, a freeze-dried product was. developed in order to minimize the levels of high molecular weight species that may form in the drug product. Various excipients such as maltose, sucrose, and amino acids such as L-arginine hydrochloride were screened as potential lyoprotectants during freeze drying of CTLA4^A29YL104E-Ig. Sucrose was found to be the most effective lyoprotectant. It was further observed that increasing the sucrose to protein ratio improved protein stability. A sucrose: protein ratio of 2:1 (wt.:wt.) was chosen for the protein solution to be freeze dried. The freeze-dried drug product has adequate stability and satisfactory constitution behavior.

[00618] Methods of Treatment

[00619] According to thisinvention, a disease mediated by T cell interactions with B7 positive cells can be treated by receiving a pharmaceutically acceptable formulation of CTLA4-Ig or CTLA4^A29Y!J94E-ig. The CTLA4-Ig or CTLA4^A29YL104E-־Ig molecules secreted by an engineered mammalian cell line (for example, a dhfr- negative Chinese Hamster Ovary cell line that harbors DNA encoding CTLA4-Ig or CTLA4^A29YLI04E-Ig) can be a population of molecules having a particular glycosylation profile. As stated herein, a particular glycoyslation profile can affect CTLA4-Ig or CTLA4^A29YL104E-Ig binding to CD80 and/or CD86 such that CTLA4-Ig or CTLA4^A29YL104E-Ig molecules can provide a greater inhibition on T cell activation and/or proliferation. As stated herein, a particular glycosylation profile can be affected by the cell line and the method of production. Thus, in certain embodiments of the invention, the invention provides CTLA4-Ig or CTLA4^A29YL,04E-Ig molecules produced by a cell line in a production method described herein in order to treat T-cell related diseases or disorders, that include but are not limited to, generally any T-cell dependent lymphoproliferative disease or disorder and any T-cell dependent autoimmune disease or disorder, and more specifically: T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, graft versus host disease (GVHD), immune disorders associated with graft transplantation rejection, psoriasis, inflammation, allergy, oophoritis, inflammatory bowel disease, glomerulonephritis, encephalomyelitis, Hashimoto's thyroiditis, Graves' disease, Addison's disease, Crohn's disease, Sjogren's syndrome, lupus erythematosus, primary myxedema, pernicious anemia, autoimmune atrophic gastritis, rheumatoid arthritis, insulin dependent diabetes mellitis, good pasture's syndrome, myasthenia gravis, pemphigus, multiple sclerosis, sympathetic ophthalmia, autoimmune uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, scleroderma, polymyositis, and mixed connective tissue disease.

[00620] The invention provides the use of any of the disclosed CTLA4-Ig or CTLA4^A29YLI04E-Ig molecules in methods for inhibiting T cell proliferation or activation, inhibiting an immune response in a subject or in vitro, and for treating an immune disorder in a subject or inducing immune tolerance to an antigen in a subject. Immune tolerance is a type of immunological response in which there develops a specific nonreactivity of the lymphoid tissues towards a specific antigen, where in the absence of tolerance, the antigen is able to induce an immune response. In one embodiment, the CTLA4-Ig or CTLA4^A29YL104E-Ig molecules and compositions of the invention can be used to treat a subject who has received a transplant in order to induce tolerance, and reduce the possibility of rejection. In another embodiment, the transplant is an organ transplant, a tissue transplant or a cell transplant. In another embodiment, the cell transplant comprises bone marrow cells or islet cells.

[00621] The invention provides the use of any of the disclosed CTLA4-Ig or CTLA4^A29YL104E-Ig molecules in the manufacture of a medicament for treating any of the above stated diseases or disorders. The invention also provides the use of any of the disclosed CTLA4-Ig or CTLA4^A29YL104E-Ig molecules in a coadministration with another agent for the treatment of the above-mentioned diseases or disorders. The CTLA4-Ig or CTLA4^A29YL104E-Ig molecules of the invention can be administered to a subject, for example, intravenously, subcutaneously, and/or by inhalation. CTLA4-Ig or CTLA4^A29YLI04E-Ig formulations applicable for intravenous or subcutaneous administration are described in U.S. Serial No. 60/752,150, filed on December 20, 2005, which is hereby incorporated by reference in its entirety. CTLA4-Ig or CTLA4^A29YL104E-Ig formulations can also include liposome-based formulations wherein the liposomes can deliver CTLA4-Ig or CTLA4^A29YLI04E-Ig molecules to target cells or tissues. CTLA4-Ig or CTLA4^A29YL104E-Ig molecules can also be delivered to target cells or tissues by administration of a virus vector that comprises a CTLA4-Ig or CTLA4^A29YLI04E-Ig gene expression cassette. Administration and dosages of a CTLA4-Ig or CTLA4^A29YL104E-Ig population of molecules are described in U.S. Patent applications published as US20030083246 and US20040022787, as well as the pending U.S. patent application serial number 60/668,774, filed on April 6, 2005, all of which are hereby incorporated by. reference in their entireties.

[00622] The CTLA4-Ig or CTLA4^A29YL104E-Ig molecules described herein may be in a variety of dosage forms which include, but are not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions. The form depends upon the mode of administration and the therapeutic application. An effective mode of administration and dosage regimen for the molecules of the present invention depends upon the severity and course of the disease, the subject's health and response to treatment and the judgment of the treating physician. Accordingly, the dosages of the molecules should be titrated to the individual subject. The interrelationship of dosages for animals of various sizes and species and humans based on mg/m of surface area is described by Freireich, E. J., et al. (Quantitative Comparison of Toxicity of Anticancer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man. Cancer Chemother, Rep., 50, No.4, 219-244, May 1966). Adjustments in the dosage regimen may be made to optimize the growth inhibiting response.

[00623] Doses may be divided and administered on a daily basis or the dose may be reduced proportionally depending upon the situation. For example, several divided doses may be administered daily or monthly or the dose may be proportionally reduced as indicated by the specific therapeutic situation. In one embodiment, the administration is monthly, quarterly, daily, twice a day, about every .10 hours, about every 6 hours, about every 4 hours, about, every 2 hours, about once an hour. In accordance with the practice of the invention an effective amount for treating a subject may be between about 0.1 and about 10 mg/kg body weight of subject. Also, the effective amount may be an amount between about 1 and about 10 mg/kg body weight of subject. The CTLA4-Ig or CTLA4^A29YLI04E-Ig molecules of the invention also have in vivo clinical application. They can be used for the enumeration of B7 positive cells in the diagnosis or prognosis of some conditions of immunodeficiency, the phenotyping of leukemias and lymphomas, and the monitoring of immunological change following organ transplantation.

[00624] The delivery of the compositions described herein may be achieved via injection, oral delivery, inhalation of a spray or other particular dispersion, subcutaneous injection, intravenous delivery, topical delivery, suppository, ocular delivery, nasal or oral delivery. The composition can be delivered via encapsulation in a liposome or other membrane-like delivery vehicle. The composition can be delivered via blood or other fluids that are previously treated with the composition and then subsequently transfused into a subject.

SEQUENCE LISTINGS

[00625] SEQ ID NO: 17 is the nucleotide sequence encoding the pcSDhuCTLA4Ig:

<td> GATCTCCCGA</td><td> TCCCCTATGG</td><td> TCGACTCTCA</td><td> GTACAATCTG</td><td> CTCTGATGCC</td><td> GCATAGTTAA</td>

<td> GCCAGTATCT</td><td> GCTCCCTGCT</td><td> TGTGTGTTGG</td><td> AGGTCGCTGA</td><td> GTAGTGCGCG</td><td> AGCAAAATTT</td>

<td> AAGCTACAAC</td><td> AAGGCAAGGC</td><td> TTGACCGACA</td><td> ATTGCATGAA</td><td> GAATCTGCTT</td><td> AGGGTTAGGC</td>

<td> GTTTTGCGCT</td><td> GCTTCGCGAT</td><td> GTACGGGCCA</td><td> GATATACGCG</td><td> TTGACATTGA</td><td> TTATTGACTA</td>

<td> GTTATTAATA</td><td> GTAATCAATT</td><td> ACGGGGTCAT</td><td> TAGTTCATAG</td><td> CCCATATATG</td><td> GAGTTCCGCG</td>

<td> TTACATAACT</td><td> TACGGTAAAT</td><td> GGCCCGCCTG</td><td> GCTGACCGCC</td><td> CAACGACCCC</td><td> CGCCCATTGA</td>

<td> CGTCAATAAT</td><td> GACGTATGTT</td><td> CCCATAGTAA</td><td> CGCCAATAGG</td><td> GACTTTCCAT</td><td> TGACGTCAAT</td>

<td> GGGTGGACTA</td><td> TTTACGGTAA</td><td> ACTGCCCACT</td><td> TGGCAGTACA</td><td> TCAAGTGTAT</td><td> CATATGCCAA</td>

<td> GTACGCCCCC</td><td> TATTGACGTC</td><td> AATGACGGTA</td><td> AATGGCCCGC</td><td> CTGGCATTAT</td><td> GCCCAGTACA</td>

<td> TGACCTTATG</td><td> GGACTTTCCT</td><td> ACTTGGCAGT</td><td> ACATCTACGT</td><td> ATTAGTCATC</td><td> GCTATTACCA</td>

<td> TGGTGATGCG</td><td> GTTTTGGCAG</td><td> TACATCAATG</td><td> GGCGTGGATA</td><td> GCGGTTTGAC</td><td> TCACGGGGAT</td>

<td> TTCCAAGTCT</td><td> CCACCCCATT</td><td> GACGTCAATG</td><td> GGAGTTTGTT</td><td> TTGGCACCAA</td><td> AATCAACGGG</td>

<td> ACTTTCCAAA</td><td> ATGTCGTAAC</td><td> AACTCCGCCC</td><td> CATTGACGCA</td><td> AATGGGCGGT</td><td> AGGCGTGTAC</td>

<td> GGTGGGAGGT</td><td> CTATATAAGC</td><td> AGAGCTCTCT</td><td> GGCTAACTAG</td><td> AGAACCCACT</td><td> GCTTACTGGC</td>

<td> TTATCGAAAT</td><td> TAATACGACT</td><td> CACTATAGGG</td><td> AGACCCAAGC</td><td> TTGGTACCGA</td><td> GCTCGGATCC</td>

<td> ACTAGTAACG.</td><td> GCCGCCAGTG</td><td> TGCTGGAATT</td><td> CTGCAGATAG</td><td> CTTCACCAAT</td><td> GGGTGTACTG</td>

<td> CTCACACAGA</td><td> GGACGCTGCT</td><td> CAGTCTGGTC</td><td> CTTGCACTCC</td><td> TGTTTCCAAG</td><td> CATGGCGAGC</td>

<td> ATGQ.CAATGC</td><td> ACGTGGCCCA</td><td> GCCTGCTG.TG .</td><td> .GTACTGGCCA</td><td> GCAGCCGAGG</td><td> CATCGCCAGC</td>

<td> TTTGTGTGTG</td><td> AGTATGCATC</td><td> TCCAGGCAAA</td><td> GCCACTGAGG</td><td> TCCGGGTGAC</td><td> AGTGCTTCGG</td>

<td> CAGGCTGACA</td><td> GCCAGGTGAC</td><td> TGAAGTCTGT</td><td> GCGGCAACCT</td><td> ACATGATGGG</td><td> GAATGAGTTG</td>

<td> ACCTTCCTAG</td><td> ATGATTCCAT</td><td> CTGCACGGGC</td><td> ACCTCCAGTG</td><td> GAAATCAAGT</td><td> GAACCTCACT</td>

<td> ATCCAAGGAC</td><td> TGAGGGCCAT</td><td> GGACACGGGA</td><td> CTCTACATCT</td><td> GCAAGGTGGA</td><td> GCTCATGTAC</td>

<td> CCACCGCCAT</td><td> ACTACCTGGG</td><td> CATAGGCAAC</td><td> GGAACCCAGA</td><td> TTTATGTAAT</td><td> TGATCCAGAA</td>

<td> CCGTGCCCAG</td><td> ATTCTGATCA</td><td> GGAGCCCAAA</td><td> TCTTCTGACA</td><td> AAACTCACAC</td><td> ATCCCCACCG</td>

<td> TCCCCAGCAC</td><td> CTGAACTCCT</td><td> GGGGGGATCG</td><td> TCAGTCTTCC</td><td> TCTTCCCCCC</td><td> AAAACCCAAG</td>

<td> GACACCCTCA</td><td> TGATCTCCCG</td><td> GACCCCTGAG</td><td> GTCACATGCG</td><td> TGGTGGTGGA</td><td> CGTGAGCCAC</td>

<td> GAAGACCCTG</td><td> AGGTCAAGTT</td><td> CAACTGGTAC</td><td> GTGGACGGCG</td><td> TGGAGGTGCA</td><td> TAATGCCAAG</td>

<td> ACAAAGCCGC</td><td> GGGAGGAGCA</td><td> GTACAACAGC</td><td> ACGTACCGTG</td><td> TGGTCAGCGT</td><td> CCTCACCGTC</td>

CTGCACCAGG ACTGGCTGAA TGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGCCCTC

CCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA ACCACAGGTG TACACCCTGC CCCCATCCCG GGATGAGCTG ACCAAGAACC AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC TCCCGTGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC GGGTAAATGA GTGCGACGGC CGGCAAGCCC CCGCTCCCCG GGCTCTCGCG GTCGCACGAG GATGCTTCTA GAGGGCCCTA TTCTATAGTG TCACCTAAAT GCTAGAGCTC GCTGATCAGC CTCGACTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGTCATT CTATTCTGGG GGGTGGGGTG GGGCAGGACA GCAAGGGGGA GGATTGGGAA GACAATAGCA GGCATGCTGG GGATGCGGTG GGCTCTATGG CTTCTGAGGC GGAAAGAACC AGCTGGGGCT CTAGGGGGTA TCCCCACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT GTGGTGGTTA CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC GCTTTCTTCC CTTCCTTTCT CGCCACGTTC GCCCTGTGGA ATGTGTGTCA GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA GGCTTTTGCA AAAAGCTTGG ACAGCTGAGG GCTGCGATTT CGCGCCAAAC TTGACGGCAA TCCTAGCGTG AAGGCTGGTA GGATTTTATC CCCGCTGCCA TCATGGTTCG ACCATTGAAC TGCATCGTCG CCGTGTCCCA AGATATGGGG ATTGGCAAGA ACGGAGACCT ACCCTGGCCT CCGCTCAGGA ACGAGTTCAA GTACTTCCAA AGAATGACCA CAACCTCTTC AGTGGAAGGT AAACAGAATC TGGTGATTAT GGGTAGGAAA ACCTGGTTCT CCATTCCTGA GAAGAATCGA CCTTTAAAGG ACAGAATTAA TATAGTTCTC AGTAGAGAAC TCAAAGAACC ACCACGAGGA GCTCATTTTC TTGCCAAAAG TTTGGATGAT GCCTTAAGAC TTATTGAACA ACCGGAATTG GCAAGTAAAG TAGACATGGT TTGGATAGTC GGAGGCAGTT CTGTTTACCA GGAAGCCATG AATCAACCAG GCCACCTCAG ACTCTTTGTG ACAAGGATCA TGCAGGAATT TGAAAGTGAC ACGTTTTTCC CAGAAATTGA TTTGGGGAAA TATAAACTTC TCCCAGAATA CCCAGGCGTC

<td> CTCTCTGAGG TCCAGGAGGA</td><td> AAAAGGCATC</td><td> AAGTATAAGT TTGAAGTCTA CGAGAAGAAA</td>

<td> GACTAACAGG AAGATGCTTT</td><td> CAAGTTCTCT</td><td> GCTCCCCTCC TAAAGCTATG CATTTTTATA</td>

<td> AGACCATGGG ACTTTTGCTG</td><td> GCTTTAGATC</td><td> TTTGTGAAGG AACCTTACTT CTGTGGTGTG</td>

<td> ACATAATTGG ACAAACTACC</td><td> TACAGAGATT</td><td> TAAAGCTCTA AGGTAAATAT AAAATTTTTA</td>

<td> AGTGTATAAT GTGTTAAACT</td><td> ACTGATTCTA</td><td> ATTGTTTGTG TATTTTAGAT TCCAACCTAT</td>

<td> GGAACTGATG AATGGGAGCA</td><td> GTGGTGGAAT</td><td> GCCTTTAATG AGGAAAACCT GTTTTGCTCA</td>

<td> GAAGAAATGC CATCTAGTGA</td><td> TGATGAGGCT</td><td> ACTGCTGACT CTCAACATTC TACTCCTCCA</td>

<td> AAAAAGAAGA GAAAGGTAGA</td><td> AGACCCCAAG</td><td> GACTTTCCTT CAGAATTGCT AAGTTTTTTG</td>

<td> AGTCATGCTG TGTTTAGTAA</td><td> TAGAACTCTT</td><td> GCTTGCTTTG CTATTTACAC CACAAAGGAA</td>

<td> AAAGCTGCAC TGCTATACAA</td><td> GAAAATTATG</td><td> GAAAAATATT CTGTAACCTT TATAAGTAGG</td>

<td> CATAACAGTT ATAATCATAA</td><td> CATACTGTTT</td><td> TTTCTTACTC CACACAGGCA TAGAGTGTCT</td>

<td> GCTATTAATA ACTATGCTCA</td><td> AAAATTGTGT</td><td> ACCTTTAGCT TTTTAATTTG TAAAGGGGTT</td>

<td> AATAAGGAAT ATTTGATGTA</td><td> TAGTGCCTTG</td><td> ACTAGAGATC ATAATCAGCC ATACCACATT</td>

<td> TGTAGAGGTT TTACTTGCTT</td><td> TAAAAAACCT</td><td> CCCACACCTC CCCCTGAACC TGAAACATAA</td>

<td> AATGAATGCA ATTGTTGTTG</td><td> TTAACTTGTT</td><td> TATTGCAGCT TATAATGGTT ACAAATAAAG</td>

<td> CAATAGCATC ACAAATTTCA</td><td> CAAATAAAGC</td><td> ATTTTTTTCA CTGCATTCTA GTTGTGGTTT</td>

<td> GTCCAAACTC ATCAATGTAT</td><td> CTTATCATGT</td><td> CTGGATCGGC TGGATGATCC TCCAGCGCGG</td>

<td> GGATCTCATG CTGGAGTTCT</td><td> TCGCCCACCC</td><td> CAACTTGTTT ATTGCAGCTT ATAATGGTTA</td>

<td> CAAATAAAGC AATAGCATCA</td><td> CAAATTTCAC</td><td> AAATAAAGCA TTTTTTTCAC TGCATTCTAG</td>

<td> TTGTGGTTTG TCCAAACTCA</td><td> TCAATGTATC</td><td> TTATCATGTC TGTATACCGT CGACCTCTAG</td>

<td> CTAGAGCTTG GCGTAATCAT</td><td> GGTCATAGCT</td><td> GTTTCCTGTG TGAAATTGTT ATCCGCTCAC</td>

<td> AATTCCACAC AACATACGAG</td><td> CCGGAAGCAT</td><td> AAAGTGTAAA GCCTGGGGTG CCTAATGAGT</td>

<td> GAGCTAACTC ACATTAATTG</td><td> CGTTGCGCTC</td><td> ACTGCCCGCT TTCCAGTCGG GAAACCTGTC</td>

<td> GTGCCAGCTG CATTAATGAA</td><td> TCGGCCAACG</td><td> CGCGGGGAGA GGCGGTTTGC GTATTGGGCG</td>

<td> CTCTTCCGCT TCCTCGCTCA</td><td> CTGACTCGCT</td><td> GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT</td>

<td> ATCAGCTCAC TCAAAGGCGG</td><td> TAATACGGTT</td><td> ATCCACAGAA TCAGGGGATA ACGCAGGAAA</td>

<td> GAACATGTGA GCAAAAGGCC</td><td> AGCAAAAGGC</td><td> CAGGAACCGT AAAAAGGCCG CGTTGCTGGC</td>

GTTT.TTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG

GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG TCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC CCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT CATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG

ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG

AAAAGTGCCA CCTGACGTCG ACGGATCGGG A

[00626] SEQ ID NO: 18 is the amino acid sequence of the extracellular domain of human CTLA4.

MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATY MMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLG IGNGTQIYVIDPEPCPDSD

[00627] Further Non-limiting embodiments:

[00628] The invention provides for a clonal Chinese Hamster Ovary cell population capable of producing CTLA4-Ig. In one embodiment, the cell population is capable of producing greater than 0.5 or more grams of CTLA4-Ig protein per liter of liquid culture, and wherein the CTLA4-Ig exhibits a molar ratio of sialic acid to CTLA4-Ig dimer is from about 6 to about 14 at a culture scale of 1,000 L or more. In one embodiment, the cell population has been adapted to serum-free, chemically defined medium. In another embodiment, CTLA4-Ig produced from culture of the cell population has an extinction coefficient of 1.00 ± 0.05 AU mL cm'¹ mg’¹. In a further embodiment, the cell population, when grown in culture, is capable of producing CTLA4-Ig polypeptides, wherein: (a) about 90% of the CTLA4-Ig polypeptides comprise an amino acid sequence of SEQ ID NO:2 beginning with the methionine at residue 27; (b) about 10% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 beginning with the alanine at residue number 26; (c) about 4% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 ending with the lysine at residue number 383; (d) about 96% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 ending with the glycine at residue number 382; and optionally, (e) about less than 1% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 beginning with the methionine at residue number 25.

[00629] The invention provides for a progeny cell of the cells described above, wherein the progeny cell produces CTLA4-Ig. In one embodiment, the progeny cell is obtained from culturing a cell over at least 5 generations. In another embodiment, the progeny cell is obtained from culturing a cell over at least 10 generations. In another embodiment, the progeny cell is obtained from culturing a cell over at least 20 generations. In another embodiment,the progeny cell is obtained from culturing a cell over at least 40 generations. In another embodiment, the progeny cell is obtained from culturing a cell over at least 50 generations. In another embodiment, the progeny cell is obtained from culturing a cell over at least 75 generations. In another embodiment, the progeny cell is obtained from culturing a cell over at least 100 generations.

[00630] The invention provides for a cell line produced from any of the cells described above. In one embodiment, the cell line is clonal. In another embodiment, the cell line is capable of producing: (a) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:8 (methionine at amino acid position 27 and glycine at amino acid position 382 of SEQ ID NO:2); (b) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:5 (methionine at amino acid position 27 and lysine at amino acid position 383 ofSEQ ID NO:2); (c) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:7 (alanine at amino acid position 26 and glycine at amino acid position 382 of SEQ ID NO:2); (d) a CTLA4-Ig fusion protein having an amino acid sequence ofSEQ ID NO: 4 (alanine at amino acid position 26 and lysine at amino acid position 383 ofSEQ ID NO:2); (e) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:4 (methionine at amino acid position 25 and lysine at amino acid position 383 ofSEQ ID NO:2); or (f) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:6 (methionine at amino acid position 25 and glycine at amino acid position 382 ofSEQ ID NO:2).

[00631] In another embodiment, the cell line is capable of producing CTLA4-Ig fusion proteins, wherein: (a) about 90% of the CTLA4-Ig polypeptides comprise an amino acid sequence of SEQ ID NO:2 beginning with the methionine at residue 27; (b) about 10% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 beginning with the alanine at residue number 26; (c) about 4% of the CTLA4-Ig polypeptides comprise the amino acid sequence ofSEQ ID NO:2 ending with the lysine at residue number 383; (d) about 96% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2 ending with the glycine at residue number 382; and optionally, (e) about less than 1% of the CTLA4-Ig polypeptides comprise the amino acid sequence ofSEQ ID NO:2 beginning with the methionine at residue number 25.

[00632] In one embodiment, the CTLA4-Ig fusion proteins, which are produced from culturing the cell line, have an extinction coefficient of 1.00 ± 0.05 AU mL cm-1 mg-1. In one embodiment, the invention provides for a cell population derived from a cell of the invention. Tn one embodiment, the cell population consists of at least one additional genetic change as compared to the originally transfected cell and wherein the derived cell population is capable of producing CTLA4-Ig. In other embodiments, the cell population consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24, 25, 26 additional genetic changes as compared to the originally transfected cell and wherein the derived cell population is capable of producing CTLA4-Ig. In one embodiment, the genetic change comprises at least one non-conservative mutation in the cellular genome or in the recombinant expression cassette encoding CTLA4-Ig.

[00633] In one embodiment, the genetic change comprises at least one additional recombinant nucleic acid within the cell. In one embodiment, the change comprises a mutation of the cellular genome. In one embodiment, the change comprises the addition of a nucleic acid to either the cell genome or as a trans nucleic acid, which encodes an antiapoptotic polypeptide. In one embodiment, the anti-apoptotic polypeptide relates to glycosylation.

[00634] In one embodiment, genetic change comprises at least one mutation of the cellular genome or of the recombinant expression cassette encoding CTLA4-Ig. In one embodiment, the cell population, when grown in culture, is capable of producing: (a) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:8 (methionine at amino acid position 27 and glycine at amino acid position 382 of SEQ ID NO:2); (b) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:5 (methionine at amino acid position 27 and lysine at amino acid position 383 of SEQ ID NO:2); (c) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:7 (alanine at amino, acid position 26 and glycine at amino acid position 382 of SEQ ID NO:2); (d) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO: 4 (alanine at amino acid position 26 and lysine at amino acid position 383 of SEQ ID NO:2); (e) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:4 (methionine at amino acid position 25 and lysine at amino acid position 383 of SEQ ID NO:2); or (f) a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:6 (methionine at amino acid position 25 and glycine at amino acid position 382 of SEQ ID NO:2).

[00635] The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of from about 6 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of from about 8 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of from about 11 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of from about 12 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of from about 13 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of from about 14 to about 18. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-lg dimer of from about 15 to about 17. The invention provides for a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of about 16.

[00636] The invention provides for a population of CTLA4-Ig molecules, wherein greater than 95% of the molecules are CTLA4-Ig dimers. In one embodiment, greater than 98% of the molecules are CTLA4-lg dimers. In one embodiment, greater than 99% of the molecules are CTLA4-Ig dimers. In one embodiment, greater than 99.5% of the molecules are CTLA4-Ig dimers. In one embodiment, from about 95% to about 99.5% of the molecules are CTLA4-Ig dimers and about 0.5% to about 5% of the molecules are CTLA4-Ig tetramers. In one embodiment, about 98.6% of the molecules are CTLA4-Ig dimers and about 1.2% of the molecules are CTLA4-Ig tetramers and about less than 0.7 % of the molecules are CTLA4-Ig monomers. The invention provides for a population consisting of CTLA4-Ig dimers. The invention provides for a population of CTLA4-Ig molecules, wherein the population is substantially free of CTLA4-Ig monomer. The invention provides for a population of CTLA4-Ig molecules, wherein the population is substantially free of CTLA4-Ig tetramer. The invention provides for a population of CTLA4-Ig monomer molecules substantially free of CTLA4-Ig dimer and tetramer. In one embodiment, each monomer of each CTLA4-Ig dimer has at least 3 sialic acid groups.

[00637] In one embodiment, each monomer of eachCTLA4-Ig dimer has from at least 3 sialic acid groups to at least 8 sialic acid groups. The invention provides for a purified population of CTLA4-Ig tetramer molecules, the population being substantially free of CTLA4-Ig dimer, and optionally wherein the population comprises an amount that is greater than about 100 grams. The invention provides for a purified population of CTLA4-Ig tetramer molecules, the population being substantially free of CTLA4-Ig monomer, and optionally wherein the population comprises an amount that is greater than about 100 grams. In one embodiment, each tetramer molecule comprises two pairs of CTLA4-Ig polypeptides, wherein each polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOS: 3-8, and wherein each member of the pair of polypeptides is covalently linked to the other member, and wherein the two pairs of polypeptides are non-covalently associated with one another. In one embodiment, each tetramer molecule is capable of binding to a CD80 or CD86. In one embodiment, each tetramer molecule has at least a 2-fold greater avidity for CD80 or CD86 as compared to a CTLA4-Ig dimer molecule. In one embodiment, each tetramer molecule has at least a 2-fold greater inhibition of T cell proliferation or activation as compared to a CTLA4-Ig dimer molecule.

[00638] The invention provides for a composition comprising CTLA4-Ig molecules, wherein the composition comprises dominant isoforms visualizable on an isoelectric focusing gel of CTLA4-Ig which have an isoelectric point, pl, less than or equal to 5.1 as determined by isoelectric focusing. In one embodiment, the pl increases after neuraminidase treatment. In one embodiment, at least 40% of the CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 5.1 as determined by isoelectric focusing. In one embodiment, at least 70% ofthe CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 5.1 as determined by isoelectric focusing. In one embodiment, at least 90% ofthe CTLA4-Ig molecules exhibit an isoelectric point less than or equal to about 2.5 as determined by isoelectric focusing. The invention provides for a population of CTLA4-Ig molecules having a pl of from about 2.0 ± 0.2 to about 5.0 ± 0.2. The invention provides for a population of CTLA4-Ig molecules having a pl from about 4.3 ± 0.2 to about 5.0 + 0.2. The invention provides for a population of CTLA4-Ig molecules having a pl of about 3.3 + 0.2 to about 4.7 ± 0.2. The invention provides for a method for preparing a composition, the composition comprising a CTLA4-Ig molecule with a pl of from about 2.0 ± 0.2 to about 5.0 ± 0.2, the method comprising: (a) subjecting a mixture of CTLA4-Ig molecules to isoelectric focusing gel electrophoresis, wherein a single band on the gel represents a population of CTLA4-Ig molecules with a particular pl, and (b) isolating the population of CTLA4-Ig molecules having a pl of from about 2.0 ± 0.2 to about 5.0 + 0.2 so as to prepare the composition. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4Ig molecules are characterized by an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer of from about 17 to about 25. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer of from about 15 to about 35. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4Ig molecules are characterized by an average molar ratio of GalNAc per mole of CTLA4-Ig dimer of from about 1.7 to about 3.6. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of galcatose per mole of CTLA4-Ig dimer of from about 8 to about 17. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4Ig molecules are characterized by an average molar ratio of fucose per mole of CTLA4-Ig dimer of from about 3.5 to about 8.3. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized by an average molar ratio of mannose per mole of CTLA4-Ig dimer of from about 7.2 to about 22. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA4Ig molecules are characterized by an average molar ratio of sialic acid per mole of CTLA4-Ig dimer of from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer from about 15 to about 35; and (b) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer from about 1.7 to about 3.6; and (c) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-lg dimer from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer from about 1.7 to about 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer from about 8 to about 17; and (d) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer from about 6 to about 12. The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer from about 1.7 to about 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer from about 8 to about 17; (d) an average molar ratio of fucose per mole CTLA4-Ig dimer from about 3.5 to about 8.3; and (e) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer from about 6 to about 12. ׳The invention provides for a composition comprising CTLA4-Ig molecules characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer from about 1.7 to about 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer from about 8 to about 17; (d) an average molar ratio of fucose per mole CTLA4-Ig dimer from about 3.5 to about 8.3; (e) an average molar ratio of mannose per mole CTLA4-Ig dimer from about 7.2 to about 22; and (f) an average molar ratio of sialic acid per mole of CTLA4Ig dimer from about 6 to about 12. \

[00639] The invention provides for a composition comprising CTLA4-Ig molecules, wherein composition exhibits an NGNA chromatogram peak of about 9.589+/-0.3 and an NANA chromatogram peak of about 10.543+/-0.3. The invention provides for a composition comprising CTLA4-Ig molecules, wherein the CTLA-Ig molecules exhibit a carbohydrate profile as shown in Figure 7. The invention provides for a composition comprising CTLA4Ig molecules, wherein the CTLA4-Ig molecules exhibit a carbohydrate profile of Domains I IV, wherein Domain I comprises peaks which represent a-sialylated oligosaccharides, Domain II comprises peaks which represent mono-sialylated oligosaccharides, Domain III comprises peaks which represent di-sialylated oligosaccharides, and Domain IV comprises peaks which represent tri-sialylated oligosaccharides. In one embodiment, the difference in retention times of N-linked oligosaccharides between a first peak in Domain I and a main peak in Domain II is from about 22 to about 28 minutes. The invention provides for a composition comprising CTLA4-Ig dimer molecules, wherein at least 0.5 % of the CTLA4-Ig dimer molecules are cysteinylated. In another embodiment, at least 1.0% of the CTLA4-Ig dimer molecules are cysteinylated. The invention provides for a population of CTLA4-Ig molecules, wherein the population exhibits a mass spectrometry profile as shown in Figures 8 and 10. The invention provides for a population of CTLA4-Ig molecules, wherein the population exhibits a capillary electrophoresis profile as shown in Figures 20 and 21. The invention provides for a composition of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of from about 6 to about 18, wherein the CTLA4-lg dimer is produced from cells of a commercial cell line. The invention provides for a CTLA4Ig composition obtained by any method of the invention. The invention provides for a population of CTLA4-Ig molecules, wherein the molecules are glycosylated at an aparagine amino acid residue at position 102 of SEQ ID NO:2, an aparagine amino acid residue at position 134 of SEQ ID NO:2, an aparagine amino acid residue at position 233 of SEQ ID

NO:2, a serine amino acid residue at position 155 of SEQ ID NO:2, or a serine amino acid residue at position 165 of SEQ ID NO:2. The invention provides for a population of CTLA4-Ig molecules, wherein the population of molecules is characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer from about 1.7 to about 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer from about 8 to about 17; (d) an average molar ratio of fucose per mole CTLA4-Ig dimer from about 3.5 to about 8.3; (e) an average molar ratio of mannose per mole CTLA4-Ig dimer from about 7.2 to about 22 ;(f) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer from about 6 to about 12; (g) a pl as determined from visualization on an isoelectric focusing gel in a range from about

2.4 ± 0.2 to about 5.0 + 0.2; (h) MCP-1 of less than or equal to 5 ppm;(i) less than 2.5 % tetramer; (j) less than 0.5% monomer; (k) CTLA4-Ig polypeptides of the population having an amino acid at least 95% identical to any of SEQ ID NOS: 2-8;(1) wherein CTLA4-Ig molecules within the population is capable of binding to CD80 and CD86. The invention provides for a population of CTLA4-Ig molecules, wherein the population of molecules is characterized by: (a) an average molar ratio of GlcNAc per mole of CTLA4-lg dimer from about 15 to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Ig dimer from about 1.7 to about 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer from about 8 to about 17; (d) an average molar ratio of fucose per mole CTLA4-Ig dimer from about 3.5 to about 8.3; (e) an average molar ratio of mannose per mole CTLA4-Ig dimer from about 7.2 to about 22; (f) an average molar ratio of sialic acid per mole of CTLA4-Ig dimer from about 6 to about 12; (g) a pl as determined from visualization on an isoelectric focusing gel in a range from about 2.4 ± 0.2 to about 5.0 ± 0.2; (h) MCP-1 of less than or equal to 5 ppm; (i) less than 2.5 % tetramer; (j) less than 0.5% monomer; (k) CTLA4-Ig polypeptides of the population having an amino acid at least 95% identical to any of SEQ ID NOS: 2-8; (1) wherein CTLA4-Ig molecules within the population is capable of binding to CD80 and CD86; or pharmaceutical equivalents thereof. The invention provides for a composition comprising an effective amount of the CTLA4-Ig molecules and a pharmaceutically acceptable carrier. The invention provides for a composition comprising an effective amount of the CTLA4-Ig molecules, wherein the composition further comprises an amount of maltose monohydrate. In one embodiment, the composition further comprises a pharmaceutically acceptable diluent, adjuvant or carrier. In one embodiment, the composition further comprises maltose, sodium phosphate monobasic monohydrate, sodium chloride, sodium hydroxide, and sterile water. In one embodiment, the composition further comprises sucrose, poloxamer, sodium phosphate monobasic monohydrate, sodium phosphate dibasic anhydrous, sodium chloride, sodium hydroxide, and sterile water.

[00640] The invention provides for a lyophilized CTLA4-Ig mixture comprising at least 95% CTLA4-Ig dimer, and not more than 5% CTLA4-Ig tetramer. In one embodiment, the mixture comprises at least 98% CTLA4-Ig dimer and no more than 2% CTLA4-Ig tetramer. In one embodiment, the mixture comprises at least 99% CTLA4-Ig dimer and no more than 1% CTLA4-Ig tetramer. In one embodiment, the mixture comprises at least 8.0 moles of sialic acid per mole of CTLA4-Ig dimer. In one embodiment, the mixture comprises from about 15.7 to about 31 moles of GlcNAc per mole of CTLA4-Ig dimer. In one embodiment, the mixture comprises from about 1.6 to about 3.2 moles of GalNAc per mole of CTLA4-Ig dimer. In one embodiment, the mixture comprises from about 9.3 to about 15.5 moles of galactose per .mole of CTLA4-Ig dimer. In one embodiment, the mixture comprises from about 3.6 to about 7.9 moles of fucose per mole of CTLA4-Ig dimer. In one embodiment, the mixture comprises from about 9.7 moles of mannose per mole of CTLA4-Ig dimer. The invention provides for a pharmaceutical kit comprising: (a) a container containing a lyophilized CTLA4-Ig mixture of claim 1; and (b) instructions for reconstituting the lyophilized CTLA4־Ig mixture into solution for injection.

[00641] The invention provides for a method for inhibiting T cell proliferation (or activation), the method comprising contacting a T cell with an effective amount of the CTLA4-Ig composition. The invention provides for a method for inhibiting an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of the composition. The invention provides methods for inducing immune tolerance to an antigen in a subject, treating inflammation in a subject, treating rheumatoid arthritis, treating psoriasis in a subject, treating lupus in a subject, treating or preventing an allergy in a subject, treating or preventing graft vs host disease in a subject, treating or preventing rejection of a transplanted organ in a subject, treating multiple sclerosis in a subject, treating type I diabetes in a subject, treating inflammatory bowel disease in a subject, treating oophoritis in a subject, treating glomerulonephritis in a subject, treating allergic encephalomyelitis in a subject, or treating myasthenia gravis in a subject by administering a composition of the invention in an amount to a subject to treat the disease or disorder. The composition may be combined with a pharmaceutically acceptable carrier. ^,The invention provides for the use of a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-Ig dimer of from about 6 to about 18 in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of an immune disorder. The invention provides for the use of a population of CTLA4-Ig molecules having an average molar ratio of sialic acid groups to CTLA4-lg dimer of from about 6 to about 18 in the manufacture of an anti-rheumatoid arthritis agent in a package together with instructions for its use in the treatment of rheumatoid arthritis. ׳The invention provides for a method for inhibiting T cell proliferation (or activation), the method comprising contacting a T cell with an effective amount of the composition of the invention in combination with methotrexate. The invention provides for a method for inhibiting an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of the composition of the invention in combination with methotrexate. The invention provides for a method for inducing immune tolerance to an antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of the composition of any of claims 1-64 in combination with methotrexate. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L, wherein the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the at least 10,000 L liquid culture. In one embodiment, the expanding of step (a) comprises: (i) culturing the cells in a serum-free, chemically defined medium with at least four passages so as to obtain a cell density of at least about 1.0 x 10⁵ viable cells per mL, wherein each seed stage starts at about 2 x 10⁵ per ml and goes to 1-2 mil cells per ml; (ii) maintaining the cells in culture for a time sufficient to produce from the culture at least about 0.5 g / L. In one embodiment, the protein is a glycoprotein. In one embodiment, the protein is a CTLA4-Ig protein. In one _r embodiment, the mammalian cells are progeny cells. In one embodiment, the mammalian cells are progeny of a CHO clonal cell line capable of producing CTLA4-Ig fusion protein, wherein the CHO cells have stably integrated in their genome at least 30 copies of a CTLA4Ig expression cassette. In one embodiment, the time sufficient is a time by which the cells’ viability does not fall below 30%. In one embodiment, the time sufficient is a time by which the cells’ viability does not fall below 40%. In one embodiment, the time sufficient is a time by which the cells’ viability does not fall below 50%. In one embodiment, the time sufficient is a time by which the cells’ viability does not fall below 60%. In one embodiment, the time sufficient is a time by which the cells’ viability does not fall below 70%, or 80% or 90% or

95%. In one embodiment, the at least four passages comprises: (i) growing the cells in a culture volume of at least 50 mL until a cell density of from about 1 million to about 2.5 mill cels per ml is reached, (ii) growing the cells in a culture volume of at least 10 L until a cell density of about 1 million to about 2.5 million cels per ml is reached; (iii) growing the cells in a culture volume of at least 100 L until a cell density of about 1 million to about 2.5 million cels per ml is reached; and (iv) growing the cells in a culture volume of200 L until a cell density of about 1 million to about 2.5 million cels per ml is reached. In one embodiment, galactose is added to the serum-free, chemically defined medium. In one embodiment, the maintaining comprises (i) lowering the temperature of the culture from 37±2°C to 34±2°C; and (ii) lowering the temperature of the culture from 34±2°C to 32±2°C. In one embodiment, the temperature is kept within the range of 32±2°C for at least 5 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 6 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 7 days. In one embodiment, the temperature is kept within the range of 32+2°C for at least 8 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 9 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 10 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 11 days. In one embodiment, the temperature is kept within the range of32±2°C for at least 12 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 13 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 14 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 15 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 16 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 17 days. In one embodiment, the temperature is kept within the range of 32±2°C for at least 18.days. In one embodiment, the temperature is kept within the range of 32±2°C for up to 18 days. In one embodiment, the temperature is kept within the range of 32±2°C until the cell density of the culture is from about 30 x 10⁵ to about 79 x 10⁵ cells per mL of liquid culture. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000 L so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the at least 10,000 L liquid culture, wherein the isolating occurs only when the liquid culture contains greater than or equal to about 6.0 moles of NANA per mole of protein. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000 L so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the at least 10,000 L liquid culture, wherein the isolating occurs only when the liquid culture has a cell density of from about 33 x 10⁵ to about 79 x 10⁵ cells per mL.

[00642] The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000 L so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the at least 10,000 L liquid culture, wherein the isolating occurs when cell viability in the liquid culture has not fallen below about 20%, or about 30%, or about 38%. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000 L so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the at least 10,000 L liquid culture, wherein the isolating occurs only when endotoxin is less than or equal to about 76.8 EU per mL of liquid culture. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000 L so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the at least 10,000 L liquid culture, wherein the isolating occurs only when bioburden is less than 1 colony forming unit per mL of liquid culture. The invention provides for a method for producing a recombinant protein, the method comprising: (a) expanding mammalian cells that secrete a recombinant protein from a seed culture to a liquid culture of at least 10,000 L so that the recombinant protein concentration is at least 0.5 grams / L of liquid culture; and (b) isolating the recombinant protein from the at least 10,000 L liquid culture, wherein the isolating occurs only if at least two of the following conditions are met: (i) the liquid culture contains greater than or equal to about 6.0 moles of NANA per mole of protein, (ii) the liquid culture has a cell density of from about 33 x 10^s to about 79 x 10^s cells per mL, (iii) cell viability in the liquid culture has not fallen below about 20%, or about 38%, or (iv) amount of CTLA4-Ig in the culture is greater than 0.5 g / L. In one embodiment, the isolating comprises: (i) obtaining a cell culture supematent; (ii) subjecting the supernatant to anion exchange chromotagraphy to obtain an eluted protein product; (iii) subjecting the eluted protein product of step (ii) to hydrophobic interaction chromatography so as to obtain an enriched protein product; (iv) subjecting the enriched protein product to affinity chromatography to obtain an eluted and enriched protein product; and (v) subjecting the eluted and enriched protein product of (iv) to anion exchange chromatography. In one embodiment, the enriched protein product obtained in step (iii) is characterized in that a percentage of any high molecular weight multimer is less than 25% by weight. In one embodiment, the anion exchange chromatography of step (ii) is carried out using a wash buffer comprising about 75 mM HEPES, and about 360 mM NaCI, and having a pH of about 8.0. In one embodiment, the anion exchange chromatography of step (ii) is carried out using an elution buffer comprising about 25 mM HEPES, and about 325 mM NaCI, and having a pH of about 7.0. In one embodiment, the hydrophobic interaction chromatography of step (iii) is carried out using a single wash buffer comprising about 25 mM HEPES, and about 850 mM NaCI, and having a pH of about 7.0. In one embodiment, the affinity chromatography of step (iv) is carried out using a wash buffer comprising about 25 mM Tris, and about 250 mM NaCI, and having a pH of about 8.0. In one embodiment, the affinity chromatography of step (iv) is carried out using an elution buffer comprising about 100 mM Glycine and having a pH of about 3.5. In one embodiment, the anion exchange chromatography of step (v) is carried out using a wash buffer comprising about 25 mM HEPES, and from about 120 mM NaCI to about 130 mM NaCI, and having a pH of about 8.0. In one embodiment, the anion exchange chromatography of step (v) is carried out using an elution buffer comprising about 25 mM HEPES, and about 200 mM NaCI, and having a pH of about 8.0. In one embodiment, the anion exchange chromatography of step (ii) is carried out using a column having an anion exchange resin having a primary, secondary, tertiary, or quartenary amine functional group. In one embodiment, the resin has a quartenary amine functional group. In one embodiment, the hydrophobic interaction chromatography of step (iii) is carried out using a hydrophobic interaction resin having a phenyl, an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group. In one embodiment, the functional group is a phenyl functional group. In one embodiment, the affinity chromatography of step (iv) is carried out using a column containing Protein A. In one embodiment, method for preparing CTLA4-Ig, the method comprising purifying CTLA4-Ig from a liquid cell culture so that the purified CTLA4-Ig (a) has about 38 ng of MCP-1 per mg of CTLA4־Ig dimer, and (b) comprises less than 2.5% of CTLA4-Ig tetramer by weight.

In one embodiment, the liquid cell culture comprises a cell of or a progeny cell of the invention. The invention provides a method for producing CTLA4-Ig, the method comprising: (a) expanding progeny cells of any of commercial cell line or CHO cells that are capable of producing CTLA4-Ig, wherein the expanding is from a seed culture to a liquid culture of at least 10,000 L, wherein the CTLA4-Ig concentration is at least 0.5 grams / L of liquid culture; and (b) isolating CTLA4-Ig from the at least 10,000 L liquid culture, wherein the chromotagraphy is on a column with hydrophobic interaction resin with at least a phenyl functional group, wherein the isolating comprises a step of hydrophobic interaction chromatography carried out using a single wash buffer comprising about 25 mM HEPES, and about 850 mM NaCl, and having a pH of about 7.0.

EXAMPLES OF THE INVENTION

[00643] A number of Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate the exemplary modes of making and practicing the present invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods may be utilized to obtain similar results.

[00644] The following Examples refer to CTLA4-Ig molecules that comprise sequences of one or more of SEQ ID NOS: 2, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or 16. These Examples are not meant to be limiting, and one skilled in the art understands that the Examples can be expanded and adapted to, for example, other CTLA4-Ig molecules, other glycoproteins, and other proteins related to or comprising portions of an Ig-superfamily protein.

[00645] The following table sets out examples that relate to CTLA4-Ig and to CTLA4^A29YL104E-Ig.

<td> CTLA44g Protisin Characteristics</td><td> :Exemplary CTLA4-ig Protein No. iK-&lt;dTLA4־Ig having SEQ: ID NO: 1, 2, 5, 6,7, 8, 9, o^r 10</td><td> Exemplary⁷ CTLA4-Ig:Protein . No, 2 - CTLA4^A'^29YL,04E-Ig having SEQ IDNQ: 3 or 4 or 1116.:</td>

<td> Binding to B7-1; on / off rates; potency, valency</td><td> Example 6</td><td></td>

<td> Bioburden</td><td> Example 49</td><td></td>

<td> Capillary Electrophoresis</td><td></td><td> Example 38</td>

<td> Carbohydrate content, N-linked HPEAC profile, Domains</td><td> Example 3 Example 44</td><td> Example 22 Example 37</td>

<td> Cell line transfection</td><td> Example 12</td><td> Example 23</td>

<td> CHO DNA</td><td> Example 58</td><td> Example 55</td>

<td> CHO Host cell protein</td><td> Example 60</td><td> Example 52</td>

<td> Genetic Characterization</td><td></td><td> Example 24</td>

<td> Disaggregation</td><td> Example 5 Example 33</td><td></td>

<td> Endotoxin</td><td> Example 48</td><td> Example 48</td>

<td> Final fill</td><td> Example 30</td><td></td>

<td> Formulations</td><td> Example 2</td><td> Example 27</td>

<td> GalNAc, GlcNAc molar ratios</td><td> Example 17, Example 63</td><td> Example 36</td>

<td> CTLA4-IgProtein Characteristics</td><td> . Exemplary CTLA4-Ig Protein .FJo/i^/CTLA^Ig having '.§EQ ΪΕ) NO: 1,2, .5,. 6, 7, 8, 9, ::pr'lO;.. . ׳</td><td> Exemplary CTLA4-Ig Protein haying!SEQ ID NO: 3 or 4 or 11- • :16.:' 'L /.</td>

<td> IEF</td><td> Example 50</td><td> Example 22</td>

<td> IL-2 bioassay</td><td> Example 45</td><td> Example 40</td>

<td> Immunogenicity</td><td> Example 31</td><td></td>

<td> Single dose healthy PK</td><td> Example 66</td><td></td>

<td> MALDI-TOF</td><td> Example 8</td><td></td>

<td> Mannose, fucose, galactose molar ratios</td><td> Example 18 Example 64</td><td> Example 35</td>

<td> Mass Spec</td><td> Example 7</td><td></td>

<td> MCP-1</td><td> Example 59</td><td> Example 54</td>

<td> Media / culturing</td><td> Example 9</td><td></td>

<td> Monkey PK</td><td> Example 32</td><td></td>

<td> Monomer</td><td> Example 4</td><td></td>

<td> NANA, NGNA molar ratios</td><td> Example 3, Example 16</td><td> Example 39</td>

<td> O-linked</td><td> Example 46</td><td></td>

<td> Oxidation and Deamidation</td><td> Example 47</td><td></td>

<td> PK Correlations</td><td> Example 42</td><td></td>

<td> Plasmid</td><td> Example !,Example 11, Example 67</td><td></td>

<td> Production</td><td> Example 14 Example 28</td><td> Example 19</td>

<td> Protein A</td><td> Example 62</td><td> Example 53</td>

<td> Purification</td><td> Example 15 Example 29</td><td> Example 20</td>

<td> Multiple Dose RA PK</td><td> Example 34</td><td></td>

<td> SDS-PAGE</td><td> Example 51</td><td> Example 26 Example 56</td>

<td> SEC - HMW, dimer, monomer, size homogeneity</td><td> Example 10</td><td> Example 25</td>

<td> SPR - binding to B7.1 (BIAcore)</td><td> Example 21</td><td> Example 41</td>

<td> Sub-cloning of cell lines</td><td> Example 13</td><td> Example 24</td>

<td> Triton-X 100</td><td> Example 61</td><td> Example 57</td>

<td> Trypic mappings</td><td> Example 3, Example 47, Example 65</td><td> Example 22</td>

[00646] Abbreviations:

15N 15 Nanometer

A280 Absorbance at 280 nm

CTLA4-Ig Cytotoxic T-Lymphocyte Antigen-4 Immunoglobulin; CTLA-4 Ig

API Active Pharmaceutical Ingredient

AU Absorbance Units

B7 CTLA-4 Receptor Ligand cfu Colony Forming Unit

CHO Chinese Hamster Ovary

CHOP Chinese Hamster Ovary Host Cell Proteins

CV Column Volume

Drug Substance Fill Drug Substance ConcentrationZDiafiltration and Fill Step

ELISA Enzyme Linked Immunosorbent Assay

EU Endotoxin Units

Fc The constant region of antibodies

GalNAc N-Acetyl-galactosamine

GlcNAc N-Acetyl-glucos amine

HEPES 4-(2-Hydroxyethyl)piperazine-l-ethanesulfonic Acid

HIC Hydrophobic Interaction Chromatography; Phenyl Sepharose™ Fast

Flow

HMW High Molecular Weight

HPLC High Performance Liquid Chromatography

IgGl Immunoglobulin in Class G1

IPC In-Process Control

LAL Limulus Amebocyte Lysate

MBR(s) Master Batch Record(s)

MCP-1 Monocyte Chemotactic Protein 1

MTX Methotrexate

MW Molecular Weight

N/A Not Applicable

NANA N-acetylneuraminic Acid; Sialic Acid; SA

NMWC Nominal Molecular Weight Cutoff

OD Optical Density

PAR Proven Acceptable Range

Planova Viral removal filters, pore size 15 nm

PP(s) Process Parameter(s)

PQ Performance Qualification psi Pounds per Square Inch psid Pounds per Square Inch, Differential psig Pounds per Square Inch; Gauge

QFF Q Sepharose™ Fast Flow

QXL Q Sepharose Extreme Load rPA Recombinant Protein A Sepharose Fast Flow

SA Sialic Acid; N-acetylneuraminic Acid; NANA

SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis

SOP Standard Operating Procedure

Tris Tris (Hydroxymethyl) Aminomethane

Triton X-100 t-Octylphenoxypolyethoxyethanol; Polyethylene glycol tert-octylphenyl ether

UF Ultrafiltration

UV Ultraviolet v/v Volume per Volume

VF Viral Filtration; Nanometer Filtration

VI Viral Inactivation

[00647]

Example 1: Confirmation of the CTLA4-Ig Coding Sequence in Plasmid pcSDhuCTLA4-Ig

[00648] The annotated nucleic acid sequence of the CTLA4-Ig gene present in pcSDhuCTLA4-lg and the corresponding deduced amino acid sequence of CTLA4-Ig are shown in FIG. 1. The pcSDhuCTLA4-Ig nucleic acid was transfected into CHO cells in order to generate stable transfectants that could express CTLA4-Ig molecules (see Example 12). The transfectants were screened and certain transfectants were subcloned or expanded to generate clonal cell lines.

[00649] Analysis of the DNA sequence data confirmed that the junctions created during the plasmid construction were as designed and that the synthetic oligonucleotide primers used in the polymerase chain reaction generated the correct oncostatin M signal sequence upstream of the CTLA4-Ig sequence. The desired cysteine to serine changes (at positions 156, 162 and 165 of SEQ ID NO:2) in the hinge region of the fusion protein were confirmed. ^,These amino acid residues are designated in bold with an asterisk in FIG. 1. An additional amino acid change of proline to serine at position 174 of SEQ ID NO:2 was also detected. This change was introduced during the IgG! cDNA synthesis by the polymerase chain reaction. This amino acid residue is also designated in bold with an asterisk in FIG. 1.

]00650] The analysis of the DNA sequence data identified one additional difference when compared to the published nucleotide sequences of the human CTLA4 and human IgG!

constant region. The codon at amino acid 110 of the CTLA4 coding region was identified as ACC (threonine) rather than GCC (alanine).

Example 2; CTLA4־Ig Formulation

[00651] CTLA4-Ig composition for Injection, 250 mg/vial, is a sterile, non-pyrogenic lyophile for intravenous (IV) administration. The population of CTLA4-Ig molecules is packaged in 15-cc Type I flint tubing glass vials. Each vial is stoppered with a 20-mm

Daikyo gray butyl D-21-7-S/B2-TR fluoro-resin coated stopper and sealed with a 20-mm aluminum, white, flip-off seal.

[00652] Each single-use vial contains 250 mg of CTLA4-Ig composition which is constituted with Sterile Water for Injection, USP and further diluted with 0.9% Sodium Chloride Injection, USP, at the time of use. The composition of CTLA4-Ig for Injection, 250 mg/vial, and the function of each component is listed in the Table below.

Table 8: Composition of CTLA4-Ig

<td> Component</td><td> Function</td><td> mg per Vial</td>

<td> CTLA4-Ig</td><td> Active Ingredient</td><td> 262.5</td>

<td> Maltose Monohydrate</td><td> Bulking Agent/ Stabilizer</td><td> 525</td>

<td> Sodium Phosphate, Monobasic, _ , D Monohydrate .</td><td> Buffering Agent</td><td> 18.1</td>

<td> Sodium Chloride*⁵</td><td> Ionic Strength Adjustment</td><td> 15.3</td>

<td> Hydrochloric Acid Sodium Hydroxide</td><td> pH Adjustment pH Adjustment</td><td> adjust pH to ~ 7.5</td>

<td colspan="3"> These components are present in the CTLA4-Ig composition solution</td>

[00653] CTLA4-Ig composition contains approximately 50 mg/mL CTLA4-Ig in 25 mM sodium phosphate buffer and 50 mM sodium chloride at pH 7.5. During early development, this buffer system was selected based on the evaluation of the physical and chemical stability of CTLA4-Ig as a function of pH, buffer type, buffer concentration and sodium chloride concentration. The stability of CTLA4-Ig solutions was investigated in the pH range of 5 to 9. The results indicated that the formation of high molecular weight species was pH dependent and the pH range of maximum stability was between 7 and 8. In a separate determination, the effect of buffer type and concentration was evaluated, where the CTLA4Ig composition was found to be equally stable in sodium phosphate or tris buffer at pH 8. Additionally, the buffer concentrations between 10 to 100 mM concentration did not have any impact on the stability of the CTLA4-Ig composition at 2°-8°C. Similarly, the presence of sodium chloride between 30 to 500 mM concentration had no effect on the solution-state stability of the CTLA4-Ig composition stored at 2°-8°C.

[00654] Based on these results, the CTLA4-Ig composition at 10 mg/mL in 25 mM sodium phosphate buffer and 50 mM sodium chloride at pH 7.5 was selected for formulation at 50 mg/vial strength. The CTLA4-Ig concentration was later changed to 50 mg/mL in the same buffer composition to allow for the development of the CTLA4-Ig compositions with 200and 250 mg/vial strengths.

[00655] CTLA4-Ig for injection is formulated with maltose in addition to sodium phosphate buffer and sodium chloride. The function of excipients used in this product is listed in the Table above.

[00656] The presence of inorganic salts, such as sodium chloride and sodium phosphate buffer components reduce the glass transition temperature (Tg’) of the frozen system. Moreover, dibasic sodium phosphate, which is formed in situ at pH 7.5 undergos preferential crystallization during freezing, which reduces the micro-environmental pH of the frozen solution. Based on these reasons, the minimum amounts of sodium chloride and sodium phosphate buffer were selected to minimize their impact on the lyophilization process.

Maltose is added as a stabilizer which acts as a cryo- and lyo-protectant during lyophilization and upon subsequent storage of the drug product, respectively. CTLA4-Ig composition for Injection, 250 mg/vial, is a sterile, single use vial without antimicrobial preservatives.

Example 3; Carbohydrate Content Analysis of a CTLA4-I״ composition

[00657] The purpose of the method is to provide chromatographic profiles of CTLA4-Ig N-linked oligosaccharides. This procedure can be used to obtain the molar ratio of N-Acetyl Neuraminic Acid (NANA) and N-Glycolyl Neuraminic Acid (NGNA) to protein in CTLA4Ig samples (total bound plus free). NANA and NGNA are two forms of sialic acid. The glycosylation on the CTLA4-Ig protein contains N-linked oligosaccharides. For example, these oligosaccharides can be liberated by enzymatic hydrolysis with PNGase F over the course of 22 hours. The free oligosaccharides are profiled using high pH anion exchange chromatography employing electrochemical detection. Carbohydrate profiles for the Nlinked oligosaccharides were evaluated using High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). These profiles were confirmed and structural information was obtained using Porous Graphitized Carbon (PGC) chromatography coupled with MS. Both techniques and results are described in this section. This procedure is exemplary for CTLA4-Ig of SEQ ID NO:

[00658] N־Linked Oligosaccharide Isolation: Cleavage of asparagine-linked (N-linked) oligosaccharides from CTLA4-Ig was performed by enzymatic hydrolysis using PNGase F. To perform the deglycosylation, CTLA4-Ig was first reduced and denatured: 1-2 mg of CTLA4-Igin 176 μΕ of 5mM sodium phosphate buffer containing 0.5% SDS and 1% beta-mercaptoethanol was heated at 100° C for 2 minutes, then allowed to cool to ambient temperature. To the cooled mixture, 16 μΕ of 10% NP-40 were added. The sample was mixed well and 40 μΕ of PNGase F (50,000 U/mL, in 50 mM sodium phosphate buffer) were added. The sample was mixed well and incubated at 38° C for 24 hrs. The enzymatically-released oligosaccharides (glycans) were purified by reversed phase high performance liquid chromatography (HPLC) using a Phenomenex Luna Cl 8 column (4.6 x 150 mm; 5 μΕ) coupled with a Thermo HyperCarb column (4.6 x 100 mm; 5 μΕ,) at a flow rate of 1.0 mL/minute; the chromatograph used for the isolation was a Waters Alliance 2695 equipped with a Waters 2996 detector. The columns were first equilibrated with 0.05% triflouroacetic acid (TFA). After injection of a sample, a gradient of acetonitrile was initiated, terminating at 15 minutes with a solvent composition of 0.05% TFA in 12% acetonitrile. The glycans were then eluted from the HyperCarb column with a step gradient to 0.05% TFA in 60% acetonitrile. The glycans were collected while monitoring by UV absorbance at 206 nm and were concentrated to dryness under vacuum. Prior to subsequent injections the Luna Cl 8 column was cleaned with 0.05% TFA in 40% acetonitrile, 40% isopropanol, 20% water.

[00659] Preparation of NANA Stock Solution (N-Acetyl Neuraminic Acid) (1 mg/mL). Important: Prior to weighing, allow the NANA standard to warm to room temperature. Failure to do so will result in water condensation in the standard. Open only long enough to obtain desired amount, then reseal bottle tightly and return to freezer, storing with desiccant. Accurately weigh between 3 and 10 mg of the N-Acetyl Neuraminic Acid. Record to the nearest 0.1 mg. Transfer to an appropriate size container if weighing is done using weighing paper/boat. Add sufficient amount of HPLC grade water to yield a concentration of Img/mL solution. Mix with a stirring bar or by vortexing until dissolved. Store between 2 and 8°C for up to 3 months in a polypropylene tube.

[00660] Preparation of NGNA Stock Solution (N-Glycolyl Neuraminic Acid) (1 mg/mL) Important: Prior to weighing, allow the NGNA standard to warm to room temperature. Failure to do so will result in water condensation in stock. Open only long enough to obtain desired amount then reseal bottle tightly and return to freezer, storing with desiccant. Accurately weigh between 3 and 10 mg (record to the nearest 0.1 mg of Nglycolyl Neuraminic Acid. Record to the nearest 0.1 mg. Transfer to an appropriate size container if weighing is done using weighing paper/boat. Add an appropriate volume of HPLC grade water to yield a target concentration of Img/mL solution. Mix with a stirring bar or by vortexing until dissolved. Store between 2 and 8°C for up to 3 months in a polypropylene tube.

(00661] System Suitability Solution. Add 0.050 mL each of 1 mg/mL of NANA and NGNA stock solutions to 0.900 mL HPLC grade water in an appropriate container. Mix by vortexing. Store between 2 and 8°C for up to 3 months. N-Acetyl Neuraminic Acid Working Solution (0.050 mg/mL). Accurately measure 0.050 mL of 1 mg/mL NANA stock solution and add to 0.950 mL of HPLC grade water. Mix by vortexing. Prepare in duplicate at the time of use. N-Glycolyl Neuraminic Acid Working Solution (0.050 mg/mL). Accurately measure 0.050 mL of 1 mg/mL NGNA stock solution and add to 0.950 mL of HPLC grade water. Mix by vortexing. Prepare in duplicate at the time of use.

[00662] Preparation of Samples and Hydrolysis Blank. Obtain the protein concentration for CTLA4-Ig material from the Certificate of Analysis (COA). Prepare a single sample of Hydrolysis Blank by adding 0.190 mL of HPLC grade water to 1.5 mL micro centrifuge tube. Prepare two 1 mg/mL solutions of samples and CTLA4-Ig Reference Material using HPLC grade water. Mix by vortexing.

[00663] Hydrolysis of Samples, CTLA4-Ig Reference Material, and Hydrolysis Blank. Perform the hydrolysis on duplicate preparations of reference material and samples in 1.5 mL micro centrifuge tubes. Note: It is important to use micro centrifuge tubes which will fit completely into the heating block. Add 0.010 mL of 1 M H2SO4 to 0.190 mL of 1 mg/mL dilutions of samples and CTLA4-Ig reference material, and hydrolysis blank. Mix by vortexing and secure lid by lid-lock or tape. Place the micro centrifuge tubes in 80°C ± 2°C heating block for 1 hour ± 2 min. After incubation, remove tubes from heating block, place tubes in micro centrifuge and spin to force sample to the bottom of the tube. Aliquot hydrolyzed solutions into autosample vials and place in autosampler for injection.

[00664] Instrument Conditions. Prepare the High Performance Liquid Chromatography (HPLC) system. Set up the following conditions. Equilibrate the column for at least one hour at the flow rate of 0.6 mL/min and a temperature of 40°C.

<td> Mobile Phase(s)</td><td> A: 5 mM. H₂SO₄ B: HPLC Grade Water (Column Wash)</td>

<td> Flow Rate</td><td> 0.6 mL/min</td>

<td> Run Time</td><td> 25 minutes</td>

<td> Detector Wavelength</td><td> 206 nm</td>

<td> Column Temperature</td><td> 40°C</td>

<td> Aurosampler Temperature Injection Volume</td><td> 4°C 5 pL</td>

<td> Retention Times NG NA NANA</td><td> 9.8 ± 1 minutes (system dependent.) 10:8+ 1 minutes (system dependent)</td>

<td> Gradient conditions</td><td> Isocratic</td>

[00665] System Suitability. Start analysis with an injection of mobile phase as the Instrument Blank to evaluate system baseline, which should be flat and stable. If the baseline is not flat and stable, additional blank injections should be made. Note: A shift of the baseline should not exceed 0.25 AU. Perform six replicate injections of system suitability solution. Calculate Resolution (R) and Theoretical Plates (N).

Using the first system suitability injection, calculate the number of theoretical plates (N) using the following equation:

־)

Number of Theoretical Plates aN=16

Where:

N — Theoretical Plate Count t = Retention time of NANA peak, in minutes

W = NANA Peak width at baseline, in minutes

[00666] Calculate the moles of NANA and NGNA in the CTLA4-Ig Reference Material and samples. NANA and NGNA standards are injected at the beginning and end of each run. Average the area counts for the replicate injections of NANA and NGNA. Use this area in the following equation:

moles of NANA or NGNA in abatacepr reference materia] or sample = (X)(Y) (Z)

X=^-Moles of NANA or NGNA in Working Solutions calculated in Section

7.3.1

Y= Ansa counts of NANA or NGNA. in abatacept reference material or sample for each preparation (Note: see Section 7.2 and Figure 3 for peaks to integrate for NANA)

Z= Averaged area of the replicate NANA or NGNA in Working Solutions

[00667] In one embodiment, the resolution between the NGNA and NANA peaks must be >1.3. The theoretical plate count must be > or = to 4000. The system suitability injection reproducibility evaluated as %RSD of the area counts of NANA peak must be < 3%. The theoretical plate count must be > 4000. The system suitability injection reproducibility evaluated as %RSD of the area counts of NANA peak must be < 3%.

[00668] N-Linked Oligosaccharide Profiling by High pH Anion Exchange

Chromatography with Pulsed Amperometric Detection (HPAEC-PAD־): HPAEC of isolated oligosaccharides was performed on a chromatography system consisting of a Waters Alliance equipped with a Waters 464 electrochemical detector utilizing a Dionex CarboPack PAI (4 x 250 mm) anion exchange column and a Dionex CarboPack guard column. The oligosaccharide samples were eluted using a sodium acetate gradient in 200 mM sodium hydroxide (increasing sodium acetate concentration from 0 mM at the time of injection to 225 mM at 60 minutes). The electrochemical detector was run under pulse mode with pulse potentials El = 0.05V (tl = 0.4 sec), S = 0.75V (t₂ = 0.2 sec), E3 = -0.15V (t3 = 0.4 sec). The detection cell was composed of a gold working electrode, a stainless steel counter electrode and a silver/silver chloride reference electrode.

[00669] This method describes the procedure to determine the HPAEC (High pH Anion Exchange Chromatography) oligosaccharide profile of N-linked oligosaccharides released from protein in CTLA4-Ig samples. The purpose of the method is to provide chromatographic profiles of CTLA4-Ig, such as CTLA4-Ig drug substance N-linked oligosaccharides which can be used for comparative analysis between different compositions of CTLA4-Ig molecules. The glycosylation on the CTLA4-Ig protein contains N-linked oligosaccharides. These oligosaccharides are liberated by enzymatic hydrolysis with PNGase F (Peptide: N-Glycosidase F) over the course of 22 hours. The free oligosaccharides are profiled using high pH anion exchange chromatography employing electrochemical detection.

CTLA4-Ig Bulk Drug

Substance

Waters Total Recovery Vials with bonded PTFE/silicone septa

RapiGest SF

Alliance HPLC system equipped with: Autosampler (refrigerated), Eluent Degas Module

Model 2465 Electrochemical

Detector

Column: CarboPac PA-1 4 x 250 mm

Guard Column: CarboPac PA-1 4 x 50 mm

CTLA4-Ig in 25 mM Sodium Phosphate, 10 mM NaCl, pH = 7.5

Waters Corporation, Catalog No. 186000234

Waters Corporation, Catalog No.

186001861

Waters Corporation

Dionex Corporation, Catalog No. 35391

Dionex Corporation, Catalog

No. 43096

Empower Data Collection system

[00670] Oligosaccharide profiles of drug substance are evaluated against concurrently run samples of reference material. Results are reported as percent deviation of selected domains and peaks from the same peaks in the reference standards.

[00671] Chromatography Conditions for Oligosaccharide Profile by Anion-Exchange Chromatography

<td> Column Temperature</td><td> 29 °C</td>

<td> Flow Rate</td><td> 1 mL/min</td>

<td> Mobile Phases and Gradient</td><td> Gradient Program</td>

<td> Conditions</td><td></td>

<td> 1: 500mMNaOAc</td><td> Time</td>

<td> 2: 400mMNaOH</td><td> (mini %1 %2 %3</td>

<td> 3: HPLC Grade Water</td><td> Initial 0 30 70</td>

<td></td><td> 0.0 0 30 70</td>

<td></td><td> 11.0 0 30 70</td>

<td></td><td> 12.0 4 30 66</td>

<td></td><td> 20.0 10 30 60</td>

<td></td><td> 80.0 45 30 25</td>

<td></td><td> 81.0 0 30 70</td>

<td></td><td> 100 0 30 70</td>

<td> Waters 2465 settings</td><td></td>

<td> Mode</td><td> Pulse</td>

<td> Empower settings</td><td> Range=5pA</td>

<td></td><td> El= +0.05V E2= +0.75V E3= -0.15V</td>

<td></td><td> 11=400 msec t2=200 msec t3=400</td>

<td></td><td> msec</td>

<td></td><td> Sampling time(ts)=100 msec</td>

<td></td><td> Time constant(f11ter)t=0.1sec</td>

<td></td><td> Range offset=5%</td>

<td></td><td> Polarity +</td>

<td></td><td> Temperature= 29 °C</td>

NOTE; Equilibrate the column and detector with the initial mobile phase at the analysis flow rate for approximately 2 hours, or until baseline is stable before making injections.

Autosampler Temperature set to:

°C >

Injection Volume 60 gL

Run Time 100 minutes

Approximate Retention Times (RT;

minutes) of dominant peaks in each Domain (see Figure 1); values may vary depending on RT of System Suitability (SS) Standard

Approximate RTs (min)

SS:18.5

Peak 1A:20.0

Peak IB:20.8

Peak 1C:21.4

Peak ID:22.4

Peak IE:23.1

Peak 231.5

Peak 3:44.8

Peak 4:58.5

[00672] Preparation of Mobile Phases for HP AEG Oligosaccharide Carbohydrate Profiling

[00673] HPAEC Eluent 1: 500 mM Sodium Acetate (NaOAc). Weigh 20.51 ± 0.05 g of Sodium Acetate (anhydrous) into a 500 mL graduated cylinder containing 400 mL of HPLC grade water. Bring volume to 500 mL with HPLC grade water and stir for 5 minutes using a plastic serological pipette until completely mixed. Filter the solution through a 0.2 gm nylon filter. Transfer to a 1 L eluent bottle. Cap the bottle loosely and sparge with helium for 20 minutes. Tighten cap and pressurize the bottle with helium. Store solution at room temperature under helium for up to three weeks.

[00674] HPAEC Eluent 2: 400 mM Sodium Hydroxide (NaOH). Using a 1 L graduated cylinder, measure 960 mL of HPLC grade water and transfer to a clean 1 L eluent bottle. Using a serological plastic pipet, add 40.0 mL of 10 N NaOH directly into the eluent bottle and mix the eluent by swirling. Cap the bottle loosely and sparge with helium for 20 minutes. Tighten cap and pressurize the bottle with helium. Store solution at room temperature under helium for up to three weeks.

[00675] HPAEC Eluent 3: HPLC grade Water. Fill a 1 L eluent bottle with approximately

L of HPLC grade water. Place eluent bottle on system, cap loosely, and sparge for approximately 20 minutes. Tighten cap and pressurize the bottle with helium. Store solution at room temperature under helium for up to three weeks.

[00676] 50 mM Sodium Phosphate Buffer, 0.02% Sodium Azide, pH = 7.5.

NaH₂PO₄*H₂O 6.9 g

NaN3 0.2 g

H2O 1.0 liter final volume

[00677] Weigh out 6.9 g ± 0.1 g of NaH₂PO₄ . H₂O and 0.2 g NaN3 and dissolve in 800 mL of HPLC grade H₂O in a 1 L reagent bottle using continuous mixing with a magnetic stirring bar. Using a pH meter, adjust the pH of the solution to 7.5 using 10M NaOH. Bring the final volume to 1.0 liter using a 1 L graduated cylinder. Store solution at room temperature for up to six months.

[00678] PNGase F Enzyme Working Stock in 50 mM Sodium Phosphate Buffer, 0.02% Sodium Azide, pH = 7.5.

mM Sodium Phosphate Buffer

0.02% Sodium Azide, pH = 7.5. 1.8 mL

PNGase F from Kit, Catalog No. P0704L 0.2 mL

[00679] Pipette 1.8 mL of 50 mM Sodium Phosphate Buffer, 0.02% Sodium Azide, pH

7.5 into a 1.8 mL cryogenic vial. Add 0.2mL of PNGase F from kit and mix thoroughly.

Store solution at -20 °C or less for up to six months. The solution may be aliquoted prior to freezing.

[00680] External System Suitability Standard. Stachyose Stock Solution (1.25 mg/mL). Weigh 0.125 g of Stachyose onto a weighing paper. Using an analytical balance and transfer to a 100 mL volumetric flask. Fill to mark with HPLC grade water and mix thoroughly. Aliquot in 2 mL portions into Nalgene cryovials. Store solution at -20 °C or less for up to six months.

[00681] Stachyose System Suitability Standard (12.5 pg/mL). Pipet 1 mL of the 1.25 mg/mL stock into a 100 mL volumetric flask. Fill to mark with HPLC grade water and mix thoroughly. Aliquot in 200 pL portions into 0.65 mL microfuge tubes. Place tubes in appropriately labeled storage box. Store system suitability solution at -20 °C or less for up to six months.

[00682] STANDARD AND SAMPLE PREPARATION

[00683] Reference Material Preparation. To a vial containing Img of lyophilized RapiGest SF, add 625 μΕ of 50 mM NaPhosphate buffer containing 0.02% NaAzide, pH 7.5. To a 0.65 mL Eppendorf tube add 120 μΕ of the RapiGest SF containing buffer. Add 40 μΕ of Reference Material (~ 50 mg/mL). The final RapiGest SF concentration should be 0.12 % w/v. Add 40 μΕ of the PNGase F working stock, mix thoroughly, spin down the sample, and place at 38 ± 2 °C for 22 + 2 hours (water bath or the Alliance autosampler compartment). Pipet sample into a microcon YM-10 centrifugal filter and centrifuge at 13,000 g for 30 minutes. Place 200 μΕ of HPLC water in the filter and rinse into the filtrate by centrifuging for an additional 30 minutes at 13,000 g. Vortex the combined filtrate for 15 seconds and centrifuge the sample for 10 seconds. Using a pipette transfer the resulting solution (~ 380 μΕ) to an HPLC total recovery autosampler vial (item 1.15).

[00684] Sample Preparation. To a 0.65 mL Eppendorf tube add 120 μΕ of the RapiGest SF containing buffer. Add 40 μΕ of protein sample ( this volume should equate to between 1 and 2 mg of CTLA4-Ig). The final RapiGest SF concentration should be 0.12 % w/v. Add 40 μΕ of the PNGase F working stock mix thoroughly by vortexing for 10 seconds. Spin down the sample, and place at 38 ± 2 °C for 22 ± 2 hours (water bath or the Alliance autosampler compartment).Pipet sample into a microcon YM-10 centrifugal filter and centrifuge at 13,000 g for 30 minutes. Place 200 μΕ of HPLC water in the filter and rinse into the filtrate by centrifuging for an additional 30 minutes at 13,000 g. Vortex the combined filtrate for 15 seconds and centrifuge the sample for 10 seconds. Transfer the resulting solution (~ 380 uL) to a total recovery HPLC autosampler vial (item 1.15).

[00685] SYSTEM SUITABILITY

[00686] Electrochemical Detector Cell Stabilization. Inject 30 pL of the external stachyose system suitability standard (12.5 pg/mL). Ensure the peak height for stachyose is >

800 nA. Ensure there is no excessive electrical noise from the cell and the baseline is flat. A representative system suitability chromatogram is shown in Figure 2. If the stachyose sensitivity or the baseline is unacceptable, check the buffer composition, clean the electrode or replace the electrode. If excessive noise is present, check cell to ensure removal of all air bubbles. Restabilize the cell and re-inject stachyose standard.

[00687] Theoretical Plates (N). Determine the number of Theoretical Plates (N) based on the Stachyose peak using the formula below. This is done through the Empower data analysis system or may also be done manually. N = 16 (t / W)² WHERE t: retention time measured from time of injection to peak elution time at maximum height

W: width of peak by extrapolation of sides to baseline.

N must be > 6000. If the plate count is less than 6000, adjust the run gradient or replace column.

[00688] Tailing Factor (T). Determine column Tailing Factor (T) based on the Stachyose peak using the formula below. This is done through the Empower data analysis system or may also be done manually. T = (W0.05 / 2f), WHERE:

Wo.05. width of peak at 5% of height (0.05h).

f: the measurement (width) from front edge of peak at W0.05 to the apex of the peak.

T must be &lt; 1.2. If the tailing factor is greater than 1.2, check buffer composition, replace the column or clean the column as indicated in and reinject system suitability standard.

[00689] Stachyose System Suitability Standard Retention Time Verification. The retention time is system dependent. The stachyose system suitability standard should exhibit a retention time of 18.5 ± 2.0 minutes. CTLA4-Ig Standard Material. Observe the carbohydrate profile from the first bracketing reference material injected prior to injection of samples. The carbohydrate profile should be similar to that shown in Figure 1. Absolute retention times are system dependent. Ensure that the difference in retention times between the first peak in Domain I (Peak 1 A) and the main peak in Domain III (Peak 3) is between 22 minutes and 28 minutes. If delineation of peaks does not resemble that obtained in Figure 1 take appropriate actions (e.g. check instrument function, clean column, check/replace buffers, replace column) and re-evaluate. The following procedure may be used to clean the column:

turn off the cell and clean the column with 80% Eluent 1,20% Eluent 2 for 5 minutes followed by 50% Eluent 1, 50% Eluent 2 for 10 minutes. Re-equilibrate the column and cell (with cell turned on) at initial conditions and re-evaluate.

[00690] INJECTION SEQUENCE

Set up the injection sequence of isolated oligosaccharides as follows: Stachyose Standard (30 pL) Reference Material (60 pL) Sample(s) (60 pL) Reference Material (60 pL)

It is recommended that &lt; five samples be run between bracketing reference material. injections.

[00691] DATA ANALYSIS

[00692] Process the Chromatograms. Process the chromatograms for the Reference Material and samples in Empower. Set integration parameters so that peak delineation and the baseline is similar to that shown in Figure 76, integration lines may need to be placed manually. Perform calculations for relative Domain areas and relative peak areas shown. Determine the average values for these parameters for the CTLA4-Ig Material and for each sample if replicate injections were made. For the Reference Material, determine relative deviation for Domains I, II, III, Peaks 1A and IB for each replicate with respect to the average of all replicates.

[00693] Comparison of Profiles of Sample to Reference Material Profiles. Visual

Comparison. Determine if both samples and Reference Material have the same number of Domains and primary peaks. Primary peaks are those peaks labeled in Figure 76 (Peaks 1 A, IB, IC, ID, 2, 3 and 4). Relative Quantitation Comparison. Compare the relative areas of samples (Domains I, II, and III and Peaks 1 A, and IB; if replicate injections were made of samples use their average values) with the average relative areas from the bracketing CTLA4-Ig injections. Determine the relative difference of these areas from the average CTLA4-Ig Material values. Calculations - % Domain Area (Relative Domain Area).

Calculate the % Domain area for the Domains of the profiles for the Reference Material and samples. Refer to Figure 76 for pattern of Domain areas. Following the example in Figure 76, calculate the Domain percent ratios by using the following information and formula (retention times are system dependent and reflect result in Figure 76):

<td> [00694]</td><td> Domain I: Sum of the peak areas at approximate retention times 18-24 minutes (Peaks 1A-1E) Domain II: Sum of the peaks from 26-38 minutes Domain III: Sum of the peaks from 39-50 minutes Domain IV: Sum of the peaks from 51 -64 minutes Domain V Sum of the peaks from 65-75 minutes NOTE: Retention time windows for Domains will shift according to variations in daily chromatographic performance. Adjust times accordingly. . . Individual Domain Area ,״״״, Domain Area %=------------------x 100% Sumofall Domain Areas For Domains I-III also calculate the average values in the bracketing reference material injections, as well as in samples if replicate injections are made. % Peak Area (Relative Peak Area). Calculate the % peak area for Peaks 1 A,</td>

IB, IC, and 3 of the profiles for the Reference Material and samples. Refer to Figure 1 for pattern of peak areas; retention times are system dependent. Calculate the peak percent ratios by using the following information and formula:

<td></td><td>_T . . ״/ IndividualPeakArea , Individual Peak Area %=-----------------------x 100% Sum of the all Domain Areas</td>

<td> [00695]</td><td> For each of Peaks 1A and IB, also calculate the average values in the</td>

bracketing reference material injections, as well as in samples if replicate injections are made. Calculation of the Percent Difference from Average Reference Material Values. Use the following formula to calculate percent differences in average relative areas of Domains I-III, Peaks 1A and IB of samples compared to Reference Material:

<td> [00696]</td><td> % Diff = |RM - S| / RM x 100 WHERE: RM = average relative area value of interest for Reference Material S = average relative area value of interest for a sample I I = absolute value</td>

[00697] Exemplary Values. For a run to be acceptable the exemplary values must be met and all injections relevant to the sample must have successfully occurred. Additionally, for each of the bracketing Reference Material injections, the % Domain Areas for Domain I, II and III and % Peak Areas for Peak 1A and IB must be within 15% of their average values.

[00698] Analysis by HPAEC-PAD: N-linked oligosaccharides were cleaved from CTLA4-Ig molecules and analyzed by HPAEC-PAD. Oligosaccharides elute into four domains based on the amount of sialic acid present. Domains were established based on the migration of oligosaccharide standards and were confirmed by MS. Domain I represents asialylated species. Domains II, III, and IV represent mono-, di-, and tri-sialylated species, respectively. In order to characterize the structure of the oligosaccharides at the three Nlinked sites, peptides T5, T7 and T14 were individually isolated (refer to Table 59 for identity of these peptides). This was performed using tryptic digestion of CTLA4-Ig and manually collecting peaks corresponding to these three peptides.

[00699] The isolated peptides were treated with PNGase E to release the oligosaccharides, which were subsequently purified and analyzed by HPAEC-PAD. Peptide T5 did not produce a good profile since it is difficult to purify due to its extreme hydrophobicity. Quantities of cleaved oligosaccharides are low due to low recovery of this peptide from the reversed phase chromatography step after tryptic digestion.

Theoretically Expected and Observed Masses of _ CTLA4-Ig

<td> Fragment No.</td><td> Residue No.</td><td> Expected Mass (Daltons)</td><td> Observed Mass (Daltons)</td><td> Peptide Sequence</td>

<td> Tl+A</td><td>-14</td><td> 1536.8</td><td> 1536.8</td><td> amHvaqpavvlassr</td>

<td> T1</td><td> 1-14</td><td> 1465.8</td><td> 1465.7</td><td> MHVAQPAVVLASSR</td>

<td> T2</td><td> 15-28</td><td> 1485.7</td><td> 1485.6</td><td> G1ASFVCEYASPGK</td>

<td> T3</td><td> 29-33</td><td> 574.6</td><td> 574.2</td><td> ATEVR</td>

<td> T4</td><td> 34-38</td><td> 586.7</td><td> 586.3</td><td> VTVLR</td>

<td> T5</td><td> 39-83</td><td> 4900.4</td><td> c</td><td> QADSQVTEVCAATYMMGNELTFLD DSIC־rGTSSGNQVNLTlQGLK</td>

<td> T6</td><td> 84-93</td><td> 1.171.4</td><td> 1.171.4</td><td> AMDTGLYICK</td>

<td> T7</td><td> 94-128</td><td> 3997.5</td><td> c</td><td> VELMYPPPYYLGIGNGTQJYVIDPEP CPDSDQEPK</td>

<td> T8^b</td><td> 129-132</td><td> 435.2</td><td> ND</td><td> SSDK</td>

<td> T9”</td><td> 133-158</td><td> 3345.7</td><td> c</td><td> THTSPPSPAPELLGGSSVFLFPPKPK</td>

<td> T10</td><td> .159-165</td><td> 834.9</td><td> 834.4</td><td> DTLMISR</td>

<td> ΊΊ 1</td><td> 166-184</td><td> 2139.3</td><td> 2138.6</td><td> TPEVTCVVVDVSHEDPEVK</td>

<td> T12</td><td> 185-198</td><td> 1677.8</td><td> 1677.2</td><td> FN W YVDG VEVHN AK</td>

<td> T13</td><td> 199-202</td><td> 500.6</td><td> 500.3</td><td> TKPR</td>

<td> TI4^״</td><td> 203-211</td><td> 1189.2</td><td> c</td><td> EEQYNSTYR</td>

<td> T15</td><td> 212-227</td><td> 1808.1</td><td> 1807.4</td><td> VVSVLTVLHQDWLNGK</td>

<td> T16</td><td> 228-230</td><td> 438.5</td><td> 438.1</td><td> :eyk</td>

<td> T17</td><td> 231-232</td><td> 307.4</td><td> ND</td><td> CK</td>

<td> T18</td><td> ־233-236</td><td> 446.5</td><td> ND</td><td> VSNR</td>

<td> T19</td><td> 237-244</td><td> 838.0</td><td> 837.4</td><td> ALPAP1EK</td>

<td> סליו-</td><td> 245-248</td><td> 447.5</td><td> 447.2</td><td> T.1SK</td>

<td> Γ2 !</td><td> 249-250</td><td> 2.17.2</td><td> ND</td><td> AK</td>

<td> T22</td><td> 251-254</td><td> 456.2</td><td> 456.2</td><td> GQPR</td>

<td> T23</td><td> 255-265</td><td> 1286.4</td><td> 1286.6</td><td> EPQVYTLPPSR</td>

<td> T24</td><td> 266-270</td><td> 604.7</td><td> 604.3</td><td> DELTK</td>

<td> Γ25</td><td> 271-280</td><td> 1161.4</td><td> 1.160.5</td><td> NQVSLTCJLVK</td>

<td> T26</td><td> 281-302</td><td> 2544.7</td><td> 2545.6</td><td> GFYPSD1AVEWESNGQPENNYK</td>

<td> Table 59. (cont.)</td><td> Theoretically Expected and Observed Masses of CTLA4-Ig</td>

<td> Fragment No.</td><td> Residue No.</td><td> Expected Mass (Daltons)</td><td> Observed Mass (Daltons)</td><td> Peptide Sequence</td>

<td> T27</td><td> 303-319</td><td> 1874.1</td><td> 1873.2</td><td> TT.PPVLDSDGSFFLYSK</td>

<td> T28</td><td> 320-324</td><td> 574.7</td><td> 574.2</td><td> LT VDK.</td>

<td>^rr29.</td><td> 325-326</td><td> 261.3</td><td> ND</td><td> SR</td>

<td> T30</td><td> 349־327</td><td> 2803.1</td><td> 2800.8</td><td> WQQGNVFSCSVMHEALHNHYTQK.</td>

<td> T31</td><td> 356־350</td><td> 659.7</td><td> 659.1</td><td> SLSLSPG</td>

<td> T3I+K</td><td> 350-357</td><td> 787.9</td><td> 787.0</td><td> SLSLSPGK</td>

<td> T2 clip</td><td> 23־15</td><td> NE</td><td> 1045.3</td><td> G1ASFVCEY</td>

<td> T12 clip</td><td> 195־185</td><td> NE</td><td> 1364.7</td><td> FNWYVDGVEVH</td>

<td> T27 clip</td><td> 317־303</td><td> NE</td><td> 1658.5</td><td> TTPPVLDSDGSFFLY</td>

<td> Ί'30 clip</td><td> 335־327</td><td> NE</td><td> 1125.3</td><td> WQQGNVFSC</td>

<td> T30 clip</td><td> 327-333</td><td> NE</td><td> 878.3</td><td> WQQGNVF</td>

ND not detected

NE not expected from trypsin digestion (these masses were not expected from the digest, non-specific cleavage).

^a Tryptic peptides T5, T7, and TI4 have N-linked glycosylation. The mass listed is that of the peptide without glycosylation.

^b Tryptic peptides T8 and T9 have O-linked glycosylation. The mass listed is that of the peptide without glycosylation.

^C Several different masses corresponding to different giycoforms of glycosylated peptides were observed.

[00700] The HPAEC-PAD profiles for all N-linked carbohydrates from CTLA4-Ig and the three peptides are shown in FIGS. 55A-D. Panel A shows the N-linked oligosaccharide profile of the CTLA4-Ig molecule, while panels B, C and D show the profiles for the T5, T7 and T14, respectively. The amount of oligosaccharides injected for each of the peptides is different due to the preparation processes. The majority of oligosaccharides detected on peptide T5 consist of the mono- and di-sialylated oligosaccharides. The profile of T7 contains primarily mono-, di-, and some a- and tri sialylated glycan species. Only a small amount of the tri-sialylated structures can be detected on T5. Peptide T14 consists of predominantly asialylated oligosaccharides and a small amount of mono- and di-sialylated oligosaccharides

[00701] The results obtained by HPAEC-PAD provide information on site-specific Nlinked glycosylation. N-linked oligosaccharides from the CTLA4 region of CTLA4-Ig contain a greater proportion of sialylated species than those from the Fc region of CTLA4-Ig.

[00702] Trypsin, Asp-N , and Trypsin/Chymotrypsin Peptide Mapping of CTLA4-Ig: CTLA4-Ig was denatured and reduced in 50 mM Tris buffer (pH 8.0) containing 6 M Guanidine and 5 mM dithiothreitol (DTT). After a 20 minute incubation at 50° C, iodoacetamide (IAA) was added to a final concentration of 10 mM to alkylate free thiols, and the incubation was continued for an additional 20 minutes at 50° C in the dark. The reduced and alkylated mixture was loaded onto a desalting column (Amersham NAP-5), then eluted into the void volume with either 50 mM Tris, 10 mM CaC12, pH 8.0 or 50 mM sodium phosphate buffer, pH 7.5. Following desalting, reduced/alkylated CTLA4-Ig was digested using two different proteases: trypsin or Asp-N. For trypsin plus chymotrypsin digestion CTLA4-Ig is neither reduced nor alkylated.

[00703] For trypsin digestion, sequence grade trypsin (Roche, 2%, w/w, enzyme/protein) was added and incubated for 4 hours at 37°C. For Asp-N digestion, sequence grade Asp-N (Roche, 4%, w/w, enzyme/ protein) was added and incubated for 16 hours at 37°C. For the trypsin/chymotrypsin digestion, sequence grade trypsin (Promega, 4%, w/w, enzyme/protein) was added and incubated for 4 hours at 37° C, then a-chymotrypsin was added (Sigma, 4%, w/w, enzyme/protein) and incubated for 16 hours at 37° C. All samples were placed in a freezer after the digestion.

[00704] The resulting peptide mixtures were separated by gradient elution from a Waters Atlantis^TM dC18 column (2.1 x 250 mm) on a Waters Alliance HPLC Workstation at 0.120 mL/minute. The column was directly connected to the Waters Micromass Q-Tof micro^TM mass spectrometer equipped with an ion-spray source for collection of mass spectra. Peptide mixtures were also separated on a Varian Polaris Cl 8 column (4.6 x 250 mm) at 0.7 mL/minute using the sameHPLC workstation. The columns were equilibrated with solvent A (0.02% TFA in water) and peptides were eluted by increasing concentration of solvent B (95% acetonitrile/0.02% TFA in water). A post-column splitter valve was used to direct 15% of the flow to the Q-Tof workstation, which was run in the positive TOF mode (m/z 1002000). The typical ion spray voltage used was 3000 V.

[00705] CTLA4-Ig Analysis by MS: CTLA4-Ig was diluted with 100 mM Tris, 25 mM NaCI, pH 8 to a final concentration of 0.7 mg/mL. PNGase F (New England Biolabs) was diluted 30-fold with 100 mM Tris, 25 mM NaCI, pH 8 to a final concentration of 17 U/gL. Equal volumes (60 gL each) of diluted purified fermentation sample and diluted glycosidase solution were mixed and incubated at 37° C for 4 hours.

[00706] The resulting deglycosylated CTLA4-Ig (2 μg) was loaded onto a polymericbased (copolymer of polystyrene and poly N-vinyl-2-pyrrolidinone) Waters Oasis® reversed phase extraction cartridge column (2.1 x 20 mm). The loaded column was washed with 5% solvent B (solvent A: .1% formic acid in water, solvent B: 1% formic acid in acetonitrile) at a flow rate of 0.2 mL/minute for five minutes to desalt, with the eluent diverted to waste. At the end of 5 minutes, a fast gradient (5% solvent B to 95% solvent B in 10 minutes) began the elution of CTLA4-Ig off the column; here the eluent was directed into the mass spectrometer (Waters Micromass Q-Tof micro^TM) at 45 gL/min after flow splitting (chromatography system used was a Waters Alliance 2695 equipped with a Waters 2996 detector).

I

[00707] The capillary voltage for the Q-Tof micro^TM was set at 3kV and the sample cone voltage at 40 V. The scans in every 0.9 second were averaged into one scan; the inter-scan time was 0.1 second. ^,The Q-Tof analyzer scans from m/z 800 to 2500. Spectra corresponding to the portion higher than half the maximum peak height (in TIC chromatogram) are combined using Waters MassLynx^TM software. The combined spectrum was subjected to Waters MaxEntl deconvolution. The resolution was set at 1 Da/Channel, and the uniform Gaussian damage model was selected with width at half height set between 0.5-1 Da. Minimum intensity ratios for the left peak and the right peak were both set at 50%.

[00708] N-Linked Oligosaccharide Analysis by Liquid Chromatography/Mass Spectrometry (LC/MS ) Using a Porous Graphitized Carbon (PGC): Isolated oligosaccharides were separated on a porous graphitized column (Thermo Hypercarb; 4.6 x 100 mm) using a Waters Alliance 2695 HPLC system equipped with a Waters 2996 photodiode array detector. Oligosaccharide separation was achieved using a.two-stage gradient of increasing proportions of acetonitrile in 0.05% TFA. In the first stage of the gradient, the acetonitrile percentage ranged from 9.6% at the time of injection to 24% at 80 minutes. In the second stage of the gradient, the acetonitrile percentage ranged from 24% at 80 minutes to 60% at 110 minutes. A flow rate of 0.2 mL/minute was used throughout. The elution stream was monitored at by UV detection at 206 nm and analyzed by mass spectrometry using a Waters MicroMass QTof micro^TM for mass identification.

[00709] Carbohydrate Analysis by LC/MS PGC method: Oligosaccharide isolation by deglycosylation of the protein was performed by enzymatic hydrolysis using PNGase F (New England Biolabs, Beverly, MA). For the deglycosylation, between 1 and 2 mg glycoprotein in 160 pL of 50 mM sodium phosphate buffer containing 0.15% (w/v) Rapigest SF (Waters Corporation) was denatured by heating at 100°C for 2 minutes. The cooled solution was mixed and 40 pL of PNGase F (50,000 U/mL, in 50 mM sodium phosphate buffer, pH 7.5) was added. The sample was vortexed followed by incubation at 38° C for 24 hours. The enzymatically-released oligosaccharides were purified by high performance liquid chromatography. Reversed phase liquid chromatography was performed on a Phenomenex Luna 5 pL Cl 8 column (4.6 x 150 mm, Phenomenex, Torrance, CA) coupled with a Thermo HyperCarb 5 pL (4.6 x 100 mm, Phenomenex, Torrance, CA) at a flow rate of 1.0 mL/min. The columns were equilibrated with 0.05% triflouroacetic acid (TFA) prior to injection. After sample injection (150 pL of the digest mixture), a gradient of acetonitrile was initiated terminating at 15 minutes and a solvent composition of 0.05% TFA in 12% acetonitrile. The glyeans were then eluted from the HyperCarb column by washing with 0.05% TFA in 60% acetonitrile. The glycans were detected by UV absorbance at 206 nm. Peaks eluted from the Hypercarb wash were collected and concentrated to dryness under vacuum. Prior to subsequent injections the Luna Cl 8 column was cleaned with 0.05% TFA in 40% acetonitrile, 40% isopropanol, 20% water.

[00710] Profiling of Isolated Oligosaccharides with PGC

[00711] The system used for PGC chromatography of isolated oligosaccharides consisted of a Waters Alliance equipped with a Waters 2996 photodiode array detector utilizing a Hypercarb column (2.1 x 100 mm). ^,The oligosaccharide samples were eluted using an acetonitrile gradient.

[00712] Acidic Mobile Phase Elution: Acetonitrile gradient in 0.05% triflouroacetic acid (TFA). A two-stage gradient of increasing acetonitrile was used for the chromatographic separation of oligosaccharides. The initial linear gradient of increasing acetonitrile volume percentage from 9.6% at the time of injection to 24% at 80 minutes is followed by a second gradient of increasing acetonitrile volume percentage from 24% at 80 minutes to 60% acetonitrile at 110 minutes. A flow rate of 0.15 ml/min was used throughout the gradient. The elution stream was monitored at 206nm with a Waters UV detector, followed by a Micromass Q-Tof Micro for mass identification. The ionization parameters for the ESI probe were set as follows: Capillary voltage = 3 kV, Cone voltage = 45 V, Source temperature 80° C, and desolvation temperature of 175° C.

[00713] Basic Mobile Phase Elution: An acetonitrile gradient in 0.4% ammonium hydroxide (NH4OH) was used for the chromatographic separation of oligosaccharides. A linear gradient of increasing acetonitrile volume percentage from 10.4% at the time of injection to 28% at 150 minutes at a flow rate of 0.15 mL/min was used to produce the profile. The elution stream was monitored at 206 nm with a Waters UV detector, followed by a Micromass Q-Tof Micro for mass identification. The ionization parameters for the ESI probe were set as follows: Capillary voltage = 3 kV, Cone voltage = 45 V, Source temperature 80° C, and desolvation temperature of 175° C.

[00714] Direct profiling of oligosaccharide digest mixtures with PGC: The system used for PGC chromatography of oligosaccharide digest mixtures consisted of a Waters Alliance fitted with both a Luna Cl 8 and a Hypercarb porous graphite column (4.6 x 100 mm, Thermo). The system was interfaced with a Waters 2996 photodiode array detector and a Q-ToF Micro (Micromass). Deglycosylation of protein was performed by enzymatic hydrolysis using PNGase F. For the deglycosylation , between 1 and 2 mg glycoprotein in 160 μΕ of 50 mM sodium phosphate buffer containing 0.15% by weight Rapigest SF (Waters Corporation), was denatured by heating at 100° C for 2 minutes. The cooled solution was mixed well and 40 μΕ of PNGase F (50,000 U/mL, in 50 mM sodium phosphate buffer, pH 7.5) was added. The sample was mixed and then incubated at 38° C for 24 hrs. The enzymatically-released oligosaccharides were profiled by high performance liquid chromatography. Reversed phase liquid chromatography was performed on a Phenomenex Luna 5|u Cl 8 column (4.6 x 150 mm) coupled with a Thermo HyperCarb column 5 μΕ (4.6 x 100 mm) at a flow rate of 1.0 mL/min. The columns were equilibrated with 0.05% triflouroacetic acid (TFA). After sample injection (150 |1L of digest mixture) a gradient of acetonitrile was initiated, terminating at 11 minutes and a solvent composition of 0.05% TFA in 9% acetonitrile. A column switch was used to isolate the hypercarb column and the glycans are then eluted from the HyperCarb column with a linear gradient of increasing acetonitrile percentage. An initial gradient of increasing acetonitrile percentage from 9% at the time of injection to 36% at 160 minutes was used. The second gradient involved increasing acetonitrile volume percentage from 36% at 160 minutes to 60% acetonitrile at 170 minutes. A flow rate of 0.15 mL/min was used throughout the elution gradients. The glycans were detected by UV absorbance at 206nm, and by MS scanning the mass range from 400 - 3000 m/z. MS parameters were set to the following values: Capillary 3 kV, Cone 45 V.

Prior to subsequent injections the Luna Cl 8 column was cleaned with 0.05% TFA in 40% acetonitrile, 40% isopropanol, 20% water.

[00715] Analysis by Porous Graphitized Carbon Chromatography: The structures of the oligosaccharides from Domains I-IV were investigated using Porous Graphitized Carbon chromatography (PGC) coupled to MS. N-linked oligosaccharides were isolated from CTLA4-Ig and purified as described in the previous HPAEC-PAD section. The oligosaccharides were analyzed using a Hypercarb (PGC) column with a UV detector followed by Q-TOF ESI/MS. The PGC profile for the N-linked oligosaccharides released from CTLA4-Ig by PNGase F digestion is shown in FIG. 56 with domains noted. The order of elution is the same as that observed in HPAEC-PAD. The structures obtained are shown in Table 60.

[00716] The nomenclature for naming the oligosaccharides displayed in Table 60 is shown below:

<img file="IL192098A_D0001.tif" />

[00717] The grey shaded area shows the N-linked oligosaccharide core structure, where P stands for PNGase F digestion, and the subsequent digits describe the number of Mannose (circle), Fucose (downwards pointing triangle), Galactose (right pointing triangle), and sialic acid residues (star), respectively. N-acetyl glucosamine (GlcNAc) is.represented bya square.

χ״ N-L1nked Oligosaccharide Structures and Masses

Table 60. _T “ . . . . _T

Detected in CTLA4-Ig

<td></td><td></td><td></td><td> Structure</td><td> Expected Mass (Daltons)</td><td> Observed Mass (Daltons)</td>

<td> Asialylated</td><td></td><td></td><td></td><td></td><td></td>

<td> P2100</td><td></td><td></td><td></td><td> 1463</td><td> 1576 [M-H]־ . TFA</td>

<td> P2110</td><td></td><td></td><td> Us 1 ׳ </td><td> 1625</td><td> 1738 [M-H.]־ . TFA</td>

<td> P2120</td><td></td><td> —Ml-</td><td> ^־3 pfe-4gj—te</td><td> 1787</td><td> 1900 [M-H]־ . TFA</td>

<td> Mono-sialylated</td><td></td><td></td><td></td><td></td><td></td>

<td> P211 1</td><td></td><td> SHi</td><td> iSHsh</td><td> 1916</td><td> 1915 [M-H]־</td>

<td> P2121</td><td> iK</td><td></td><td></td><td> 2078 .</td><td> 2077 [M-H]־</td>

<td> P3131</td><td></td><td></td><td> j *</td><td> 2443</td><td> 1279 [M-21-1]²־</td>

Table 60. (cont.) N-Linked Oligosaccharide Structures and Masses

Detected in CTLA4-Ig

<td> Di-sialylated</td><td colspan="2"></td><td></td><td></td>

<td> P2.122</td><td colspan="2"></td><td> 2369</td><td> 1184 [M-2H]²־</td>

<td> P3132</td><td></td><td></td><td> 2734</td><td> 1367 [M-2H]²־</td>

<td> P4142</td><td colspan="2"> τέ־ ־# A .A 4¾. A ffl. i® ש ® 111 Jb</td><td> 3099</td><td> 1549 [M-2H]²־</td>

<td> Tri-sialylated</td><td colspan="2"></td><td></td><td></td>

<td> P3133</td><td colspan="2"> A &amp; A ffl B</td><td> 3025</td><td> 1512 [M-2H]²־</td>

<td> P4133</td><td>^י</td><td> 4־0-</td><td> 3389</td><td> !695 [M-2.FI]'²</td>

★ NANA ► Galactose Hi GlcNAc ®> Mannose W Hi cose

[00718] In Domain I, six peaks were identified, corresponding to three asialylated oligosaccharides (structures P2100, P2110, P2120). The 113 Dalton mass difference between the predicted structure and observed mass is due to detection of a trifluoroacetic acid (TFA) adduct. Different peaks with the same mass of 1,463 corresponding to P2100, indicates they are likely different anomers.

[00719] In Domain Π, six peaks were identified, corresponding to three biantennary and triantennary oligosaccharides (structures P2111, P2121, and P3131, respectively), each containing one sialic acid residue (N-acetylneuraminic acid, NANA).

[00720] In Domain III, six peaks were identified, corresponding to three biantennary, triantennary and tetraantennary oligosaccharides (structures P2122, P3132, and P4142, respectively), each containing two sialic acid residues (NANA).

[00721] In Domain IV, two peaks were identified, corresponding to two triantennary and tetraantennary oligosaccharides (structures P3133 and P4133), each containing three sialic acid residues (NANA).

[00722] Measurement of molar ratio of moles sialic acid to moles CTLA4-Ig molecules or dimer by acid hydrolysis treatment of CTLA4-Ig molecules (see FIGS. 25 and 26): In this method, NANA and NGNA are cleaved from the protein by acid hydrolysis. The released NANA and NGNA are separated by HPLC on a Rezex Monosaccharide RHM column and detected by UV absorbance (206 nm). NANA and NGNA are quantitated based on the response factors of concurrently run NANA and NGNA standards. The test results are reported as molar ratios (MR) of NANA and NGNA respectively, to protein. This assay determines the total number of moles, bound and unbound, of sialic acid.

[00723] Reagent, instrumentation and chromatographic conditions used in the assay: IM sulphuric acid H2SO4 (stock) and 5mM H2SO4 mobile phase running buffer; NANA standard solution of Img/ml; NGNA standard solution of Img/ml. Alliance chromatographic system Waters Corporation 2695 Separations Module with integrated autosampler; Rezex monosaccharide RHM column—8micrometer, 7.8 x 300mm, Phenomenex, equipped with 7.8 x 50 mm guard column, Phenomenex; Detector— Waters Corporation 996 photodiode array detector or Waters Corporation 2487 dual wavelength absorbance detector. Chromatographic parameters: Flow—0.600mL/min; Mobile phase—5mM H2SO4; Injection volume— 5microL; Target concentration— Img/ml; Run time— 25min; Column temperature— 40°C;

Autosampler temperature— 4°C; Wavelength - 206nm; Retention time NANA (system dependent) -10.8 min (+ or - Imin), Retention time NGNA (system dependent) - 9.8 min (+ or — lmin.).

[00724] System Suitability Standard: Add 50 pL each of 1 mg/mL of NANA and NGNA to 900 pL H2O in an appropriate container. Store at 4°C for up to 3 months; N-Acetyl Neuraminic Acid Working Standard (0.05 mg/mL); N-Glycolyl Neuraminic Acid Working Standard (0.05 mg/mL).

[00725] Hydrolysis of Samples and CTLA4-Ig standard material: Samples and CTLA4-Ig standard material are diluted to 1 mg/mL in H2O for hydrolysis. CTLA4-Ig samples and CTLA4-Ig standard material are hydrolyzed by adding 10 pL 1 M H2SO4 to 190 pL of 1 mg/mL diluted samples and CTLA4-Ig. Hydrolysis is performed in duplicate in 1.5 mL micro centrifuge tubes. Lids are secured by lid-lock or tape. Tubes are mixed by vortexing and placed in 80°C heating block for 1 h. After incubation, tubes are removed from the heating block, cooled down at room temperature for 3 min, and placed in centrifuge for a quick spin to collect sample to the bottom of the tube. From the tubes, 50 pL are aliquoted of the hydrolyzed solution into sample vial, which is placed in cooled autosampler for injection. A hydrolysis blank is made as a single preparation by adding 10 pL 1 M H₂SO4t0 190 pL water in 1.5 mL micro centrifuge tubes. The blank is processed as the samples.

[00726] System Suitability: To check the system suitability six replicate injections of the system suitability standard (5 pL each) were injected followed by one injection of the hydrolysis blank (5 pL). Using the last system suitability standard injection, Resolution (R), acceptable values are higher than 1.3, and Theoretical Plates (N), accepatable theoretical plate count must be at least 4000, were calculate respectively. Using the last five system suitability replicates, reproducibility of NANA counts were calculated, and the hydrolysis blank was evaluated.

[00727] Resolution: Using the last system suitability standard injection, peak Resolution was calculated using the following equation: R=2(T2-T1)/(W2+W1), where R is resolution, T2 is the retention time of the NANA peak (peak 2), T1 is the retention time of the NGNA peak (peak 1), W2 is the width at the baseline of lines drawn tangent to the sides of peak 2, W1 is the width at the baseline of lines drawn tangent to the sides of peak 1. FIG. 25 depicts a typical system suitability injection.

[00728] Theoretical plates (N): Using the last system suitability standard injection, the theoretical plate count (N) was calculated using the following equation: N=16(RT²/W), where

N is the theoretical plate count, RT is the retention time of the NANA peak in minutes, W is the width at the baseline of lines drawn tangent to the sides of the NANA peak.

[00729] The last five injections of the system suitability standard were used to calculate the average area counts and their standard deviation for NANA. The relative standard deviation was equal or less than 3%. The hydrolysis blank was free of any significant peaks with the retention time of NANA and NGNA.

[00730] Injection sequence: One injection each of the NANA and NGNA working standards was injected, followed by the hydrolyzed CTLA4-Ig sample material (duplicate samples), followed by the hydrolyzed CTLA4-Ig material. After the CTLA4-Ig runs were completed, one injection each of the NANA and NGNA working standards was injected.

[00731] To determine the moles of NANA or NGNA injected in the working standard the following equation is used: mole NANA or NGNA= (C)(P)(I)/MW, where C is the concentration of NANA and NGNA in the working standard, P is the purity of the standard, I is the injection volume, MW is the molecular weight (309.2 g/mole for NANA, and 325.3 g/mole for NGNA).

[00732] To determine the moles of NANA and NGNA in the CTLA4-Ig samples, the following equation is used: moles NANA or NGNA in sample=(X)(Y)/Z, where X is the number of moles in the working standard of NANA and NGNA, Y is the average counts of NANA and NGNA in the CTLA4-Ig sample, Z is the averaged area of the duplicate NANA and NGNA in Working Standards. From the duplicate injections of the standards, the area counts of the NANA and NGNA standard must have less than 10% RSD.

[00733] To determine the amount injected in each sample, the following equation is used: moles protein=(C)(D)(I)/MW, where C is the concentration of CTLA4-Ig dimer in g/ml (obtained from UV analysis), D is the dilution for hydrolysis (0.95), I is the injection volume (0.005ml) and MW is the molecular weight of CTLA4-Ig dimer as determined from mass spectrometry (92,439g/mol).

[00734] Molar ratio (MR) of NANA or NGNA to CTLA4-Ig protein is calculated by the following equation: MR=AZB, where A is the number of moles of NANA or NGNA, and B is the number of moles of CTLA4-Ig molecules or dimer.

[00735] Molar ratio (MR) of sialic acid, NANA and NGNA, to CTLA4-Ig protein is calculated by the following equation: MR=(A+B)/C, where A is the number of moles of NANA, B is the number of moles of NGNA, and C is the number of moles of CTLA4-Ig molecules or dimer. Duplicates of CTLA4-Ig samples and CTLA4-Ig standard material must have less than 10% RSD in molar ratios for NANA.

[00736] Linearity of responses determined by hydrolysis method of measuring sialic acid content: NANA responses were demonstrated to be linear to with respect to NANA standard concentrations in the range from 0.5 |Xg/mL (~0.1 % nominal NANA standard = NANA—QL) to 98.7 μg /mL (-200% nominal NANA standard). NGNA responses were demonstrated to be linear to NGNA standard concentrations in the range from 5.0 μg /mL (-10% nominal NGNA standard) to 82.0 pg /mL (-160% nominal NGNA standard).

[00737] Responses of NANA from hydrolyzed CTLA4-Ig material are linear with respect to protein concentrations in the range from 0.25 mg/mL (25 % nominal CTLA4-Ig load) to 2.0 mg/mL CTLA4-Ig (200 % nominal CTLA4-Igload). Responses of NGNA from hydrolyzed CTLA4-Ig material are linear to protein concentrations in the range from 0.25 mg/mL (25% nominal CTLA4-Ig load) to 2.0 mg/mL CTLA4-Ig 200% nominal CTLA4-Ig load).

[00738] Accuracy of the hydrolysis method of measuring sialic acid content: Accuracy was demonstrated for CTLA4-Ig material (1 mg/mL) spiked with NANA or NGNA standards.

[00739] Precision of the hydrolysis method of measuring sialic acid content: Validation experiments demonstrated instrument precision (%RSD &lt; 3%), repeatability for sample preparations (%RSD &lt; 4%) and reproducibility across different sample preparations, different days, different instruments and different analysts (%RSD &lt; 6% for NANA, % RSD &lt; 12% for NGNA). NANA and NGNA molar ratios were considered to be precise within a range of 10% and 20%, respectively, of the reported results.

[00740] Range of the hydrolysis method of measuring sialic acid content: The working range for this assay was shown to be from 0.49 mg/mL to 3.87 mg/mL CTLA4-Ig material.

[00741] Detection Limit (DL) of the hydrolysis method of measuringsialic acid-content: . The individual DL values for NANA and NGNA standards using a photodiode array detector (PDA; HPLC System 1) were 0.464 μg/mL and 0.402 μg/mL_ג respectively; the individual DL values for NANA and NGNA using dual wavelength detector (HPLC system 2) were 0.131 μg/mL and 0.111 μg/mL, respectively. The method DL, for both sialic species and based on use of the least sensitive detector, was 0.5 μg/mL for NANA and NGNA.

[00742] Quantitation Limit (QL) of the hydrolysis method of measuring sialic acid content: The individual QL values for NANA and NGNA standards using a photodiode array detector (PDA; HPLC System 1) were 1.68 μg/mL and 1.52 μg/mL, respectively; the QL values for NANA and NGNA using a dual wavelength detector (HPLC System 2) were 0.48 μβ/ιηΕ and 0.41 μ£/η!Ε, respectively. The method QL, for both sialic acid species and based on use of the least sensitive detector, was 1.7 pg/mL for NANA and NGNA.

[00743] Ruggedness/Robustness: The method was demonstrated to be robust with respect to the sample 48 hours refrigerated solution stability, the use of difference columns, the use of different NANA and NGNA lots and the use of mobile phases with ± 5 % alteration of concentration.

[00744] IEF gel electrophoresis: The pl of the glycoprotein can also be measured, before and after treatment with neuraminidase, to remove sialic acids. An increase in pl following neuraminidase treatment indicates the presence of sialic acids on the glycoprotein. An IEF gel can be use to determine the isolecetrc point, the numer of isoforms of CTLA4-Ig. A suitable system for running IEF gel is the Multiphore II Electrophoresis System, and an IEF gel of pH 4.0 to 6.5 (Amersham Biosciences). The anode buffer has the following composition: 0.1M Glutamic Acid in 0.5M Phosphoric acid. The cathode buffer has the following composition: 0.1 M beta-Alanine. The IEF was gel is prefocused under constant power (25watts) and current (25mAmps) until the voltage reaches > 300V. IEF calibration standards and samples of the appropriate concentration were loaded and the gel was run under constant power (25watts) and current (25mAmps) with maximum of 2000V for 2.5 hours. After fixing and Coomassie blue staining of the IEF gel, protein bands are visualized by densitometer. A typical IEF gel of CTLA4-Ig dimer preparation is shown in FIG. 11.

Example 4: Isolation and Characterization of Single Chain (Monomer) of CTLA4־Ig

[00745] Preparation of native single chain CTLA4-Ig: Samples of CTLA4-Ig recombinant protein prepared by the methods of the invention was separated by nondenaturing SEC using a 2695 Alliance HPLC (Waters, Milford, MA) on two 21.5 x 300 mm TSK Gel® G3000SWxlpreparative columns (Tosoh Bioscience, Montgomery, PA) in tandem. Thirty injections (1.0 mL each) of the sample at ~50 mg/mL were separated under isocratic conditions using a mobile phase consisting of 0.2 M NaH₂PO4, 0.9% NaCI, pH 7.0, at a flow rate of 1.0 mL/min. Samples were monitored at an absorbance of 280 nm using Water's 2996 PDA detector. Analysis was performed using Waters Millennium 4.0™ and Empower Pro© Software. Fractions were collected (1.0 mL each) on a Foxy 200 automated fraction collector from 90 to 150 minutes. Fractions 16 to 39 (starting at 105 mL and ending at 129 mL) were pooled and concentrated using centricon concentrators with a cutoff of 3500 MW.

[00746] The sample (2.0 mL at -4 mg/mL) was further chromatographed under denaturing conditions using a HiLoad 26/60 Superdex 200 prep grade column (Amersham Biosciences, Piscataway, NJ) at an isocratic flow rateof 2.0 mL/min using 200 mM NaH₂PO4, 6.0 M guanidine hydrochloride at pH 6.0 as mobile phase on an AKTAexplorer™ (Amersham Biosciences). Fractions 12-16 were collected, pooled, buffer-exchanged into 200 mM NaH₂PO4, pH 6.0 using a HiPrep 26/10 desalting column (Amersham Biosciences), and finally concentrated.

[00747] Preparation of induced single chain CTLA4-Ig: Induced single chain CTLA4-Ig was prepared through denaturation, reduction, and alkylation of CTLA4-Ig recombinant protein prepared by the methods of the invention. Guanidine hydrochloride (0.684 g) was weighed into a 1.5 mL Eppendorf centrifuge tube, and 512 gL of 200 mM NaH₂PO4, pH 6.0 was added and vortexed until the guanidine hydrochloride was completely dissolved. CTLA4-Ig recombinant protein was denatured by adding 238 gL of CTLA4-Ig material (concentration: 50 mg/mL) into the above tube and vortexed, resulting in a CTLA4-Ig final concentration of-10.0 mg/mL in 6.0 M guanidine hydrochloride. The denatured protein was reduced by adding 2.6 gL of 1.0 M DTT and incubating at 37°C for 90 minutes. The reduced protien was then alkylated by the addition of 0.047 g iodoacetamide solid into the sample mixture, followed by vortexing, and incubation at 37°C for 60 minutes in the dark. The sample (2.0 mL at -4.0 mg/mL for each injection) was chromatographed under denaturing conditions using a HiLoad 26/60 Superdex 200 prep grade column at an isocratic flow rate of 2.Ό mL/min using 200 mM NaH₂PO4,6.0 M guanidine hydrochloride at pH 6.0 on an AKTAexplorer™. The resulting single chain fractions (9-12) were collected, pooled, buffer-exchanged into 200 mM NaH₂PO4, pH 6.0 on a HiPrep 26/10 desalting column, and concentrated.

[00748] MALDI-TOF mass spectrometry analysis of naive and induced single chain CTLA4-Ig: The single chain samples (20 pL) were desalted and concentrated with C4 ZipTips (Millipore, Billerica, MA), then eluted with 20 pL of 80% acetonitrile with 0.1% TFA saturated with sinnapinic acid. The mixture (1.0 μί.) was spotted onto a well of the MALDI sample plate and allowed to air dry before being placed in the mass spectrometer. The MALDI-MS spectra were acquired on an OmniFlex mass spectrometer (Bruker Daltonics, MA) using a nitrogen laser (337 nm). Samples were analyzed in the reflective, positive-ion mode by delayed extraction using an accelerating voltage of 20 kV and a delay time of 200 ns. A total of 250 single-shot spectra were summed for each sample. External calibration was achieved using a mixture of Trypsinogen (23982 m/z), Protein A (44613 m/z), and Bovine Albumin (66431 m/z).

[00749] Single Chain Analysis using Denaturing (Guanidine HC1) Size Exclusion Chromatographv: CTLA4-Ig supernatant from cell culture growth collected at different points during the time course are prepared for HPLC analysis. The samples are prepared by weighing 0.114 g guanidine hydrochloride (Mallinckrodt Baker Inc.) in a 0.65 mL Eppendorf microcentrifuge tube; adding 125 uL of time course CTLA4-Ig sample, and vortexing to completely dissolve the guanidine HO. Then immediately adding 1.8 uL of250 mM iodoacetamide and mix, and incubating at 37°C for 30 minutes.

[00750] A tandem TSK-GEL®G3000SWxl size exclusion column (7.8 mm ID x 30 cm) with a TSK column guard (SWXL, 6.0 mm ID x 4.0 cm) is used for the single chain SEC analysis performed on the Waters 2695 separations module with a 2996 photodiode array detector. 25 rL of each sample is injected onto the column equilibrated with 200 mM sodium phosphate, 6.0 M guanidine hydrochloride pH 6.0 as mobile phase. The proteins are separated on the column with a flow rate of 0.5 mL/min, and the resulting chromatogram is collected over a 60 minutes window. The integration and quantitation of individual peaks (monomer, single chain, etc.) are performed using the Empower Pro software. To ensure the HPLC system is working properly, injections are also made on the mobile phase, the protein sample buffer, the system suitability standard, and the current CTLA4-Ig material before and after the sample injections. The peak resolution and plate count on the system suitability standard chromatogram are calculated.

[00751] Analysis of cysteinvlation of CTLA4-Ig single chain by LC/MS Peptide Analysis: CTLA4-Ig was denatured and reduced in 50 mM Tris buffer (pH 8.0) containing 6 M Guanidine and 5 mM dithiothreitol (DTT). After a 20 minute incubation at 50 °C, iodoacetamide (IAM) was added to a final concentration of 10 mM to alkylate free thiols and the incubation was continued for an additional 20 minutes at 50 °C in the dark. The reduced and alkylated mixture was loaded onto a desalting column (Amersham, NAP-5), then eluted into the void volume with either 50 mM Tris, 10 mM CaC12, pH 8.0 or 50 mM sodium phosphate buffer, pH 7.5. Following desalting, reduced/alkylated CTLA4-Ig was digested using two different proteases: trypsin or Asp-N. CTLA4-Ig material was also subjected to trypsin / chymotrypsin digestion without reduction and alkylation.

’ [00752] For trypsin digestion, sequence grade trypsin (Promega, 2%, w/w, enzyme/protein) was added and the mixture was incubated for 4 hours at 37 °C. For Asp-N digestion, sequence grade Asp-N (Roche, 2%, w/w, enzyme/protein) was added and the mixture was incubated for 16 hours at 37 °C. For the trypsin/chymotrypsin digestion, sequence grade trypsin (Promega, 4%, w/w, enzyme/protein) was added and the mixture was incubated for 4 hours at 37 °C, then a-chymotrypsin was added (Sigma, 4%, w/w, enzyme/protein) and the mixture was incubated for 16 hours at 37 °C. All samples were frozen (-20 °C) after the digestion.

[00753] The resulting peptide mixtures were separated by gradient elution from a Waters Atlantis^TM dC18 column (2.1 x 250 mm) on a Waters Alliance HPLC Workstation at 0.12 mL/minute. The column was directly connected to the Waters Micromass Q-Tof micro^TM mass spectrometer equipped with an ion-spray source for collection of mass spectra. Peptide mixtures were also separated on a Varian Polaris Cl 8 column (4.6 x 250 mm) at 0.70 mL/minute using the same HPLC workstation. The columns were equilibrated with solvent A (0.02% TFA in water) and peptides were eluted by increasing concentration of solvent B (95% acetonitrile/0.02% TFA in water). A post-column splitter valve was used to direct 15% of the flow to the Q-Tof workstation, which was run in the positive TOF (time of flight) mode (m/z 100-2000). The typical ion spray voltage used was 3000 V.

[00754] The loss of 113 ± 4 u upon reduction suggests that there is a covalent disulfide modification to the protein. The predicted shift for cysteinylation is 119.14 u. However, an actual mass loss of 111.14 u is expected upon reduction of the single chain species. Loss of 119.14 u results from the removal of a cysteine and a gain of 8 u results from the addition of 8 protons upon reduction of the eight intra chain cysteines (Cys ⁴⁷’ ⁷⁴’ ^92303,361 ’¹⁵⁷ ’^{197 &lt;8״}׳ of SEQ ID NO:2). Thus, the additional 113 ± 4 u on the intact mass corresponds to cysteinylation with a cysteine amino acid. The cysteine most likely to be modified is Cys¹⁴⁶ since the interchain disulfide linkage is absent in the single chain species based on the intact MALDI data. Using LC/MS peptide analysis to examine the peptides, which contain Cys¹⁴⁶, it is found that reduced and non-reduced material display different retention times and masses than the single chain material. To confirm cysteinylation, the single chain peptide containing Cys¹⁴⁶was collected and analyzed using MALDI.

[00755] The collected peak containing Cys¹⁴⁶ has a mass of 1787.48 u, in agreement with the predicted mass of 1787.51 u for the peptide with a cysteinylation of Cys¹⁴⁶ (FIG. 27, panel A). This peptide, after being subjected to reduction, loses 119.11 u in agreement with a predicted loss of 119.14 u; the loss of cysteine (FIG. 27, panel B). The material is then further manipulated with iodoacetamide producing a shift of 56.99 u in agreement with a predicted mass gain of 57.03 u (FIG. 27, panel C).

Example 5: Manipulating Monomer or Multimer CTLA4-Ig Formation

[00756] Agonistic Effects of Media And Media Components on Single Chain Formation:

The agonistic affects of different media and media components are determined for single chain formation. CTLA4-Ig molecules can be incubated at 37°C with various media and individual media constituents over a period of 60 hours and analyzed for single chain formation by tandem column denaturing SEC. An overwhelming agonist response for single chain formation is found upon the addition of 10 mM cysteine to formulation buffer. There is a rapid rise in single chain formation which peaks around six hours following the addition of cysteine. This response gradually decreases over the remaining 56 hours. In addition, the +30% gal feed affected a more gradual but still relatively rapid increase in single chain formation. The +30% gal feed is a composition of galactose and 117E. This mixture is added every day to feed the cells. While in this experiment, cysteine was introduced at artificially large quantities independent of 117E, there is a need to determine which of the 117E components can be involved in single chain formation.

[00757] Specific components of 117E and other media are incubated with CTLA4 Ig over a period of 60 hours and analyzed for single chain formation by tandem column denaturing SEC. Investigation into this medium centers around possible disulfide reducing components and/or inhibitors, which would effect single chain formation based on previous experiments showing the interchain disulfide is not present. The constituents of 117E which are known to have some reducing affects on disulfides were tested; lipoic acid, cystine, cysteine, methionine, and glutathione. Again, an overwhelming agonist response for single chain formation is found upon the addition of cysteine to formulation buffer. There is a rapid response in single chain formation, which peaks at around six hours, following the addition of cysteine. This response gradually decreases over the remaining 56 hours. The major single chain formation occurs with cysteine containing media: cysteine, yeastolates and fermentation media. The other sulfur containing components such as methionine, and glutathione have no to very little affect on single chain formation. There are no affects of ammonium chloride observed.

[00758] The present invention therefore encompasses a method for providing a ratio of single chain: dimer form of a protein, such protein capable of existing in dimer as well as in single chain form, comprising the steps of (1) providing and/or maintaining (such as during step (2)) a liquid cell culture medium for the culture of cells expressing said protein, in which the concentration of an agent capable of reducing or inhibiting dimer formation (such as cysteine) is selected to provide said ratio, and (2) culturing said cells to express said protein. Adding and/or increasing the concentration of such an agent (for example, cysteine) in a liquid cell culture medium provides a higher ratio of single chain:dimer form of such protein, while removing, decreasing or eliminating the concentration of such an agent (for example, cysteine) in a liquid cell culture medium decreases the ratio of single chain:dimer form of said protein.

[00759] One particular embodiment of this method is where said protein is a glycoprotein capable of dimer formation through the formation of an interchain disulfide bond, such as the CTLA4-Ig molecules of the present invention.

[00760] Agonistic Effects of High Salt on High Molecular Weight Formation: During the purification process, CTLA4-Ig is exposed to high salt concentrations for varying amounts of time. The affects of high salt concentrations are determined for high molecular weight formation. CTLA4-Ig (at ~ 50 mg/mL) is incubated in the presence of 500 mM sodium phosphate, pH = 6.0, 37°C. There is an agonistic affect at high concentrations of sodium phosphate; a sustained, rapid increase in high molecular weight forms, mostly tetramer, is observed over a period of 100 hours.

[00761] The present invention therefore encompasses a method for reducing the ratio of aggregate: dimer form of a protein, such protein capable of existing in aggregate as well as in dimer form, during processing (such as during purification) of such protein, comprising the use of one or more liquids which are non-aggregate salt solutions. A “non-aggregate salt . solution” refers to a liquid containing a concentration of salt dissolved therein which is, relative to the same liquid containing a higher concentration of such salt, less agonistic in the formation of aggregate.

[00762] One particular embodiment of this method is where said protein is a glycoprotein capable of dimer formation through the formation of an interchain disulfide bond, such as the CTLA4-Ig molecules of the present invention.

[00763] Antagonistic Effects on Single Chain Formation: The previous modeling experiments demonstrated a large and rapid affect on single chain formation by the addition of cysteine containing components. Cysteine is an amino acid which contains a free sulfhydryl. If the sulfhydryl is involved in the formation of single chain it should be blocked through the use of antagonistic compounds. One such compound is iodoacetamide, lodoacetamide is a water-soluble compound that reacts in a rapid fashion with any free sulfhydryl to form an irreversible thioether bond. CTLA4-Ig is incubated at 37°C with various medias, cysteine, and iodoacetamide over a period of 60 hours and analyzed for single chain formation by tandem column denaturing SEC and HMW by tandem column non-denaturing SEC. lodoacetimide not only blocks single chain formation but also blocks aggregate formation in both a CTLA4-Ig composition and high salt. lodoacetamide does not block the aggregate formation in low sialic acid monomer. However, the amount of aggregate formed in low sialic acid material is comparable to the amount formed in CTLA4Ig composition.

[00764] The model provides insight to a mechanism that has not previously been well understood. It appears that there are at least two major pathways of aggregate formation in the CTLA4-Ig process that have been identified. The first pathway, which produces the large amount of aggregate, free sulfhydryl cysteine acts as an agonist for the formation of single chain species. The agonistic affect of cysteine can be blocked by the addition of iodoacetamide. Surprisingly, iodoacetamide is not only an antagonist for single chain formation but also high molecular weight formation. It should be noted that the process is designed to produce a composition that contains an increased amount of sialic acid as compared to fermentation during the downstream purification. In a second path, which produces much less aggregate, a subspecies which contains low amounts of sialic acid is not affected by iodoacetamide for the formation of single chain or aggregate.

[00765] The present invention therefore encompasses a method for decreasing the ratio of single chaimdimer form of a protein, such protein capable of existing in dimer as well as in single chain form, comprising the steps of (1) providing and/or maintaining (such as during step (2)) a liquid cell culture medium for the culture of cells expressing said protein, such medium containing an agent antagonistic to single chain formation (such as iodoacetamide), and (2) culturing said cells to express said protein.

[00766] The present invention also encompasses a method for decreasing the ratio of aggregate: dimer form of a protein, such protein capable of existing in aggregate as well as in dimer form, comprising the steps of (1) providing and/or maintaining (such as during step (2)) a liquid cell culture medium for the culture of cells expressing said protein, such medium containing an agent antagonistic to aggregate chain formation and/or antagonistic to single chain formation (such as iodoacetamide), and (2) culturing said cells to express said protein.

[00767] One particular embodiment of this method is where said protein is a glycoprotein capable of dimer formation through the formation of an interchain disulfide bond, such as the CTLA4-Ig molecules of the present invention.

[00768] Based on these data, it is not difficult to imagine at least two pathways to aggregate formation are induced in CTLA4-Ig. In the major pathway, single chain is involved in the formation of aggregate through a yet not completely clear mechanism. A second minor pathway, which is independent of single chain formation, is involved in the formation of aggregate. These pathways can help to explain why there are at least three forms of high molecular weight species that can be chromatographically separated. These are models and must be tested during the actual fermentation process in order to determine the actual affects if any and magnitude of the affects. Based on this data consideration should be given to testing not only the current fermentation process but also fermentation devoid of cysteine.

Example 6: CTLA4-Ig Dimer and Tetramer

[00769] CTLA4-Ig dimer and tetramer binding to B7-1 Ig

[00770] The invention provides methods for evaluation of CTLA4-Ig dimer and tetramer binding to B7-lIg. Physical characteristics (e.g. diffusion coefficient, molecular weight, binding valency) and instrument operational parameters (e.g. flow rate, chip density) can influence the Biacore assay results. Under mass transfer limitation; the binding rate of tetramer is approximately 20% slower than dimer for the same molar concentration. At a specified flow rate, it is possible that tetramer molecules penetrated the matrix less efficiently compared to the dimer. Under a high density B7-1 Ig immobilized chip, competitive binding of tetramer and dimer exhibits comparable inhibition curves. This indicates that the theoretical valency of tetramer (four) versus dimer (two) has no influence on binding to B7-1 Ig. Molar concentrations of tetramer can be calculated based on a dimer standard curve. Using this approach, the binding of tetramer to B7-lIg was found to have equivalent dose dependent response compared to dimer.

[00771] Comparison of the binding potency of a tetramer and a dimer is influenced by the unit of measurement used for the preparation of standards and samples. Tetramer and dimer samples can be compared at a concentration of 2000 ng/mL. On a mass basis (ng/mL), each species shows a binding potency of approximately 100% using a standard curve of the same species. However, the binding potency of tetramer was halved when determined on a dimer standard curve, and the binding potency of a dimer was more than doubled when determined on a tetramer standard curve. Since the Biacore instrument detects molecular interactions based on mass, the signal resonance units (RU) from identical concentrations (ng/mL) of tetramer and dimer should be the same. Although the detection system is a function of mass, the interaction between molecules occurs on a molar basis. Therefore, the molar concentrations of CTLA4-Ig dimer and CTLA4-Ig tetramer are 21.6 nM and

10.8 nM, respectively, at 2000 ng/mL. Using the molar concentration, both CTLA4-Ig dimer and CTLA4-Ig tetramer samples show comparable binding potency on a dimer standard curve. Using the same molar concentration approach on a CTLA4-Ig tetramer standard curve, the CTLA4-Ig dimer sample shows an additional 30% increase in binding potency compared to the tetramer sample. This observation is due to a decrease in the slope , of the tetramer standard curve at high concentrations, resulting in a higher calculated concentration for a given initial binding rate. One explanation for this observation is the..

effect of mass transfer.

[00772] Mass transfer limitation experiment indicates that the initial binding rate of the CTLA4-Ig tetramer is approximately 20% slower than the CTLA4-Ig dimer for the same number of molecules (i.e. the binding rate of the tetramer differs from dimer by a factor of 0.8). This observed difference is due to the molecular weight of the two species and its effect on diffusion of the molecules to the surface of the chip. The diffusion coefficient of a molecule is inversely proportional to the cube-root of the molecular weight. A lower diffusion coefficient would indicate slower movement of the molecules. Based on respective molecular weights, the calculated diffusion coefficient of CTLA4-Ig tetramer is 0.8 times that of the CTLA4-Ig dimer, or conversely the dimer is 1.25 times that of the tetramer. Experimental data are consistent with this observation where the CTLA4-Ig dimer shows a potency of 133% as calculated from a CTLA4-Ig tetramer standard curve using molar concentrations. Mass transfer limitations on high-density chips are more pronounced at lower flow rates and lower analyte concentrations. As the flow rate increases, the dimer shows faster initial binding rates compared to tetramer. Therefore, the increased molecular weight and lower diffusion coefficient of the tetramer contribute to initial binding rate differences compared to dimer.

[00773] Competitive binding of CTLA4-Ig tetramer and dimer to B71Ig indicates that tetramer and dimer show similar binding valency under mass transfer limited conditions. The effects of additional tetramer onto a B7-lIg chip initially bound with either dimer or tetramer indicate low binding potential. This observation is not due to limited binding site availability because additional dimer could bind to B7-lIg chips initially bound with CTLA4-Ig dimer or CTLA4-Ig teramer. Limited penetration into the matrix can explain the observed decrease in tetramer binding.

[00774] The nature in which molecules bind on the Biacore affects the interpretation of the results. The tetramer and dimer molecules diffuse.at different rates due to their differences in molecular size under the condition of mass transfer limitation. In addition, steric hindrance on the surface of a high density chip affects penetration of subsequent molecules to the matrix.

[00775] Physical characteristics such as the molecular weight, diffusion coefficient, and binding of each species need to be considered when performing concentration analyses on the Biacore. Standards used for comparison to the analyte of interest should be of the same material. However, tetramer can still be analyzed against dimer standards if both the standards and samples are expressed on a molar basis where molecular size is taken into consideration. The data presented indicate that the binding of CTLA4-Ig dimer and CTLA4Ig tetramer to B7-lIg is comparable.

[00776] Both CTLA4-Ig dimer and tetramer show similar association rates (k_on) However, the tetramer shows a slower dissociation rate (k_Qff) which is attributed to avidity due to the increase in the number of binding sites.

[00777] Binding kinetic analysis between two proteins such as a ligand and receptor can be performed on the Biacore using a chip immobilized with a low density of ligand of approximately 600-800 RUs such that the maximum binding capacity (R_max) is in the range of 50-150 RU. The purpose of a low-density chip is to minimize the effects of avidity and mass transport. Avidity is observed when multivalent analytes remain bound to the surface of the chip due to close proximity of ligands available as dissociation of individual binding sites occurs. Mass transport is observed when there is a significant difference in the analyte concentrations between the surface of the chip and the bulk solution.

[00778] In one embodiment, the CTLA4-Ig molecule is a dimer consisting of two singlechain molecules linked by a single interchain-disulfide bond, and contains two binding sites for B7 molecules. In another embodiment, formation of CTLA4-Ig tetramer, as confirmed by light-scattering, SEC and SDS-PAGE, results in a molecule with potentially four binding sites. The binding kinetics of purified monomer and dimer are statistically compared to CTLA4-Ig dimer material. There are no significant differences in the k_on rates (p values > 0.05). The Aerate of tetramer is significantly different from the Aerate of the dimer. The Aerate of the dimer purified from the HIC cleaning peak is not significantly different from the kqffreAe of the CTLA4-Ig dimer material. Therefore, the tetramer dissociates slower than the dimer, indicating an avidity effect due to the increased number of binding sites per molecule.

[00779] Tetramer can be unfolded and dissociated into dimer by guanidine treatment. This guanidine-treated CTLA4-Ig dimer was analyzed and the results indicate that its binding kinetic characteristics were similar to those of the CTLA4-Ig dimer formed under physiological conditions. This observation supports the hypothesis that the observed difference in the Aerate between dimer and tetramer is related to binding valency of the molecules and the inherent nature of the specific Biacore method where avidity plays a role in the binding kinetics.

[00780] Affinity purification of CTLA4-Ig material from HIC cleaning peak:

[00781] Protein A-Sepharose affinity chromatography: 500 mL of HIC cleaning peak was filtered through a 0.22 micron IL filter system (Coming, Coming, NY, Part no. 430517) and loaded onto a rProtein A-Sepharose column (5 cm x 15 cm), which was pre-equilibrated with phosphate buffered saline (Sigma, St. Louis, MO, P-4417) at pH 7.4. The column was washed with 700 mL PBS, pH 7.4 and eluted with 100 mM glycine, pH 3.5. Fractions of 50 mL each were neutralized during collection by the addition of 0.5 mL of 2.0 M tris, pH 10 to the collection tubes. Fractions were assayed at 280 nm absorbance, pooled and concentrated using a centriprep YM-3 cartridge (Millipore Corporation, Bedford, MA, Part no. 4203). The purified protein solution was stored at -70° C .

[00782] PROSEP- rA (recombinant Protein A) affinity chromatography: 500 mL of HIC cleaning peak was filtered through a 0.22 micron 1L filter system (Coming, Coming, NY, Part no. 430517) and loaded at a flow rate of 25 mL/minute on a PROSEP-rA High Capacity (Millipore Corporation, Bedford, MA) column (25 mm x 28 cm), which was pre-equilibrated with 25 mM Tris, pH 8.0 containing 250 mM sodium chloride. Waters PrepLC system equipped with Waters 2767 Sample Manager and Waters 2996 Photodiode Array Detector was used for this chromatography. The column was washed with 25 equilibration buffer for 30 minutes at a flow rate of 25 mL/minute and eluted with 100 mM acetate, pH 3.0 at a flow rate of 25 mL/minute for 30 minutes. Fractions of 10 mL each were neutralized during elution by collecting over 50 ul of 2.0 M tris, pH 10, which was previously added to the tubes. Fractions having high absorbance at 280 nm were pooled and concentrated using a centriprep YM-3 cartridge (Millopore Corporation, Bedford, MA, Part No. 4203). The purified protein solution was stored at -70° C

[00783] Size Exclusion Chromatography: Size exclusion chromatography was performed on a Waters Alliance 2695 separations module equipped with a Waters 2996 Photodiode Array Detector (Milford, MA), and a Foxy 200 fraction collector controlled by Millennium³² version 3.20 or Empower software. Tandem TOSOH BIOSCIENCE (Montgomery, PA) TSK

G3000 SW (21.5 mm x 300 mm) and tandem TSK G3000 SWxL (7.8 mm x 300 mm) columns were used for preparative and analytical SEC, respectively. Eluted proteins were monitored by UV absorbance at 280 nm.

[00784] Preparative isolation of dimer, tetramer and multimer was achieved by SEC of Protein A purified HIC cleaning peak material. Thirteen samples (~10 mg each) of Protein A eluate were injected onto a preparative tandem SEC column using a mobile phase of 100 mM monobasic sodium phosphate buffer pH 7.0 containing 0.9% sodium chloride at a flow rate of 1 mL/min. Fractions were collected at every minute from 90-160 minutes for each of the thirteen injections. The run time for a single injection was 180 minutes.

[00785] Each fraction was examined by tandem column analytical size exclusion HPLC. Fractions 13-15 (containing multimer), fractions 22 and 23 (containing teramer), and fractions 43-49 (containing dimer) were pooled. Purified multimer, tetramer and dimer fractions were examined on analytical two column SEC with dynamic light scattering detection (DSL).

[00786] Biospecific binding analysis of CTLA4-Ig component of the HIC cleaning peak and purified components to immobilized B7-1 Ig: The biospecific binding of CTLA4-Ig to immobilized B7-lIg (on a CM5 chip) was measured using a SPR based BIAcore C biosensor (BIAcore, AB, Piscataway NJ). CTLA4-Ig material was used to generate the standard curve. B7-lIg was immobilized at a density of 5000 to 10,000 resonance units (RU's) on an activated CM5 sensor chip. CTLA4-Ig reference standards, quality controls, and samples were injected at a flow rate of 20 pL/min. over the B7-lIg sensor chip surface to generate sensorgrams. The initial binding rate (RU/s) of CTLA4-Ig onto immobilized B7-lIg was measured under diffusion-limited conditions on a high density B7-lIg surface, and this correlates directly to the active concentration of CTLA4-Ig in the samples. Standard, quality control sample, and unknown sample concentrations were interpolated from the standard curve generated by plotting the RU's versus CTLA4-Ig concentrations in the nominal range of 125-8000 ng/mL. The final results were expressed as percent binding (Mean Concentration of unknown / sample / 2000) xlOO.

[00787] Determination of molar mass and hydrodynamic radius: The SEC separation was performed with a TSK3000 SWXL column and corresponding guard column. The mobile phase consisted of 25mM HEPES, 850mM NaCI, pH 7.0, using isocratic conditions for elution at 0.8 mL/min. HPLC analyses were performed at ambient temperatures and samples maintained at 4°C during analysis. Molar mass determination incorporated the Wyatt Dawn EOS utilizing 15 distinct scattering angles to measure the angular variation of light scatter for each species. A Zimm plotting formalism was used for molar mass determination where slices for each species were averaged for molar mass. The specific refractive index increment (dn/dc) value used to calculate absolute molar mass was 0.189 obtained using a) and an Optilab DSP Interferometer (RI. Hydrodynamic radius (Rh) determination was performed inline with a Photon Correlator QELS detector positioned at an angle approximately 90° to the flow cell. The translational diffusion constant is measured from this signal and Rh is calculated using the Einstein-Stokes relationship. Data analysis was accomplished using Astra software version 4.90 from Wyatt Technology.

[00788] The molecular weight and hydrodynamic radius values for dimer and tetramer species were found to be 86 - 91 kDa and 172- 199 kDa, respectively. The cleaning peak samples were observed to contain additional HMW species corresponding to hexamer and decamer by molecular weight. The range of the hydrodynamic radii for the dimer species is

3.8 - 4.7 nm. The ranges of the hydrodynamic radii of the heterogeneous tetramer species were 5.7 nm to 6.2 - 6.3 nm.

[00789] Binding of CTLA4-Ig dimer and tetramer to B7-lIg using surface plasmon resonance: Concentration Analyses: Concentration analyses of the various CTLA4-Ig species to B7-Hg were performed on a Biacore 3000 instrument (Biacore, Piscataway, NJ) according to Method 7441-4² with minor modifications. Modifications include the following: Biacore 3000 was used instead of Biacore C. The flow rate was 10 μΕ/min instead of 20 / μΕ/min. Sample was injected for 60 seconds as opposed to 15 seconds. The sensor chip surface was regenerated at 30 μΕ/min by three short 30-second (15 μΕ) pulses of 10 mM sodium citrate, pH 4.0, containing 100 mM NaCI (regeneration buffer), followed by one 30-second pulse of water.

[00790] A CM5 sensor chip was immobilized with B7-lIg at a concentration of 20 gg/mL in acetate buffer, pH 5.0, aiming for a target density of 6000-12000 resonance unit (RU). Standards were prepared by serially diluting CTLA4-lg material to concentrations of

62.5-8000 ng/mL (0.675-86.3 nM) and dimer to concentrations of 125-16000 ng/mL (0.67586.3 nM) in HBS-EP buffer. Test samples, consisting of either monomer or dimer, were diluted to a target concentration of ~2000 ng/mL and analyzed on the Biacore.

Concentrations were determined by the BIAevaluation software (version 4.0.1) using standard curves of either CTLA4-Ig material (> 98% monomer) or purified dimer. The binding potency is calculated as the percentage of theconcentration determined on the

Biacore divided by the concentration determined by A280 nm.

[00791] The binding rate of a molecule to a given surface is a function of concentration, which allows for the determination of unknown concentrations. CTLA4-Ig dimer exhibits a higher binding rate as a function of concentration (ng/mL) as compared to CTLA4-Ig tetramer.

[00792] The binding potencies of CTLA4-Ig dimer and tetramer are summarized in Table?. Based on ng/mL, the binding potency of a dimer sample is calculated from a dimer standard curve and found to be 99.5%; whereas, an equivalent concentration of a tetramer sample is 47.2%. Conversely, the binding potency of a dimer sample is calculated from a tetramer standard curve and found to be 266%; whereas, an equivalent concentration of a tetramer sample is 103%. However, when dimer and tetramer are expressed as molar concentrations (nM), the binding potencies of both species are comparable. The binding potency of dimer and tetramer samples calculated from a dimer standard curve are 99.4% and 94.3%, respectively. On a tetramer standard curve, it is 133% and 103%, respectively. Standard curves of dimer and tetramer are comparable based on molar concentrations.

Table 9: Binding Potencies of CTLA4-Ig Dimer and Tetramer.

<td></td><td colspan="2"> Dimer Standard Curve</td><td colspan="2"> Tetramer Standard Curve</td>

<td> Sample</td><td> ng/mL</td><td> nM</td><td> ng/mL</td><td> nM</td>

<td> Dimer</td><td> 99.5%</td><td> 99.4%</td><td> 266%</td><td> 133%</td>

<td> Tetramer</td><td> 47.2%</td><td> 94.3%</td><td> 103%</td><td> 103%</td>

[00793] Binding Valency: CTLA4-Ig dimer (25-1600 nM) or tetramer (25-400 nM) was pre-mixed at various molar ratios with B7-lIg for three minutes before 30 (μΕ of the mixture was injected at a flow rate of 10 μΕ/min over a B7-1 Ig chip immobilized with a density of 9392 RU. The chip was regenerated after each injection by three 30-second pulses of regeneration buffer followed by one 30-second pulse of water. The RU's obtained at the end of each injection were compared.

[00794] Theoretically, the binding valency of the dimer molecule is two; each single chain consists of one binding site. A tetramer molecule consists of two dimer molecules, and thus has a binding valency of four. To determine the apparent binding valency of dimer and teramer, a competitive assay was designed and conducted on the Biacore 3000. In the experiment, analytes were pre-mixed with B7-Hg at various molar ratios for three minutes before the mixture was injected onto a B7-lIg chip (9392 RU) at a flow rate of 10 μΕ/min for one minute. Table 10 shows the percentage of either dimer or tetramer that was competitively inhibited with increasing molar amounts of B7־llg. At a molar ratio of 1.25 (B7-lIg) to 1 (dimer or tetramer), a significant difference was observed in competitive inhibition with monomer at 96.1% and dimer at 84.9%. However, dimer and tetramer exhibit similar inhibition curves, this suggests that the valencies are approximately equal. In addition, both dimer and teramer also showed similar inhibition profiles using lower density chips.

Table 10: Inhibition of Dimer and Tetramer with B7-lIg.

<td></td><td colspan="3"> Dimer (% Inhibition)</td><td colspan="3"> Tetramer (% Inhibition)</td>

<td> B7-lIg Molar Ratio</td><td> N</td><td> Avg</td><td> S.D.</td><td> N</td><td> Avg</td><td> S.D.</td>

<td> 0.02 to 1</td><td> 1</td><td> 1.5</td><td> n/a</td><td> 1</td><td> 3.4</td><td> n/a</td>

<td> 0.04 to 1</td><td> 2</td><td> 1.2</td><td> 0.2</td><td> 2</td><td> 5.1</td><td> 0.3</td>

<td> 0.08 to 1</td><td> 3</td><td> 2.9</td><td> 1.1</td><td> 3</td><td> 7.4</td><td> 0.8</td>

<td> 0.16 to 1</td><td> 4</td><td> 5.5</td><td> 0.7</td><td> 4</td><td> 12.6</td><td> 0.9</td>

<td> 0.32 to 1</td><td> 5</td><td> 14.7</td><td> 3.3</td><td> 5</td><td> 20.9</td><td> 2.2</td>

<td> 0.64 to 1</td><td> 5</td><td> 38.1</td><td> 6.1</td><td> 4</td><td> 40.8</td><td> 3.5</td>

<td> 1.25 to 1</td><td> 5</td><td> 96.1</td><td> 2.6</td><td> 3</td><td> 84.9</td><td> 4.6</td>

<td> 2.5 to 1</td><td> 4</td><td> 99.7</td><td> 0.1</td><td> 2</td><td> 97.5</td><td> 0.5</td>

<td> 5.0 to 1</td><td> 3</td><td> 99.9</td><td> 0.1</td><td> 1</td><td> 99.4</td><td> n/a</td>

<td> 10.0 to 1</td><td> 2</td><td> 100.0</td><td> 0.0</td><td> n/a</td><td> n/a</td><td> n/a</td>

<td> 20.0 to 1</td><td> 1</td><td> 100.0</td><td> n/a</td><td> n/a</td><td> n/a</td><td> n/a</td>

[00795] Saturation of B7-lIg Chip: CTLA4-Ig dimer or tetramer (200, 1000, or 8000 ng/mL) was initially injected at 10 μΕ/min for one minute over a high density B7-lIg chip (6738 RU) followed by a series of seven 1-minute injections with either monomer or dimer (200,1000, or 8000 ng/mL). The chip was regenerated after each condition by four 25 μΕ injections of regeneration buffer followed by 25 μΕ injection of water. The RU's obtained at the end of each injection were compared.

[00796] Either dimer or tetramer was repeatedly injected over a B7-lIg surface pre-coated with either dimer or tetramer without surface regeneration. Initial binding with tetramer does not impede subsequent injections of dimer from binding, however, additional tetramer injections result in a significantly decreased rate of binding as compared to dimer injection. Initial binding with dimer followed by subsequent injections of dimer results in an increased binding towards saturation. Subsequent injection of tetramer to the dimer pre-coated chip shows a gradual decrease in binding, indicating a dissociation of molecules from the chip and a lack of tetramer penetration into the matrix. Similar results are observed with initial injection of either 200 ng/mL or 8000 ng/mL of CTLA4-Ig molecules.

[00797] CTLA4-Ig tetramer has higher avidity to the B7-lIg receptor: CTLA4-lg species, including, the discarded portions of purification columns such as the cleaning peaks of HIC and QFF columns were purified and their binding kinetics were analyzed on the Biacore.

[00798] CTLA4-Ig Species from HIC Cleaning Peak: The HIC column is used in the CTLA4-Ig process to remove high molecular weight species such as DNA. CTLA4-Ig species from the cleaning peak from the HIC column can be used for subsequent purification and kinetic analysis. The HIC cleaning peak was passed through a Protein A column to capture all CTLA4-Ig species and to remove other impurities that can be present. The eluate from the Protein A column, which consisted of a mixture of all CTLA4-Ig species (i.e., dimer, tetramer, hexamer multimer), showed apparent k_on and k^ff rates which were comparable to the CTLA4-Ig dimer standard. Separation of the Protein A eluate by 2column SEC resulted in three CTLA4-Ig species: dimer, tetramer, and hexamer/multimer, with k_on and kfr rates as summarized in Table 11. Sialic acid content of the purified dimer from the HIC cleaning peak was low compared to CTLA4-Ig dimer.

[00799] In Table 11, sample concentrations of 75-200 nM were tested on B7-lIg chip (694 RU). CTLA4-Ig species purified from the HIC cleaning peak showed lower binding compared to CTLA4-Ig reference. Tetramer which was disaggregated gave comparable k_onand gyrates to CTLA4-Ig reference.

Table 11: Kinetic Analysis of CTLA4-Ig Species from HIC Cleaning Peak

<td> Species</td><td> konX 10⁵(1/Ms)</td><td> AojT-xIO’Q/s)</td><td> ׳K_Axl0 . (1/M)</td><td> Kd (nM)</td>

<td> CLTA4-Ig dimer standard</td><td> 4.03</td><td> 9.65</td><td> 4.18</td><td> 23.9</td>

<td> Protein A Eluate</td><td> 3.77</td><td> 9.73</td><td> 3.87</td><td> 25.8</td>

<td> Dimer</td><td> 1.52</td><td> 5.88</td><td> 2.59</td><td> 38.7</td>

<td> Tetramer</td><td> 1.65</td><td> 8.07</td><td> 2.04</td><td> 48.9</td>

<td> Hexamer</td><td> 1.54</td><td> 12.3</td><td> 1.25</td><td> 79.9</td>

<td> Dimerized Tetramer</td><td> 3.12</td><td> 9.88</td><td> 3.16</td><td> 31.7</td>

[00800] Statistical analysis of the data was performed using the Student's T-test based on 7 observations of the CTLA4-Ig dimer standard and 14 observations of purified dimer and tetramer. There was no difference in the k_on rates comparing the CTLA4-Ig dimer standard with either purified dimer or tetramer. However, the kqffrate and Kd were statistically significant when the reference was compared with purified tetramer (Table 12).

Comparing the purified tetramer to purified dimer, both the k_on and kfif rates as well as the Kd were statistically different. It should be pointed out that although the data were grouped into dimer and tetramer, individual classifications of samples (i.e., frontal, backside, etc) can have slightly different characteristics that can affect their binding kinetics. For the dimer, the k_on and Aerates averaged 3.3 ± 1.0 x 10⁵M-ls-' and 8.8 ± 3.5 x 103s-¹, respectively. The the k_on and Aerates of the tetramer were 2.6 ± 0.8 x 10⁵M-ls-* and 3.1 ± 1.4 x 103s-¹, respectively.

[00801] In Table 12, Dimer (n=14) and tetramer (n=14) and CTLA4-Ig dimer standard (n=7) were analyzed.

Table 12: Statistical Analysis of CLTA4-Ig Species.

<td> Student's T-test</td><td colspan="3"> p-values</td>

<td></td><td> k.״.</td><td> k_orr</td><td> K_D</td>

<td> Dimer standard vs. Dimer</td><td> 0.9118</td><td> 0.8678</td><td> 0.7893</td>

<td> Dimer standard vs. Tetramer</td><td> 0.1044</td><td> 0.0000002</td><td> 0.0002</td>

<td> Dimer vs. Tetramer</td><td> 0.0372</td><td> 0.000006</td><td> 0.0004</td>

[00802] CTLA4-Ig Snecies from OFF Cleaning Peak: The QFF column is the last purification column used to clean up residual impurities from the product. CTLA4-Ig' species were isolated from the cleaning peak of the QFF column using the 2-column SEC method and analyzed on the Biacore 3000. The data showed that the binding kinetics of this QFF cleaning peak sample was similar to the CTLA4-Ig dimer standard. In addition, both dimer and tetramer־purified from the QFF cleaning peak gave similar binding kinetics compared to those purified from the composition. Furthermore, sialic acid contents of the purified monomer fractions were greater than that of CTLA4-Ig dimer standard.

[00803] Guanidine Treatment (Dimerization of the tetramer): The tetramer can be converted to dimer by treatment with guanidine followed by dialysis into phosphate buffer and confirmed to exist as a dimer by analytical SEC. Kinetic analysis of the dimerized tetramer from the HIC cleaning peak showed that its k_on and Aerates were similar to those of CTLA4-Ig dimer.

Example 7: Intact Analysis by MS Electrospray Ionization (ESI)

[00804] CTLA4-Ig was diluted with 100 mM Tris, 25 mM NaCl, pH 8 to a final concentration of 0.7 mg/mL. PNGase F (New England Biolabs) was diluted 30-fold with 100 mM Tris, 25 mM NaCl, pH 8 to a final concentration of 17 units/pL. Equal volumes (60 pL each) of diluted sample and diluted glycosidase solution were mixed and incubated at 37°C for 4 hours.

[00805] The resulting deglycosylated CTLA4-Ig (2 pL) was loaded onto a Waters Oasis® reversed phase extraction cartridge column (2.1 x 20 mm). The loaded column was washed with 5% solvent B (solvent A: 1% formic acid in water, solvent B: 1% formic acid in acetonitrile) at a flow rate of 0.2 mL/minute for five minutes to desalt, with the eluant diverted to waste. At the end of 5 minutes, a fast gradient (5% solvent B to 95% solvent B in 10 minutes) began the elution of CTLA4-Ig off the column; here the eluant was directed into the mass spectrometer (Waters Micromass Q-Tof micro^TM) at 45 pL/min after flow splitting (chromatography system used was a Waters Alliance 2695 equipped with a Waters 2996 detector).

[00806] The capillary voltage for the Q-Tof micro^TM was set at 3kV and the sample cone voltage at 40 V. The scans (every 0.9 second) were averaged into one scan; the inter-scan time was 0.1 second. The Q-Tof analyzer scans from m/z 800 to 2500. Spectra corresponding to the portion higher than half the maximum peak height (in TIC chromatogram) were combined using Waters MassLynx^TM software. The combined spectra were subjected to Waters MaxEntl deconvolution. The resolution was set at 1 Da/Channel, and the uniform Gaussian damage model was selected with width at half height set between 0.5-1 Da. Minimum intensity ratios for the left peak and the right peak were both set at 50%.

Example 8: Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF)

[00807] MALDI-MS spectra were acquired on an OmniFlex™ (Broker Daltonics, MA) using a nitrogen laser (337 nm). Protein samples were used without desalting or desalted using solid phase extraction in the form of C4 ZipTip® pipette tips (Millipore, Bedford MA). The pipette tips were wetted with acetonitrile-water (1:1 v/v) and equilibrated with 0.1% tri fluoroacetic acid (TFA) prior to use. The pipette tips were then loaded by drawing and expelling 10 μΕ of sample from the pipette three times. The loaded sample was washed three times with 10 μΕ of 0.1% TFA. Desalted protein samples were eluted from the pipette with 10 μΕ of acetonitrile to water (1:1 v/v). Samples were spotted by mixing a 1 μΕ sample (either desalted or buffer containing) with 1 μΕ of matrix solution and placing 1 μΕ of the mixture on the stainless steel target. The matrix solution was a saturated solution of sinapic acid in 1:1 water to acetonitrile (v/v) containing 0.1 % TFA. The mixture was spotted onto the MALDI sample plate and allowed to air dry before being placed in the mass spectrometer. All protein samples were analyzed in the linear, positive-ion mode by delayed extraction using an accelerating voltage of 20 kV and a delay time of 200 nanoseconds. A total of 400 single-shot spectra were accumulated from each sample. External calibration was achieved using a mixture of standard proteins containing trypsinogen (23982 m/z), Protein A (44613 m/z), and bovine albumin (66431 m/z).

[00808] MALDI -TOF MS Analysis of Peptides

[00809] Peptide mixture was separated by reversed phase chromatography and fractions from chromatographic peaks were collected and evaporated to dryness. Sample was reconstituted in 50 μΕ of 25mM phosphate buffer pH 7.5. DTT was added to a final concentration of 5 mM and the fractions were incubated at 50 °C for 20 minutes. After reduction־, IAM was added to a 10 mM final concentration and incubated in darkness at 50 °C for an additional 20 minutes.

[00810] MALDI-MS spectra were acquired on an OmniFlex (Broker Daltonics, MA) using a nitrogen laser (337 nm). Samples were prepared by mixing a 1 μΕ sample with 1 μΕ of matrix solution. The matrix solution was a saturated solution of a-cyano-4hydroxycinnamic acid in 1 to 1 water: acetonitrile with 0.1% TFA. The mixture was spotted onto a well of the MALDI sample plate and allowed to air dry before being placed in the mass spectrometer. All peptides were analyzed in the reflective, positive-ion mode by delayed extraction using an accelerating voltage of 20 kV and a delay time of 200 nanoseconds. A total of 100 single-shot spectra were accumulated from each sample. External calibration was achieved using a mixture of standard peptides containing angiotensin II (1046.54 m/z), angiotensin I (1296.68 m/z), substance P (1347.74 m/z), bombesin (1619.82 m/z), ACTH clip 1-17 (2093.09 m/z), ACTH clip 18-39 (2465.20 m/z), and somatostatin (3147.47 m/z).

Example 9: Analysis of Media And Media Constituents Effects On CTLA4-Ig Single Chain and Multimer Species

[00811] CTLA4-Ig dimer, a low sialic acid sub-fraction, a high sialic acid sub-fraction, monomer frontal, and monomer species of CTLA4-Ig are prepared and purified. These samples are used in a series of modeling experiments designed to determine the affects of media and media constituents on single chain and high molecular weight formation over times between zero to at least 60 hours. The affects of formulation buffer, iodo acetamide, sodium phosphate, ammonium chloride, basal medium, fermentation broth, medium 177e, medium Concentrated Acid Solution I, medium concentrated acid solution II, insulin, EDTA, cysteine, lipoic acid, glutathione, methionine, and yeastolates alone and in combinations are tested.

Example 10: Analyzing CTLA4-Ig Molecules by Size

[00812] A method of size exclusion chromatography (SEC) which uses denaturing conditions can be employed for the quantitation of protein species of different size. In one embodiment tandem SEC method using denaturing conditions can be employed for the quantitation of CTLA4-Ig single chain species. Single chain CTLA4-Ig can be a species lacking the inter-chain disulfide bridge. Single chain CTLA4-Ig species isolated during the purification process is referred to as native single chain CTLA4-Ig. Purified single chain produced by reduction and alkylation of CTLA4-Ig dimer is referred as induced single chain. Native and induced single chain CTLA4-Ig have the same characteristics.

[00813] Materials:

Potassium Phosphate Monobasic (KH2PO4) ACS grade Potassium Hydroxide (KOH) 45% w/w Solution ACS grade Sodium Chloride (NaCl) ACS grade Calibrated Adjustable Single Pipettor, 100 _L Rainin, (Catalog No. P-100) Water (H2O) HPLC grade

2.0 mL Cryogenic Vials Nalgene, (Catalog No. 5000-0020)

Concentrated HydrochloricAcid (HCI) Fisher (Catalog No. A144-212)

Sodium Hydroxide, (NaOH) 10N Solution

J.T.Baker, (Catalog No. 5674-02)

1000 mL Filter Unit 0.22 mm Coming, (Catalog No.430517)

Polypropylene 15 mL test tube Falcon, (Catalog No. 352097)

Sodium Azide (NaN3) ACS grade

Sodium Phosphate Monobasic, Monohydrate (NaH2PO4*H2O)

Potassium Hydroxide (KOH) Pellets

Instrumentation and Conditions

HPLC System Waters 2695 Separations Module

Column Toso Haas 5 gTSK 3000 SWXL, 300 mm x 7.8 mm I.D. (Part No. 08541) Guard Column Toso Haas 5 gTSK 3000 SWXL, 40 mm x 6.0 mm I.D. (Part No. 08543)

Detector Waters 2487 Dual Wavelength Detector Wavelength 280 nm Flow Rate 1 mL/min

Integration System Empower

Injection Volume 20 gL

Assay Target Cone. 10 mg/mL

Mobile Phase 0.2 Μ KH2PO4, 0.9% NaCI, pH 6.8 with KOH

Assay Run Time 20 min

Column Temperature Ambient

Sample Temperature 4°C

Retention Time Monomer 8.7 min ± 1.0 min, HMW species at 7.5 min ± 1.0 min, if present MW species will elute after the Monomer peak.

Reagents

4N Potassium Hydroxide (4N KOH) (100 mL)

Use one of the following preparations as described:

Add 40 mL of HPLC grade water and 11.6 mL of 45% w/w solution of KOH to a 100 mL volumetric flask. Bring volume up to 100 mL with HPLC grade water.

In a 100 mL volumetric flask add 80 mL of HPLC grade water, weigh 22.4 grams of KOH pellets, and stir magnetically until completely dissolved. Bring to volume of 100 mL with HPLC grade water.

Transfer solution into a 250 mL glass reagent bottle. Mix well by invertion. Store at room temperature for up to 1 year.

Mobile Phase (0.2 Μ KH2PO4, 0.9% NaCI, pH 6.8)

Weigh out 27.2 grams of KH2PO4 and 9.0 grams of NaCI into a 1000 mL beaker.

Dissolve the solids in 800 mL of HPLC grade water using continuous mixing with a magnetic stirring bar.

Using a pH meter, adjust the pH of the solution to 6.8 using 4N KOH solution. If the pH exceeds 6.8, adjust it with concentrated HCI.

Bring the final volume to 1 liter using a 1000 mL graduated cylinder. Filter the solution through a 0.22 gm filter unit.

Transfer into a 1000 mL glass reagent bottle and sonicate while vacuum degassing for +/- 1 minute. Degas before each use.

Store at room temperature for up to 1 month.

2N Sodium Hydroxide (2N NaOH)

Transfer 20 mL of 10N NaOH into a 100 mL glass graduated cylinder.

Bring volume up to 100 mL with HPLC water.

Transfer solution into a 250 mL glass reagent bottle. Mix well by invertion. Store at room temperature for up to 1 year.

Weigh 3.45 grams of NaH2PO4־H2O and 2.92 grams of NaCl and dissolve with mixing with a stir bar in 900 mL of HPLC grade water. Using a pH meter, adjust the pH of the solution to 7.4 with 2N NaOH. If the pH exceeds 7.4, adjust it with concentrated HCI.

Bring the final volume to 1 liter using a 1000 mL graduated cylinder. Filter the solution through a 0.22 pm filter unit.

Store at 2°-8°C for up to 6 months.

Column Storage Buffer (0.05% w/v NaN3 in Water) Weigh 50 ± 5 mg of Sodium Azide and add it to a 1000 mL beaker with a magnetic stir bar.

Add 500 mL of HPLC grade water to the beaker and stir until completely dissolved. Bring the volume to 1 L with water. Filter the solution through a 0.22 pm filter unit and pour into a 1000 mL HPLC reagent bottle.

Store at room temperature for up to 6 months.

Preparation of High Molecular Weight Species and System Suitability Standard

[00814] This will provide for a three-fold dilution of heated to unheated CTLA4-Ig reference material and a 15% - 30% High Molecular Weight Species area percent amount. In a 15mL Falcon test tube, prepare approximately 3 mL of reference material at 10.0 ±1.0 mg/mL using CTLA4-Ig dilution buffer. The initial concentration of CTLA4-Ig is from the COA. Make the 10.0 ± 1.0 mg/mL dilution from that value. Transfer 1.0 mL of the diluted reference material, made in into a 2.0 mL cryovial using a 1 mL pipettor, and heat it in a water bath at 67 ± 2°C for 45 ± 2 minutes. Transfer the heated reference material made in back into the 15 mL test tube using a 1 mL pipettor. Gently pipet up and down a 1 mL volume of the contents of the test tube for a total of 10 times. This is the system suitability standard. Storage conditions: Prepare 150 pL aliquots of the system suitability standard in 2.0 mL cryovials for storage at -80 ± 5°C for up to 1 year. Obtain the initial concentration of Reference Material, and make the 10.0 ±1.0 mg/mL dilution from that value. Use a minimum of 100 μΐ of reference material and an appropriate amount of the dilution buffer to achieve a final concentration of 10.0 ±1.0 mg/mL. Refer to the following equation for dilutions

Dilution Buffer to Add = (Aliquot Volume x Starting Concentration) _ Aliquot volume Target Concentration

[00815] For CTLA4-Ig Drug Substance make the 10.0 ±1.0 mg/mL dilution from the protein concentration using a minimum of a 100 pL aliquot of sample and an appropriate amount of the dilution buffer to achieve a final concentration of 10.0 ± 1.0 mg/mL. Refer to the following equation for dilutions:

(0.1 mLx 50 mg/mL) - 0.1 mL = 0.4 mL dilution buffer .1.0 mg/mL

[00816] Once diluted to 10.0 ±1.0 mg/mL, the samples can be stored at 2°- 8°C for up to 24 hours. For CTLA4-Ig Drug Product make the 10.0 ± 1.0 mg/mL dilution from the protein concentration using a minimum of a 200 gL aliquot of sample, and an appropriate amount of the dilution buffer to achieve a final concentration of 10.0 ±1.0 mg/mL. Refer to the following equation for dilutions:

(0.2 mL x 25 mg/ni.L) - 0.2 mL = 0.3 mL dilution buffer mg/m’L

[00817] Place the Mobile Phase in one solvent reservoir and HPLC grade water in another. Sonicate and vacuum degas prior to run. Turn on detector and allow 15 minutes to warm up prior to the run. Before a new or current column is used for analysis, flush the column with HPLC grade water for at least 20 minutes followed by mobile phase buffer equilibration for at least 20 minutes. Use a flow rate of 1.0 mL/min. Thaw a 150 gL aliquot of system suitability standard, add it to an autosampler vial and place the vial in the autosampler. Inject 20 gL of the standard under the conditions.

[00818] Determine the percentage of High Molecular Weight Species eluting at approximately 7.5 minutes according to the following formula: (In the formula below, Monomer actually refers to dimer.) (A)

Area % High Molecular Weight Species = ——— x 100 (A) + (B)

Where:

A = Peak area of High Molecular Weight Species B = Peak area of Monomer

[00819] The Area Percent High Molecular Weight Species should not be less than 15%. If it is less than 15%, add additional concentrated High Molecular Weight Species to the system suitability standard above. Resolution (R) Determination and Retention Time Evaluation. Inject 20 pL of system suitability standard. Calculate the resolution between the High Molecular Weight Species (retention time approximately 7.5 minutes) and the Monomer peak (retention time approximately 8.7 minutes) using the following equation: (In the formula below, “monomer” actually refers to dimer.) ו ס t2 ץ? ׳(t2-t1) Resolution (R) =-------W2 + W1

Where:

t! = Retention time of the high molecular weight species t₂ = Retention time of Monomer

W! = Peak width of high molecular weight species

W₂ = Peak w idth of Monomer

[00820] Peak width is measured in minutes at the base of the peak after extrapolating the relatively straight sides of the peak to the baseline. Retention time and peak width are measured in the same units. In one embodiment, (R) must be >1.2 and the retention time for _sthe peak should be 8.7 ±1.0 minutes.

Number of Theoretical Plates Determination (N)

From the system suitability standard chromatogram, determine the efficiency of the column by calculating the number of theoretical plates (N) according to the following equation:

²ו

W = 76 ־ k«׳ J

Where:

t = is the retention time of Monomer (in minutes) w = is the width (in minutes) at baseline of the Monomer obtained by extrapolating the sides of the peak to the baseline.

[00821] A total of six (6) injections of CTLA4-Ig material will be made. Aliquot 200 gL of 10 mg/mL reference material into an autosampler vial, and place the vial in the autosampler and inject six times. Process the chromatograms, and calculate the dimer peak area for each chromatograph. Sum the dimer peak areas from the six chromatographs and calculate the average, standard deviation and %RSD according to the following equations:

X1+X2 + X3..X״

Where:

X 12,3..= a specific value in a set of data n_x = a set of values (x)

5,4.4.2 Calculate the standard deviation as follows:

(1-מ)מ

Where:

n - number of values (x) in a set of data x = one value in a set of data

5.4.4.3 Calculate the % Relative Standard Deviation (RSD) as follows:

%RSD = Standard Deviation X 100

Mean

Example of an Injection Sequence:

<td> SAMPLE</td><td> INJECTION</td>

<td> Dilution Buffer Blank</td><td> 1 Injection</td>

<td> Systeme Suitability</td><td> 1 Injection</td>

<td> Reference Material</td><td> 6 Injections</td>

<td> Sample #1</td><td> 2 Injections</td>

<td> Sample #2</td><td> 2 Injections</td>

<td> Sample #3</td><td> 2 Injections</td>

<td> Reference Material</td><td> 1 Injection</td>

<td> Dilution Buffer Blank</td><td> 1 Injection</td>

Integration of Peaks

[00822] Integrate all peak areas in the chromatogram from 5.5 to 11.8 minutes.

Enlarge baseline of chromatogram to ensure total area of all LMW and HMW species are included in the integration. Disregard peaks in the sample that correspond to peaks in the control. The inclusion volume peak (11.8-13.5 minutes) and peaks after are not considered in this calculation. However, if the area at the inclusion volume is 0.1 % or greater of the total area, it should be noted. The dimer peak elutes at 8.7 ±1.0 minutes, the high molecular weight species peak elutes at 7.5 ±1.0 minutes, and the low molecular weight species (e.g., monomer, if present) will elute after the dimer peak. Any peak other than the high molecular weight species, dimer or low molecular weight species that has 280 nm absorbance in excess of 0.1 area % of the total peak area should be noted. Calculate the area percentages as follows: (The reference to “Abatacept Monomer” in this example refers to CTLA4-Ig dimer.)

7.2.1 Area % Hieh Molecular Weieht Species =-----------— x 100 (A) + (B) + (C)

CO

7.2.2 Area % Low Molecular Weight Species =-----—------x :100 ^{s P} (A) + (B) + (C)

Area % Monomer = 100-(Area % HMW + Area % LMW)

Where:

A = Abatacept Monomer peak area.

B = Total area of all peaks with retention times less than Abatacept Monomer.

C = Total area of all peaks with retention limes greater than Abatacept Monomer peak (excluding inclusion volume).

[00823] Samples were separated by size exclusion chromatography using a Water's Alliance® 2695 (Milford, MA) on two 7.8 x 300mm TSK Gel G3000SWXL™ columns (Tosoh Biosep, Montgomery, PA) placed in series employing a 6.0 x 40mm guard column. 25 microliters of each purified sample was injected and separated under isocratic conditions using 0.1M Na2HPO4, 0.1M Na2SO4, pH 6.8 at 1 .OmL/min. Samples were detected at 280nm using a 996 PDA (photodiode array) detector (Waters, Milford, MA) and analyzed using Millennium 4.0™ chromatography software (Waters, Milford, MA).

[00824] The overlaid chromatograms of native and induced single chain material from the denaturing analytical tandem size exclusion chromatography show an average retention time (6 replicates) of 27.96 ± 0.02 and 27.99 ± 0.02, respectively. The average peak area for native single chain was found to be 495525.0 ± 9589.6 and for induced single chain to be

463311.8 ± 7997.2 (Table 13). The overlaid MALDI-TOF spectra of native and induced single chain material have peaks that are quite broad with a baseline width of about 15000 mass units. This is expected due to the heterogeneity of the glycosylation in both samples. The apex point of the single chain peak in each mass spectrum was used to calculate the average mass. The average masses for native and induced CTLA4-Ig single chain are 45694.426 ± 297.735 and 45333.086 ± 264.778 mass units respectively based on the analysis of six replicates (Table 14). The native single chain mass is expected to be higher than that of induced single chain, because on the native single chain there is an extra cysteine (residue mass 103 dalton) on Cys¹⁴⁶ of SEQ ID NO:2, whereas the induced single chain is a result of selectively reducing a single interchain disulfide bridge followed by alkylation that adds an acetyl group (mass 58 dalton).

[00825] These data show that the native and induced materials produce equivalent results by the tandem denaturing SEC chromatography and MALDI-TOF analyses. These results demonstrate comparability between the native and induced CTLA4-Ig single chain materials.

Table 13: HPLC SEC Data of Native and Induced Single Chain

<td rowspan="3"> Replicate s</td><td colspan="2"> Induced</td><td colspan="2"> Native</td>

<td> Retention Time</td><td> Peak</td><td> Retention</td><td> Peak Area</td>

<td> (min)</td><td> (|xV*sec)</td><td> (min)</td><td> (|xV*sec)</td>

<td> 1</td><td> 27.985</td><td> 470199</td><td> 27.968</td><td> 505576</td>

<td> 2</td><td> 27.979</td><td> 464424</td><td> 27.941</td><td> 499800</td>

<td> 3</td><td> 27.977</td><td> 469509</td><td> 27.934</td><td> 505708</td>

<td> 4</td><td> 27.998</td><td> 469081</td><td> 27.954</td><td> 482472</td>

<td> 5</td><td> 28.027</td><td> 453103</td><td> 27.991</td><td> 490434</td>

<td> 6</td><td> 28.016</td><td> 453555</td><td> 27.985</td><td> 489160</td>

<td> Average</td><td> 27.997</td><td> 463311.8</td><td> 27.962</td><td> 495525.0</td>

<td> Stdev</td><td> 0.021</td><td> 7997.2</td><td> 0.023</td><td> 9589.6</td>

<td> %RSD</td><td> 0.074</td><td> 1.726</td><td> 0.083</td><td> 1.935</td>

Table 14: MALDI-TOF Mass Spectrometry Data of Native and Induced Single Chain

<td> Replicates</td><td> Induced SC</td><td> Native SC mass</td>

<td> 1</td><td> 45397.896</td><td> 45597.475</td>

<td> 2</td><td> 45432.199</td><td> 45621.300</td>

<td> 3</td><td> 45256.929</td><td> 45543.839</td>

<td> 4</td><td> 45433.849</td><td> 45555.893</td>

<td> 5</td><td> 45381.812</td><td> 45634.620</td>

<td> 6</td><td> 45348.376</td><td> 45340.712</td>

<td> Average</td><td> 45375.177</td><td> 45548.973</td>

<td> Stdev</td><td> 66.265</td><td> 108.043</td>

<td> %RSD</td><td> 0.15</td><td> 0.24</td>

[00826] The presence of cysteines within a polypepetide chain permits formation of disulphide bonds, which can be intramolecular or intermolecular leading to formation of dimer or multimer protein complexes. CTLA4-Ig exists as a dimer (wherein the dimer is made up of two monomers having any one of the following sequences: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2; (iii) 27-383 of SEQ ID NO:2, (iv) 26-382 of SEQ ID NO:2), (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2 held together by one disulfide bond between C¹⁴⁶ on each chain. The reduction of this disulfide bond can result in the formation of two equivalent protein chains held together by non-covalent electrostatic forces. When subjected to denaturing conditions that overwhelm or outweigh the attracting electrostatic forces, such CTLA4-Ig can completely dissociate resulting in two identical protein structures of approximately 46 kDa. The resulting structure is referred to as single chain or monomer. The presence of the single chain can be monitored by tandem column size exclusion chromatography run under denaturing conditions.

[00827] A subpopulation of CTLA4-Ig molecules contains a modification on Cys¹⁴⁶: the disulfide linkage present in the majority population is changed to a free cysteine amino acid (referred to as a cysteinylation). CTLA4-Ig dimer is predominantly a protein with a molecular weight of approximately 92,000. It is comprised of two glycosylated polypeptide chains which are held together by one inter-chain disulfide bond at Cys¹⁴⁶ and non-covalent interactions (also referred to as CTLA4-Ig dimer). The purified protein exists as a heterogeneous population and contains modifications such as glycosylations and variations at the N- and C-termini.

[00828] A distinct population of CTLA4-Ig molecules exists, which lacks the interchain disulfide bond linkage. This non-covalently linked population exists within the frontal dimer peak generated by size exclusion chromatography. The frontal dimer was found to lack the interchain disulfide bond. The CTLA4-Ig species which lack the interchain disulphide link are modified by cysteinylation at Cys , which modification occurs on >99% of the single chain species based on the ESI-MS intact data. Cys¹⁴⁶ cysteinylation and the enrichment of O-linked carbohydrates are the two major modifications on the CTLA4-Ig single chain species. The frontal dimer is subjected to denaturing size exclusion chromatography resulting in isolation of the CTLA4-Ig single chain peak. The purified frontal monomer material was compared to CTLA4-Ig with and without solid phase extraction (SPE) and analyzed on MALDI. The purified frontal monomer contains two dominant species: a major species at either 47005 u for SPE-treated or 46897 u for nontreated; a minor species at either 95070 u for SPE-treated or 96172 u for non-treated. The CTLA4-Ig material also contains two dominant species: a major species at either 91518 u for SPE-treated or 91143 u for non-treated; a minor species at either 45660 u for SPE-treated or 46014 u for non-treated.

example example

<td> Size Homogeneity (HPLC) Dimer</td><td> &gt; 95.5 Area % &gt; 95.5 Area % &gt; 97.0% Area % &gt; 97.0% Area %</td>

<td> Size Homogeneity (HPLC) High MW Species</td><td> &lt;3.0 Area %. &lt;3.0 Area %. &lt;2.0 Area %. &lt; 2.0 Area %.</td>

<td> Size Homogeneity (HPLC) Low MW Species (monomer)</td><td> &lt;0.5 Area % &lt; 0.5 Area %</td>

[00829] The CTLA4-Ig composition, in one embodiment, have the following characteristics as to Size Homogeneity (HPLC) analysis of dimer, High MW Species, and Low MW Species (monomer, single chain):

• >97.0% Dimer • <2.0% HMW species • < 0.5% LMW species (e.g., monomer, single chain)

In another embodiment, the CTLA4-Ig composition has the following characteristic amounts of each species:

• >95.5% dimer • <3.0% HMW species • < 0.5% LMW species (e.g., monomer, single chain)

Summary of SE-HPLC Analysis of Process CDCHOI Batches

<td colspan="3"> Process CD-CHO1 n = 109</td>

<td></td><td> dimer</td><td> HMW</td>

<td> Average (%)</td><td> 99.4</td><td> 0.6</td>

<td> %CV</td><td> 0.3%</td><td> 45.7%</td>

<td> Minimum (%)</td><td> 98.4</td><td> 0.2</td>

<td> Maximum (%)</td><td> 99.8</td><td> 1.6</td>

<td> 95% Tolerance Interval</td><td> &gt;98.7</td><td> &lt;1.3</td>

[00830] The percent monomer ranged from 98.4 to 99.8% with an average value of 99.4% for drug substance manufactured using Process CD-CHO1. The percentage HMW species varied from 0.2 to 1.6%. The average value was 0.6% with a CV of 45.7%. The 95% tolerance interval was >98.7% for the dimer and <1.3% for the HMW species. The percent HMW species of the batches varied from a minimum value of 0.4% to a maximum value of 2.1%. The average percent HMW species was 0.8% with a % CV of 40%. In all cases, the LMW or monomer species were below the detection limit (DL = 0.1%). The 95% tolerance interval (to provide coverage for 99 area % of the population) for CTLA4-Ig manufactured by Process CD-CHOl were <1.3 and <1.8% respectively for the HMW species. The 95% tolerance intervals for the dimer in drug substance from Process CD-CHOl were 98.7% and 96.5% respectively.

Summary of combined data for CTLA4-Ig

<td></td><td> dimer (n=143)</td><td> HMW (11=141)³</td>

<td> Average (%)</td><td> 99.3</td><td> 0.6</td>

<td> %CV</td><td> 0.5</td><td> 46.9</td>

<td> Minimum (%)</td><td> 94.8</td><td> 0.2</td>

<td> Maximum (%)</td><td> 99.8</td><td> 2.1</td>

<td> 95% Tolerance Interval</td><td> &gt;97.3</td><td> &lt;1.8</td>

<td> % CV (between-site)</td><td> 0.3%</td><td> 26.4%</td>

<td> % CV (within-site)</td><td> 0.5%</td><td> 44.2%</td>

<td> % CV (total-site)</td><td> 0.6%</td><td> 51.4%</td>

[00831] The above Table shows that the percent HMW species ranged from 0.2 to 2.1% with an average of 0.6%. The dimer ranged from 94.8% to 99.8% with an average value of 99.3% and a precision of 0.3%. The variation between-site, within-site and total-site variation for the dimer was within 0.3 to 0.5%. The between site variation for the HMW was 26.4%. The within-site and total-site variation was for the percent HMW species 44.2 and 51.4% respectively. The 95% tolerance interval for the dimer (97.3%) and the HMW species (1.8%) were within the specification.

Example 11: Vector Construction

[00832] Construction of the pcSD Expression Vector: The expression vector, pcSD was constructed from the commercially available pcDNA3 vector (Invitrogen, Carlsbad, CA) as shown in FIG. 28. The neomycin resistance gene cassette was removed from plasmid pcDNA3 by digestion with restriction endonuclease Nae I. The restriction endonuclease Nae I creates blunt ends. The DNA fragments were separated by agarose gel electrophoresis and the 3.821 kb pcDNA3 vector backbone was purified from the gel. The DNA fragment containing the gene coding for mouse dihydrofolate reductase (dhfr) and an SV40 promoter was isolated from plasmid pS V2-dhfr by digestion of the plasmid with restriction endonucleases Pvu II and BamH I. The 1.93 kb fragment corresponding to the dhfr gene cassette was separated and purified by agarose gel electrophoresis. The 3-prime recessed ends generated by BamH I digestion were filled in using the Klenow fragment of DNA polymerase I to generate blunt ends. This isolated fragment was ligated to the blunt-ended

3.8 kb pcDNA3 vector backbone to create the expression vector pcSD. ^,This expression vector has the following features: a cytomegalovirus (CMV) promoter followed by a multiple cloning site, a bovine growth hormone (BGH) polyad enylation signal and transcriptional termination sequence, a mouse dhfr cDNA sequence for selection and amplification, and an ampicillin resistance gene and pUC origin of replication for selection and maintenance in Escherichia coli.

[00833] Construction of the pcSDhuCTLA4-Ig Expression Vector: A 1.2 kilobase (kb)

DNA fragment containing a sequence encoding a CTLA4-Ig protein was isolated from plasmid pCDM8-CTLA4-Ig by digestion with the restriction enzymes Hind III and Xba I. The 1.2-kb Hind W/Xba I fragment was ligated into vector piLN previously digested with the restriction enzymes Hind III and Xba I. The resulting plasmid construct, designated piLN-huCTLA4-Ig, is shown in FIG. 21. The piLN-huCTLA4-Ig plasmid was used as the source of the CTLA4-Ig coding sequence used in the construction of the final expression vector pcSDhuCTLA4-Ig.

[00834] The final vector for expression of the CTLA4-Ig gene was constructed as shown in FIG. 29. A 1.2 kb DNA fragment containing the CTLA4-Ig gene was isolated from plasmid piLN-huCTLA4-Ig by a two step restriction digest procedure. Plasmid piLNhuCTLA4-Ig was first digested with restriction enzyme Hind III. The resulting 3-prime recessed ends were filled in by treatment with the Klenow fragment of DNA polymerase I. The plasmid was then digested with the restriction enzyme Xba I to release the 1.2 kb fragment containing the CTLA4-Ig gene. This fragment was purified and ligated to the EcoR. V and Xba I fragment isolated from the restriction digestion of pcSD. The EcoR. V and Xba I restriction sites are located in the multiple cloning site of pcSD between the CMV promoter and a cassette containing the bovine growth hormone polyadenylation signal and transcriptional termination sequence. This placed the CTLA4-Ig gene fragment under the control of the CMV promoter. This plasmid is designated pcSDhuCTLA4-Ig, and comprises

SEQ ID NO:1.

Example 12: Transfection of CTLA4־Ig Expression Vector to Obtain Stable Cell Lines

[00835] This Example and Example 13 describe a newly transfected population of cells from which individual clones were selected and expanded, and thus the expanded clones are different than the cells deposited with the ATCC as Accession No. CRL-10762. The previous CHO cell line harboring an expression vector containing DNA encoding the amino acid sequence corresponding to CTLA4-Ig (DNA having ATCC Accession Number 68629) is described in U.S. Patent No. 5,434,131. Briefly, an expression plasmid (for example, pCDM8) containing cDNA that was deposited under ATCC Accession No. 68629, was transfected by lipofection using standard procedures into dhfr negative-CHO cells to obtain cell lines that stably express CTLA4-Ig. Screening B7 positive CHO cell lines for B7 binding activity in the medium using immunostaining resulted in a stable transfectant that expressed CTLA4-Ig. This heterogenous population of transfected cells was designated Chinese Hamster Ovary Cell Line, CTLA4-Ig-24 and was deposited with the ATCC under the Budapest Treaty on May 31,1991 having ATCC Accession Number CRL-10762.

[00836] The Chinese hamster ovary cell line, DG44, contains a deletion of the gene coding for the enzyme dihydrofolate reductase. The expression plasmid pcSDhuCTLA4-Ig contains a copy of the dihydrofolate reductase gene (dhfr). Insertion of plasmid pcSDhuCTLA4-Ig into the DG44 genome results in functional complementation of the dhfr deletion. This functional complementation can be used for selection of transfectants in the presence of methotrexate (MTX) and amplification of dhfr and adjacent genes.

[00837] The human CTLA4-Ig-secreting cell line 1D5 was constructed by transfection of cell line DG44 with the pcSDhuCTLA4-Ig expression plasmid as shown in FIG. 29. The plasmid DNA was introduced into DG44 cells by electroporation using standard procedures known in the art. Transfectants were selected using a minimal essential medium (MEM; JRH Biosciences, Inc., Kansas) supplemented with 5% (v/v) dialyzed Fetal Bovine Serum.

Culture supernatants from the transfectants were screened for human IgG production using a sandwich ELISA method. An Fc-specific goat anti-human IgG was used as the capture antibody. Goat anti-human IgG antibody conjugated to horseradish peroxidase was used to defect human IgG. Transfectants expressing higher levels of the human CTLA4-Ig gene were selected for further amplification.

[00838] Gene amplification of the selected transfectants was accomplished by addition of MTX to the culture medium at a final concentration of 100 nM. MTX is a folic acid analogue that acts as a competitive inhibitor of dihydro folate reductase. Addition of MTX to the medium allowed selection of transfectants containing multiple copies of the dhfr gene and elevated levels of dihydrofolate reductase. Transfectants also containing multiple copies of the adjacent CTLA4-Ig gene were identified using the human IgG specific ELISA method. A CTLA4-Ig-producing clone, designated 1D5, was selected for further development, in part described in Example 13.

Example 13: Subcloning of Stably Transfected Cells

[00839] Cell line 1D5 was subjected to soft agar cloning. Subclones derived from the soft agar cloning were analyzed for human IgG production using the ELISA method. Selected subclones were evaluated for CTLA4-Ig production and growth properties. The lead subclone, designated 1D5-100A1, was selected.

[00840] The lD5-100Alcell line was adapted from DE medium (JRH Biosciences, Inc., Kansas, which contains animal-sourced raw materials, to a chemically defined medium designated CD-CHO (Table 15). CD-CHO medium is a proprietary, animal component-free medium manufactured by Invitrogen Corporation, Carlsbad, CA.

Table 15: Composition of Process Y Medium

<td> Component</td><td> Concentration</td>

<td> CD-CHO 25x Acid Solubles I</td><td> 40.0 mL/L</td>

<td> CD-CHO 25x Acid Solubles II</td><td> 40.0 mL/L</td>

<td> CD-CHO 25x Salts I</td><td> 40.0 mL/L</td>

<td> CD-CHO 25x Salts II</td><td> 40.0 mL/L</td>

<td> L-Glutamine</td><td> 0.585 g/L</td>

<td> r-human Insulin (10 mg/mL solution)</td><td> 0.1 mL/L</td>

<td> Methotrexate (20 mM solution)</td><td> 5 pL/L</td>

<td> Sodium Bicarbonate</td><td> 2.22 g/L</td>

<td> Water</td><td> As required</td>

<td> 1 N HC1 Solution</td><td> 0-5 mL/L to adjust pH</td>

<td> IONNaOH Solution</td><td> 0-10 mL/L to adjust pH</td>

[00841] Cell line 1D5-100A1 was cultured and passaged in DE medium according to standard tissue culture protocol. The cells were then transferred to a medium composed of 50% DE medium and 50% CD-CHO medium. After several passages in this medium, the cells were transferred to T-flasks containing 100% CD-CHO medium. The cells were grown in 100% CD-CHO medium for several passages. The adapted cells were then subjected to cloning by limiting dilution.

[00842] Cells from the CD-CHO-adapted 1D5-100A1 cell line were cloned by limiting dilution using serum-free media. The 1D5-100A1 cells were seeded at a target of 1 cell/well into 96-well microtiter plates containing supplemented MCDB medium. MCDB is a chemically defined medium formulation distributed by Sigma-Aldrich, St. Louis, MO. The MCDB medium was supplemented with 4 mM glutamine, 500 pg/mL recombinant human insulin, 100 nM MTX and 10% conditioned medium. The conditioned medium was a filter-sterilized supernatant from a culture of the CD-CHO adapted 1D5-100A1 cell line grown in MCDB medium.

[00843] Wells containing a single colony were identified and the clones evaluated for CTLA4-Ig production using the ELISA method. Selected clones were expanded from 96well microtiter plates to 6-well cell culture plates. The cultures were further expanded into 2 cm T-flasks and then roller bottles.

[00844] The roller bottle cultures were evaluated for CTLA4-Ig titer, CTLA4-Ig sialic acid content, and growth. Three clones were selected for further evaluation in bioreactors and further characterization. A frozen vial research stock of clonal cell line 1D5-100A1.17 stored at —80°C was used to generate a cell bank.

Example 14: Production of CTLA4-Ig in Bio reactors via a Fed-Batch Process

[00845] Commercial Scale Culturing of Suspension Mammalian Cells Expressing CTLA4Ig: This Example describes the production of CTLA4-Ig molecules comprising SEQ ID NO:2 monomers, from suspension cultured &lt;7/2/r־negative CHO cells. The methods described in this Example can be adapted and extended for the production of other recombinant proteins, including but not limited to, secreted proteins such as cytokines and other hormones,

ל secreted proteins that are members of the Ig superfamily or comprise a portion of an Ig superfamily protein, and generally any protein expressed in CHO cells. A process flow diagram for the CTLA4-Ig culturing steps is shown in FIG. 10.

[00846] The culture flasks (for example, T-175 and Erlenmyer flasks), roller bottles, and cell bags were used for the inoculum expansion steps of the CTLA4-Ig culturing process to serially propagate cells from a frozen vial to provide a sufficient number of viable cells to inoculate a 20,000-L bioreactor.

[00847] A single vial of cells is removed from the vapor phase of a liquid nitrogen storage freezer and thawed in a water bath at 37°C. The entire contents of the vial are aseptically transferred into a sterile 15-mL conical centrifuge tube. CD-CHO medium is added to bring the final volume to 10 mL. The cell suspension is centrifuged, the supernatant discarded and the cell pellet resuspended in 10 mL of CD-CHO cell culture medium. The resuspended cells are transferred to a T-175 flask containing 10 mL of CD-CHO medium. The viable cell density and the percent viability of the culture in the T-175 flask is determined. A criterion for the percent viability at this step of > 84% was established. CD-CHO medium is added to the T-175 flask to achieve a target viable cell density of 1.7-2.6 x 10⁵ cells/mL.

[00848] The T-175 flask is incubated at 37°C in an atmosphere of 6% carbon dioxide for a maximum of four days to achieve a target final cell number of >6 x 10⁶ viable cells.

Following the T-175 flask step, the culture is expanded using a series of shaker flasks, 1-L, and 2-L Roller Bottles, as shown in FIG. 10. At each passage, the cells are seeded at a target density of 2.0x10⁵ viable cells/mL, wherein cultures targeted at having a final culture cell viability ^0%. The cultures are incubated in CD-CHO medium at 37°C in an atmosphere of 6% carbon dioxide for a maximum of four days.

[00849] Expansion of the culture occurs in a series of cell bags (20-L, 100-L, and 200-L) in order to further inoculate a 1000-L bioreactor. Cell culture material from the 2-L roller bottles inoculum expansion step is pooled to inoculate a 20-L cell bag at a target seeding density of 2.0x10⁵ viable cells/mL. A condition for the final viable cell density at the 2-L roller bottle inoculum expansion step of 1.0 to 2.0 x 10⁶ cells/mL and a minimum percent cell viability of > 80% were established. Upon inoculation, the 20-L cell bag culture is incubated in CD-CHO medium at 37°C in an atmosphere of 6% carbon dioxide for a maximum of four days. For each subsequent passage (100-L and 200- L cell bags), the cells are seeded at a target density of 2.0x10⁵ viable cells/mL, wherein cultures targeted at having a final culture cell viability 330%. The cultures are incubated in CD-CHO medium at 37°C in an atmosphere of 6% carbon dioxide for a maximum of four days. Exemplary values for the final viable cell density at the 20-L, 100-L, and 200-L cell bag inoculum expansion step of

1.0 to 2.0 x 10⁶ cells/mL and a minimum percent cell viability of > 80% were established.

These exemplary values ensure that a sufficient number of viable cells is used to inoculate the

1000-L bioreactor.

[00850] The objective of the 1000-L and 4000-L seed bioreactor inoculum expansion steps of the CTLA4-Ig process is to provide a sufficient number of viable cells to inoculate the 20,000-L production bioreactor.

[00851] The seed bioreactors are operated in batch mode using CD-CHO cell culture medium. Temperature, pH, dissolved oxygen, pressure, agitation and gas flow rates for air, oxygen, and carbon dioxide are controlled by a distributed control system (DCS) and provide conditions for optimal growth of the culture in the seed bioreactors. The seed bioreactors are operated at 37°C. Culture samples are removed from the seed bioreactors for the determination of viable cell density, percent viability, and metabolite concentrations.

[00852] The 1000-L seed bioreactor is inoculated with inoculum from the 200-L cell bag expansion step to a target initial viable cell density of 1.0 to 3.0xl0⁵ viable cells/mL. The culture is incubated in CD-CHO medium at 37°C for a maximum of 5 days. Exemplary values for the final viable cell density at the 1000-L seed bioreactor inoculum expansion step is 1.0 to 2.0 x 10 cells/mL and a minimum percent cell viability is > 80%.

[00853] The 4000-L seed bioreactor is inoculated with inoculum from the 1000-L seed bioreactor expansion step to a target initial viable cell density of 1.0 to 3.0x10⁵ viable cells/mL. The culture is incubated in CD-CHO medium at 37°C for a maximum of 6 days. Exemplary values for the final viable cell density at the 4000-L seed bioreactor inoculum expansion step is 1.0 to 2.0 x 10⁶ cells/mL and a minimum percent cell viability is > 80%. These exemplary values ensure that a sufficient number of viable cells is used to inoculate the 20,000-L production bioreactor.

[00854] The 20,000-L seed bioreactor is inoculated with inoculum from the 4000-L seed bioreactor expansion step to a target initial viable cell density of 1.0 to 1.8 xl 0⁵ viable cells/mL. The culture is incubated in CD-CHO medium at 37°C for a maximum of 6 days.

Exemplary values for the final viable cell density at the 20,000-L seed bioreactor inoculum expansion step is 1.0 to 2.0 x 10⁶ cells/mL and a minimum percent cell viability is > 80%.

These exemplary values ensure that a sufficient number of viable cells is used prior to initiating the production phase in the 20,000-L production bioreactor.

[00855] Commercial Scale Production of CTLA4-Ig׳. The production phase of this invention occurring in a 20,000-L production bioreactor produces both high quantity and high quality CTLA4-Ig protein, which involves culture runs having a two-step temperature shift. The 20,000L culture that is incubated in CD-CHO medium at 37°C for a maximum of 6 days (as described above) is subjected to a temperature shift (T-shift) from 37°C to 34°C on day 6 (the end of logarithmic growth phase). Twelve hours after the 37°C to 34°C temperatureshift, CD-CHO medium is supplemented with a modified eRDF feed medium (Invitrogen Corp., Carlsbad, CA; Tables 16, 17), and this feed is provided daily to the production reactor as a bolus (1% w/w).

Table 16: Composition of eRDF Feed Medium

<td> Component</td><td> Concentration</td>

<td> eRDF־l Medium (Invitrogen Corp.)</td><td> 16.47 g/kg</td>

<td> Dextrose</td><td> 30.29 g/kg</td>

<td> D-Galactose</td><td> 12.38 g/kg</td>

<td> L-Glutamine</td><td> 4.02 g/kg</td>

<td> r-human Insulin (10 mg/mL solution)</td><td> 0.98 mL/kg</td>

<td> TC Yeastolate</td><td> 4.90 g/kg</td>

<td> Water</td><td> As required</td>

<td> 1 N HC1 Solution</td><td> 0-5 mL/kg to adjust pH</td>

<td> 10 N NaOH Solution</td><td> 0-2 mL/kg to adjust pH</td>

Tablel7: Composition of eRDF-1 Medium

<td> Component</td><td> Concentration (mg/L)</td>

<td> Cupric Sulfate 5 ¾0</td><td> 0.0008</td>

<td> Ferrous Sulfate 7 ¾0</td><td> 0.220</td>

<td> Magnesium Sulfate (MgSO4)</td><td> 66.20</td>

<td> Zinc Sulfate 7 H2O</td><td> 0.230</td>

<td> Sodium Pyruvate</td><td> 110.0</td>

Tablel7: Composition of eRDF-1 Medium

<td> Component</td><td> Concentration (mg/L)</td>

<td> DL-Lipoic Acid Thioctic</td><td> 0.050</td>

<td> Linoleic Acid</td><td> 0.021</td>

<td> L-Alanine</td><td> 6.68</td>

<td> L-Arginine</td><td> 581.44</td>

<td> L-Asparagine</td><td> 94.59</td>

<td> L-Aspartic Acid</td><td> 39.93</td>

<td> L-Cystine 2 HC1</td><td> 105.38</td>

<td> L-Glutamic Acid</td><td> 39.7</td>

<td> Glycine</td><td> 42.8</td>

<td> L-Histidine HC1-H₂O</td><td> 75.47</td>

<td> L-Isoleucine</td><td> 157.40</td>

<td> L-Leucine</td><td> 165.30</td>

<td> L-Lysine HCI</td><td> 197.26</td>

<td> L-Methionine</td><td> 49.24</td>

<td> L-Phenylalanine</td><td> 74.30</td>

<td> L-Proline</td><td> 55.3</td>

<td> L-Hydroxyproline</td><td> 31.5</td>

<td> L-Serine</td><td> 85.10</td>

<td> L-Threonine</td><td> 110.8</td>

<td> L-Trypt ophan</td><td> 18.40</td>

<td> L-Tyrosine 2 Na 2H₂O</td><td> 108.10</td>

<td> L-Valine</td><td> 108.9</td>

<td> Para Amino Benzoic Acid</td><td> 0.51</td>

<td> Vitamin Bl2</td><td> 0.339</td>

<td> Biotin</td><td> 1.00</td>

<td> D-Ca Pantothenate</td><td> 1.29</td>

<td> Choline Chloride</td><td> 12.29</td>

<td> Folic Acid</td><td> 1.96</td>

<td> i-Inositol</td><td> 46.84</td>

<td> Niacinamide</td><td> 1.47</td>

<td> Pyridoxal HCI</td><td> 1.00</td>

<td> Pyridoxine HCI</td><td> 0.420</td>

<td> Riboflavin</td><td> 0.21</td>

<td> Thiamine HCI</td><td> 1.59</td>

Tablel7: Composition of eRDF-1 Medium

Component Concentration (mg/L)

Putrescine 2HC1 0,020

[00856] The 20,000L culture is incubated in CD-CHO medium supplemented daily with eRDF feed medium at 34°C for a maximum of 4 days. On day 10, the 20,000L culture is subjected to a second T-shift from 34°C to 32°C. The 20,000L production culture in the production bioreactor was maintained at 32°C for a maximum of 8 days. On day 18, a culture sample was analyzed for the following exemplary values: viable cell density at the 20,000-L seed bioreactor production step is 3.0 to 8.0 χ 10⁶ cells/mL; minimum percent cell viability is > 38%; final sialic acid molar ratio (described elsewhere) is > 6; and final CTLA4-Ig protein product titer is 0.5 to 1.3 g/L. These exemplary values ensure that a protein product of sufficient quality and quantity is being produced by the recombinant CHO cell line and that the 20,000-L mammalian cell culture is ready to be harvested.

[00857] The culture in the bioreactor during the production phase is given a daily bolus feed using modified eRDF medium (Table 16, 17), as follows: starting 12 hours after the initial temperature shift (37°C to 34°C), a minimum of 1% culture volume was added as feeding medium; if the glucose level fell below 3 g/L, a calculated volume is added to bring the glucose level back to 3 g/L.

[00858] The production phase had duration of 18 days at the 20,000 L scale. Samples were taken on a daily basis from the production bioreactor for analysis. For example, a sample used for cell counting was stained with trypan blue (Sigma, St. Louis, Mo.). Cell count and cell viability determination was performed using a hemocytometer to count viable stained cells under the microscope. For analysis of metabolites, an additional sample aliquot was centrifuged for 20 minutes at 2000 rpm (4°C) to pellet the cells. The supernatant was analyzed for protein titer, sialic acid, glucose, lactate, glutamine, glutamate, pH, pO2, pCO2, ammonia, and LDH, using techniques and protocols conventionally practiced in the art.

Example 15: Purification of Recombinant CTLA4-Ig

[00859] QXL Anion Exchange Chromatography for CTLA4-Ig Purification: The anion exchange chromatography step in the CTLA4-Ig process uses Q Sepharose Extreme Load (QXL) anion exchange chromatography resin. This resin is supplied by GE Healthcare,

Waukesha, WI (formerly Amersham Biosciences). The QXL chromatography step is to capture and concentrate the CTLA4-Ig dimer from the in-process material from the harvest operation steps for further downstream processing.

[00860] A 1.0-2.0 m inner diameter column is packed with QXL resin to a height of 17 to 30 cm, representing a volume of about 643 L to 1018 L. The column is qualified for use by determining the height equivalent to a theoretical plate (HETP) and asymmetry (A_s) of the packed column. A HETP of 0.02 to 0.08 cm and an A_s of 0.8 to 1.2 are employed for qualification of the QXL column.

[00861] The QXL column operation is carried out at ambient temperature. The clarified cell culture broth is loaded onto an equilibrated QXL column. The QXL chromatography step is performed using a maximum flow rate of 99.4 L/min. The column inlet pressure is maintained below 35 psig. The maximum CTLA4-Ig protein load for the QXL column is 28 grams of CTLA4-Ig per liter of resin.

[00862] The QXL chromatography column is first sanitized with a 1 N sodium hydroxide solution. The sanitization is performed using 2 to 4 column volumes (CV) of the 1 N sodium hydroxide solution. The sanitization is complete when the conductivity of the column effluent equals 169 ± 33 mS/cm and the column is held for 60 to 120 minutes.

[00863] After the sanitization step, the column is equilibrated with a 75 mM HEPES, 360 mM sodium chloride, pH 8.0 buffer. The equilibration is complete when a minimum of 3 CV of equilibration buffer have been passed through the column and the pH of the effluent is 8.0 ± 0.2 and the conductivity of the effluent is 13.4 ±1.0 mS/cm.

[00864] The in-process material from the harvest operation step is loaded onto the QXL column. The column is washed with a minimum of 10 CV of wash buffer (75 mM HEPES, 360 mM NaCl, pH 8.0), and the absorbance at 280 nm (A280) of the column effluent is measured at the end of the wash step. CTLA4-Ig is then eluted from the column with a 25 mM HEPES, 325 mM NaCl or 850 mM NaCl, pH 7.0 buffer. The eluate is diverted into a collection vessel when the A280 increases to > 0.02 absorbance units (AU) above the AU value at the end of the wash step. The eluate is collected until the A280 of the trailing edge of the elution peak decreases to a value of < 1.0 AU.

[00865] . A CTLA4-Ig dimer product with a molar ratio of moles sialic acid to moles

CTLA4-Ig protein that is >8 is collected, and a pool of CTLA4-Ig high molecular weight material is present at <25.7%. The CTLA4-Ig high molecular weight material, which includes tetramers, can then be further purified for use as a separate substance for the methods of treatment described herein.

[00866] Phenyl Sepharose FF HIC for CTLA4-Ig Purification: The hydrophobic interaction chromatography (HIC) step uses Phenyl Sepharose Fast Flow resin (GE Healthcare, Waukesha, WI (formerly Amersham Biosciences)). The HIC step reduces the level of CTLA4-Ig high molecular weight material present in the QXL product pool. The CTLA4-Ig dimer does not bind to the HIC resin under the loading conditions used for the HIC step.

[00867] A 1.0 to 2.0 m inner diameter column is packed with Phenyl Sepharose Fast Flow resin to a height of 18 to 22 cm, representing a volume of about 680 to 852 L. The column is qualified for use by determining the HETP and A_s of the packed column. A HETP of 0.02 to 0.08 cm and an A_s of 0.8 to 1.2 are employed for qualification of the HIC column.

[00868] The HIC column operation is carried out at ambient temperature. The eluate pool from the QXL column step is loaded without further treatment onto the equilibrated HIC column. The HIC step is operated at a maximum flow rate of 65.4 L/min and at a operating pressure of &lt; 13 psig. The maximum CTLA4-Ig protein load applied to the HIC column is 10.0 g of CTLA4-Ig protein per liter of resin. Multiple cycles of the HIC step can be employed based on the amount of CTLA4-Ig protein present in the QXL eluate pool.

[00869] The HIC column is first sanitized with a 1 N sodium hydroxide solution. The sanitization is complete when 2 to 4 CV of the 1 N sodium hydroxide solution have been passed through the column. The column is then held for 60 to 120 minutes to ensure sanitization.

[00870] After the sanitization step, the column is equilibrated with a 75 mM HEPES, 2.55 M sodium chloride, pH 7.0 buffer. The equilibration is complete when a minimum of 3 CV of equilibration buffer have been passed through the column and the pH of the effluent is 7.0 ± 0.3 and the conductivity is 71.5 to 75.5 mS/cm.

[00871] The eluate from the QXL step is applied to the equilibrated HIC column. The column is then washed with the chase equilibration buffer until the A280 of th® effluent decreases to between 0.8 and 1.0 AU. The CTLA4-Ig protein-containing effluent from each cycle of the HIC step is filtered through a 0.2 gm cellulose acetate filter into a common stainless steel collection vessel. This HIC product pool is held in the collection vessel at 2° to 8°C. The maximum hold time in the collection vessel is 3 days.

[00872] A CTLA4-Ig dimer product with a molar ratio of moles sialic acid to moles CTLA4-Ig protein that is 33 is collected, and a pool of CTLA4-Ig high molecular weight material is present at ^!.5%.

[00873] Recombinant Protein A Affinity Chromatography for CTLA4-lg Purification׳. The recombinant Protein A Sepharose Fast Flow affinity resin (rPA) used in the downstream CTLA4-Ig production process is obtained from GE Healthcare (Waukesha, WI (formerly Amersham Biosciences)). The rPA column chromatography step further purifies the CTLA4Ig protein. This step removes DNA and host cell proteins including monocyte chemotactic protein 1 (MCP-1).

[00874] An 80 to 140 cm inner diameter column is packed with rPA resin to a height of 18 to 25 cm, representing a volume of about 339 to 372 L. The column is qualified for use by determining HETP and A_s of the packed column. A HETP of 0.02 to 0.08 cm and an A_s of 0.8 to 1.2 are employed for qualification of the column. The maximum number of uses for the rPA resin established in a resin lifetime study is 60.

[00875] The rPA column operation is carried out at ambient temperature. The viral inactivation product pool is loaded onto the equilibrated rPA column. The rPA step is operated at a maximum flow rate of 26.7 L/min and an operating pressure of &lt; 13 psig. The maximum CTLA4-Ig protein load applied to the rPA column is 25 g of CTLA4-Ig protein per liter of resin.

[00876] The rPA column is equilibrated with a 25 mM Tris, 250 mM NaCI, pH 8.0 buffer. Equilibration is complete when a minimum of 3 CV of equilibration buffer have been passed through the column and the pH and conductivity values of the effluent are between 7.8 to 8.2 and 23.0 to 27.0 mS/cm, respectively.

[00877] The viral inactivation step product pool is applied to the equilibrated rPA column. The rPA chromatography step includes two wash steps. The first wash step is performed using a minimum of 5 CV of a 25 mM Tris, 250 mM NaCl, 0.5 % Triton X-100, pH 8.0 buffer to remove weakly bound material from the rPA column. The second wash step is performed using a 25 mM Tris, 250 mM NaCl, pH 8.0 buffer. The second wash step uses a minimum of 5 CV to remove the residual Triton X-100 from the rPA column.

[00878] The CTLA4-Ig protein is eluted from the rPA chromatography column with a 100 mM glycine, pH 3.5 buffer. The eluate is diverted into a collection vessel when the A280 increases to > 0.2 AU above the baseline. The column effluent is filtered through a 0.2 pm cellulose acetate filter into a collection vessel equipped with an agitator. The eluate is collected until the A280 of the trailing edge of the elution peak decreases to a value of < 0.2 AU. ׳The pH of the eluate pool is adjusted to pH 7.5 ± 0.2 with a 2 M HEPES, pH 8.0 buffer. The rP A chromatography step product pool is held at 2° to 8°C for a maximum of 3 days.

[00879] A CTLA4-Ig dimer product with a molar ratio of moles sialic acid to moles CTLA4-Ig protein that is 28 is collected; a pool of CTLA4-Ig high molecular weight material is present at ^.5%; and a pool of MCP-1 &lt;38 ng/mL is present.

[00880] QFF Anion Exchange Chromatography for CTLA4-Ig Purification׳. The anion exchange chromatography step in the downstream CTLA4-Ig production process uses Q Sepharose Fast Flow (QFF) anion exchange chromatography resin (GE Healthcare (Waukesha, WI (formerly Amersham Biosciences). The objective of the QFF chromatography step is to reduce the residual Protein A levels and provide additional reduction of host cell DNA from the viral filtration step product pool. The QFF column step is also used to control the sialic acid to CTLA4-Ig protein molar ratio of the QFF chromatography step product pool and to provide additional control of in-process CTLA4-Ig HMW material levels. The primary in-process control point for the reduction of CTLA4-Ig HMW material is the HIC step.

[00881] A 60 to 140 cm inner diameter column is packed with QFF resin to a height of 28 to 35 cm, representing a volume of about 536 to 667 L. The column is qualified for use by determining the HETP and A_s of the packed column. A HETP of 0.02 to 0.08 cm and an A_sof 0.8 to 1.2 are employed for qualification of the column.

[00882] The QFF column operation is carried out at ambient temperature. The viral filtration step product pool is loaded onto the equilibrated QFF column. The QFF step is operated at a maximum flow rate of 38.7 L/min and an operating pressure of &lt; 35 psig. The maximum CTLA4-Ig protein load applied to the QFF column is 25 g of CTLA4-Ig protein per liter of resin.

[00883] The QFF chromatography column is first sanitized with a 1 N sodium hydroxide solution. The sanitization is performed using 2 to 4 CV of the 1 N sodium hydroxide solution. The sanitization is complete when the conductivity of the column effluent equals 136 to 202 mS/cm and the column is held for 60 to 120 minutes.

[00884] After the sanitization step, the column is equilibrated with a 25 mM HEPES, 100 mM sodium chloride, pH 8.0 buffer. The equilibration is complete when a minimum of 4 CV of equilibration buffer have been passed through the column and the pH of the effluent is 7.7 to 8.3 and the conductivity is 10.5 to 12.9 mS/cm.

[00885] The viral filtration step product pool contained in bioprocess bags is transferred into a sterile stainless steel collection vessel.

[00886] The viral filtration step product pool is applied to the equilibrated QFF column. The QFF chromatography step includes two wash steps. The first wash step is performed using a minimum of 5.0 CV of a 25 mM HEPES, 120 mM NaCl, pH 8.0 buffer. The second wash step is performed using a minimum 5.0 CV of a 25 mM HEPES, 130 mM NaCl, pH 8.0 buffer.

[00887] The CTLA4-Ig dimer is eluted from the QFF chromatography column using a 25 mM HEPES, 200 mM NaCl, pH 8.0 buffer. The eluate collection is initiated when the A280 of the effluent begins to increase. During elution, the column effluent is filtered through a 0.2 pm cellulose acetate filter into the stainless steel collection vessel. The eluate is collected until the absorbance of the trailing edge of the elution peak decreases to &lt; 0.2 AU above the baseline. The collection vessel is then cooled to 2° to 8°C. The maximum hold time for the QFF’chromatography step product pool at 2° to 8°C is 3 days.

[00888] A CTLA4-Ig dimer product with a molar ratio of moles sialic acid to moles CTLA4-Ig protein that is 35 is collected; a pool of CTLA4-Ig high molecular weight material is present at &lt;ί.5%; a pool of CTLA4-Ig low molecular weight material (for example

CTLA4-Ig monomer) is present at ^).5%; and a pool of MCP-1 &lt;9.5 ng/mL is present.

[00889] The Pall Filtron TFF system is used in the concentration and diafiltration step of the downstream CTLA4-Ig production process. The objective of this step is to concentrate the QFF chromatography step product pool to 45 to 55 g/L and to exchange the elution buffer used in the QFF chromatography step with the final buffer used for CTLA4-Ig compositions. The concentrated CTLA4-lg protein product pool is transferred through a 0.2 pm polyvinylidene fluoride filter and into a 50-L bioprocess bag.

[00890] Example 16: CTLA4-Ig-M01ar Ratio Determination of Amino Monosacch arides

[00891] This example provides methods to obtain molar ratios of amino monosaccharides (N-acetyl galactosamine, N-acetyl glucosamine) to protein in CTLA4-Ig samples.

[00892] Instrumentation: Capillary Electrophoresis System Beckman P/ACE

MDQ CE System; Detector Beckman Laser-Induced-Fluorescence (LIF) detection system(coupled with P/ACE MDQ); Uncoated capillary (i.d. 25 pm;o.d. 360 pm), 27-31 cm total length to accomodate either P/ACE MDQ or 5510 PolyMicro Technologies, Cat. No. TSP025375; Maxi-Mix mixer Thermolyne, (VWR Catalog No. 58810-185)

[00893] Reagents:

Hydrolysis Solution (4 N HCI aqueous solution)

Add 160 mL of 6 N HCI and 80 mL of HPLC grade water to a 250 mL glass bottle. Stir to mix well.

Store at 2-80 C for up to 6 months.

Derivatization Solution I (0.1 M APTS aqueous solution)

Add 192 pL of HPLC grade water to 10 mg powder of APTS in a glass vial.

Vortex the vial 5-10 seconds to completely dissolve the APTS.

Store at —20 oC for up to one year.

Derivatization Solution II (1 M acetic acid and 0.25 M NaBH3CN)

Dilute 20 pL acetic acid with 320 pL HPLC grade water (17 fold dilution) in a 0.4 mL centrifuge tube to make a 1 M acetic acid solution.

Weigh 2.0±0.5 mg of NaBH3CN into a cryogenic vial.

Using the following formula, add an appropriate volume of the 1 M acetic acid solution to make 0.25 MNaBH3CN. Volume (pL) = 103 * (weight 0fNaBH3CN in mg)/(62.84 g/mol 0.25 א mol/L)

[00894] . Sodium cyanoborohydride (NaBH3CN) should be stored in dark in a desiccator. . Subdividing of the reagent into a series of 2.0 mL cryovials for storage is recommended to avoid repeated opening of the original reagent bottle as follows: . Weigh 1.0 g ± 0.2 mg of Sodium Cyanoborohydride into 2.0 mL cryovial. Aliquot out the entire contents of Sodium Cyanoborohydride from the original bottle in this manner. . Cap tightly and label cryovials sequentially (1,2,3, etc.) along with reagent name, lot number, and a 6 month expiration date. . The vials should be sealed with parafilm to avoid moisture. . Weigh out Sodium Cyanoborohydride for Derivatization Solution II no more than three times from the same cryovial. Make note of this and the cryovial sequence number on the lab worksheet. • Either a reagent peak observed in the CE profile or poor labeling may occur after repeated opening of the cryovial or with that particular lot of Sodium Cyanoborohydride. If this effects the results, discard the cryovial being used and either weigh out reagent from a cryovial with the next sequence number or from a new lot of Sodium Cyanoborohydride.

Re-acetylation Buffer (25 mM sodium bicarbonate, r>H 9.5) Weigh 0.210 ± 0.02 g of sodium bicarbonate into a clean 100 mL clean glass beaker.

Add 90 mL of HPLC grade water, and mix on a stir plate until salts are completely dissolved.

Adjust the pH to 9.5 ±0.1 with 10N NaOH.

Add HPLC grade water to make the final volume 100 mL. Filter (step 1.26) the solution and store at room temperature for up to 3 months. Running Buffer (60 ± 5 mM Sodium tetraborate, pH 9.25) Weigh 1.21 ± 0.02 g sodium tetraborate into a 100 mL clean glass beaker. Add 90 mL of HPLC grade water, and mix on a stir plate until salts are completely dissolved.

Adjust the pH to 9.25 ±0.10 with 10N NaOH.

Add HPLC grade water to make the final volume 100 mL for a final concentration of 60 ± 5 mM.

For a 55 mM solution, weigh 1,11 g (± 0.02) sodium tetraborate and follow above instructions for dissolving and titrating.

For a 65 mM solution, weigh 1.31 g (± 0.02) sodium tetraborate and follow above instructions for dissolving and titrating.

Store at room temperature for up to 3 months. Prepare fresh buffer if peak resolution is effected (R value &lt;1.0).

Optional: Dilute tetraborate buffer solution (MicroSolv) by adding 120 mL of ultra pure water to 80 mL of 150 mM sodium tetraborate buffer for a final concentration of 60 mM (± 5 mM). Titrate with 1 ON NaOH to bring the solution pH to 9.25 (± 0.1).

For a 55 mM tetraborate solution, dilute 66 mL of 150 mM sodium tetraborate buffer into 114 mL of ultra pure water. Titrate as above. For a 65 mM tetraborate solution, dilute 78 mL of 150 mM sodium tetraborate buffer into 102 mL of ultra pure water. Titrate as above.

Store the solution at room temperature for a maximum of 3 months.

Prepare fresh buffer if peak resolution is effected (R value &lt; 1.0).

Capillary Rinsing Solutions

N NaOH solution

Add 1 mL of 10 N NaOH solution to a 15 mL graduated plastic tube containing 9 mL of HPLC grade water. Mix well by vortexing 5-10 sec.

Store the solution at room temperature for up to 6 months.

N HC1 solution:

Add 1 mL of 6 N HC1 solution to a 15 mL graduated plastic tube containing 5 mL of HPLC grade water. Mix well by vortexing 5-10 sec.

Store the solution at room temperature for up to 6 months.

3.6.3 80% methanol solution:

Add 8 mL HPLC grade methanol to a 15 mL graduated plastic tube containing 2 mL HPLC grade water. Mix well by vortexing 5-10 sec.

Store the solution at room temperature for up to 6 months.

Monosaccharide Standard Stock Solutions

N-Acetyl Glucosamine (GalNAc־)

Accurately weigh 5 ± 1 mg of GalNAc into a 2.0 mL cryogenic vial.

Add 1 mL of HPLC grade water and mix well by vortexing until dissolved. Record the accurate concentration of the solution (mg/mL).

N-Acetyl Galactosamine (GlcNAc)

Accurately weigh 5 ± 1 mg of GlcNAc into a 2.0 mL cryogenic vial.

Add 1 mL of HPLC grade water and mix well by vortexing until dissolved. Record the accurate concentration of the solution (mg/mL).

N-Acetvl Mannosamine (ManNAc)

Accurately weigh 5 ± 1 mg of ManNAc into a 2.0 mL cryogenic vial.

Add 1 mL of HPLC grade water and mix well by vortexing until dissolved. Record the accurate concentration of the solution (mg/mL).

Store Monosaccharide Standard Stock Solutions at — 200 C for up to 1 year.

Monosaccharide Working Solution I: Internal Standard Working Solution Dilute stock solution of ManNAc 100 fold with HPLC grade water by adding 20 pL of ManNAc stock solution into a 2 mL cryogenic vial which already contains 1980 pL of HPLC grade water. Vortex approximately 5 to 10 seconds.

Store the internal standard working solution at 2-8״C for up to 6 months.

Monosaccharide Working Solution II: Amino Mix Standard Working Solution

In a 2.0 mL cryogenic vial containing 1960 pL of HPLC grade water, add 20 pL of stock solutions of GalNAc and GlcNAc, respectively. Vortex approximately 5 to 10 seconds.

Store the amino mix standard working solution at 2-80C for up to 6 months. Sample and reference material solutions.

Thaw frozen protein samples at 2-80 C, and gently mix by inversion.

Dilute both samples and reference material with HPLC grade water to about 1.0 mg/mL. Make note of concentration out to three significant figures.

[00895] CE Running Conditions

Running Buffer (step 2.5) 60 mM sodium tetraborate, pH 9.25

Capillary Cartridge temperature 250 C

Voltage 25-30 kV, positive mode

Detector condition LIF detector, Excitation: 488nm, Emission: 520 nm.

Sample injection Pressure injection mode, 20 s at 0.5 PSI

Run Time 10 minutes

Sample storage 10° C

[00896] Procedure

Note: Use a 10 pL Pipettor and micro tips to transfer 10 pL sample volumes and appropriately sized Pipettors to transfer other reagents (see ranges in steps 2.10 through 2.14).

Hydrolysis ’

In a 0.65 mL centrifuge tube, add 10 pL of ManNAc working solution and 200 pL 4 N Hydrolysis Solution (step 3.1). This serves as a system blank.

In a 0.65 mL centrifuge, tube, add 10 pL of ManNAc working solution and 10 pL of Amino Mix Standard Solution (step 3.9). Further add 200 pL of 4N Hydrolysis Solution. ־This serves as monosaccharide standard for quantitation and System Suitability. Prepare in duplicate.

In a 0.65 mL centrifuge tube, add 10 pL of ManNAc working solution and 10 pL of CTLA4-Ig reference material solution (approximately 1 mg/rnL).

Further add 200 pL of 4N HC1 solution. Prepare in duplicate.

In a 0.65 mL centrifuge tube, add 10 pL of ManNAc working solution and 10 pL of sample solution (approximately 1 mg/mL). Further add 200 pL of 4N HC1 solution. Prepare in duplicate.

Vortex samples for approximately 10 seconds and centrifuge for approximately 5-10 seconds. Place samples in a 96-position vial rack and incubate in an oven at 950 C for 6 hr.

After hydrolysis, place hydrolyzed samples at — 200 C for 10 minutes to cool down.

Briefly centrifuge the hydrolyzed samples until any condensate is forced to the bottom of the tube (5-10 seconds at high speed). Evaporate samples to dryness in SpeedVac.

Note: Turn off SpeedVac heat, and set the evaporating rate to ^,,Low”.

Reconstitute each sample with 100 pL of HPLC grade water and vortex 10-15 sec. Evaporate samples to dryness in SpeedVac.

Note: Turn off SpeedVac heat, and set the evaporating rate to Low”.

Re-acetylation

Reconstitute each sample with 10 pL of M6 re-acetylation buffer and vortex 5-10 sec. to mix well. Add 4 pL of M3 re-acetylation reagent into each tube. Vortex for approximately 5-10 seconds. Incubate on ice for 30 minutes.

Note: The re-acetylation buffer (M6) and reagent (M3) can be replaced respectively with 25 mM NaHCO3 (add 20 pL) prepared in house and acetic anhydride (add 4 pL).

Evaporate samples to dryness in SpeedVac.

Note: Turn off SpeedVac heat, and set the evaporating rate to Low.

Reconstitute each sample with 100 pL of HPLC grade water and vortex

10-15 sec. Evaporate samples to dryness in SpeedVac.

Note: Turn off SpeedVac heat, and set the evaporating rate to Low”.

Derivatization

Place the micro centrifuge in the oven to equilibrate to .the oven temperature of 550 C.

Reconstitute each sample with 10 pL of Derivitization Solution I (0.1 M APTS solution, step 3.2). Vortex approximately 5-10 seconds.

Add 5 pL of the Derivatization Solution II (IM HAc and 0.25 M NaBH3CN, step

3.3). Vortex approximately 5-10 seconds and centrifuge.

Quickly load the sample vials into the pre-warmed centrifuge, and place the centrifuge back in the 550 C oven. Incubate for 3 hr while centrifuging at 2000 rpm. This prevents the condensation of solvent on vial surface.

Instrumentation Preparation

Installing a new capillary, rinse in high pressure mode (80 PSI) using the following steps:

N NaOH for 20 minutes.

HPLC grade water for 10 minutes.

mM sodium tetraborate buffer for 10 minutes.

Operation

Before each operation, run the washing/rinse sequences to rinse the capillary.

Then run the System Suitability Standard (monosaccharide standard) to ensure the system is suitable.

Using IN NaOH may etch the inside of capillaries from different vendors and cause a shift in migration times throughout the run. If this causes the migration time of the last peak (GlcNAc) to be more than 10.0 minutes, it may be necessary to replace IN NaOHwith 0.1N NaOH or HPLC grade water for the step 2 rinse.

When using an equivalent capillary and the above washing procedure is not adequate using 80% methanol and/or IN HCI may be necessary for the last peak (GlcNAc) to be within the exemplary values of 10.0 minutes.

Preparation for injection

After derivatization, let samples cool down to room temperature. Centrifuge approximately 10 seconds at room temperature, until condensate is forced to the bottom of the tube.

Add 85 pL of HPLC grade water to each tube to bring the final volume of each sample to 100 pL. Vortex for 5-10 seconds.

Transfer lOpL of sample from each tube to a CE micro vial and add 190 pL of HPLC grade water to each tube. Vortex for 5-10 seconds.

Rinse steps and Injection sequence:

Note: For every four injections, change the CE running buffer with newly prepared CE running buffer (due to ionic depletion effect). Perform capillary rinse at 40 psi.

<td> Step</td><td colspan="2">^;״&lt;: (beicripiliin?׳'׳ '</td><td> ίίϊηη Time) (min); .</td>

<td> 1 (Rinse)</td><td colspan="2"> .HPLC grade water</td><td> 1</td>

<td rowspan="3"> 2 (Rinse)</td><td rowspan="3"> refer to section 5.4.2.3</td><td> IN NaOH or 0.1 N NaOH</td><td> 3</td>

<td colspan="2"> OR</td>

<td> HPLC grade water Note: When using HPLC water for the step 2 rinse, steps 1,2, and3 maybe combined in a single 3 minute run.</td><td> 1</td>

<td> 3 (Rinse)</td><td colspan="2"> HPLC grade water</td><td> 1</td>

<td> 4 (Rinse)</td><td colspan="2"> 60 mM sodium Tetraborate Run Buffer</td><td> 5</td>

<td> 5 (Rinse)</td><td colspan="2"> Blank (Internal Standard Marker)</td><td> IO</td>

<td> 6 (Rinse)</td><td colspan="2"> Repeat 1-4</td><td> 10</td>

<td> 7 (Rinse)</td><td colspan="2"> Sys tern Suitabi lity (Mono Std prep 1)</td><td> 10</td>

<td> S (Rinse)</td><td colspan="2"> Repeat 1-4</td><td> 10</td>

<td> 9</td><td colspan="2"> System Suitability (Mono Std prep 1)</td><td> 10</td>

<td> 10 (Rinse)</td><td colspan="2"> Repeat 1-4</td><td> 10</td>

<td> 11</td><td colspan="2"> Sys tern Suitabi lity (Mono Std prep 2)</td><td> 10</td>

<td> 12 (Rinse)</td><td colspan="2"> Repeat 1-4</td><td> 10</td>

<td> 13</td><td colspan="2"> System Suitabi lity (Mono Std prep 2)</td><td> 10</td>

<td> 14 (Rinse)</td><td colspan="2"> Repeat 1-4</td><td> 10</td>

<td> 15</td><td> CTLA4-Ig</td><td> prep 1</td><td> 10</td>

<td> 16 (Rinse)</td><td colspan="2"> Repeat 1-4</td><td> 10</td>

<td> 17</td><td> CTLA4-Ig</td><td> prep 2</td><td> 10</td>

<td> 1S (Rinse)</td><td colspan="2"> Repeat 1-4</td><td> 10</td>

<td> 19</td><td colspan="2"> Sample 1 prep 1</td><td> 10</td>

<td> 20 (Rinse)</td><td colspan="2"> Repeat 1-4</td><td> 10</td>

<td> 21</td><td colspan="2"> Sample 1 prep 1</td><td> 10</td>

<td> 22 (Rinse)</td><td colspan="2"> Repeat 1-4</td><td> 10</td>

<td> 23</td><td> Sample 1 prep 2</td><td> 10</td>

<td> 24 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 25</td><td> Samplel prep 2</td><td> 10</td>

<td> 26 (Rinse)</td><td> :Repeat 1-4</td><td> 18</td>

<td> 27</td><td> Sample 2 prepl</td><td> 10</td>

<td> 28 (Rinse)</td><td> 4־1 Repeat.</td><td> 10</td>

<td> 29</td><td> Sample 2 prep 1</td><td> 10</td>

<td> 30 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 31</td><td> Sample 2 prep 2</td><td> 10</td>

<td> 32 (Rinse)</td><td> .Repeat 1-4</td><td> 10</td>

<td> 33</td><td> Sample 2 prep 2</td><td> 10</td>

<td> 34 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 35</td><td> Sample 3 prep 1</td><td> 15</td>

<td> 36 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 37</td><td> Sample 3 prep 1</td><td> 10</td>

<td> 38 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 39</td><td> Sample 3 prep 2</td><td> 10</td>

<td> 40 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 41</td><td> Sample 3 prep 2</td><td> 10</td>

<td> 42 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 43</td><td> CTLA4-Ig a Material prepl</td><td> 10</td>

<td> 44 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 45</td><td> CTLA4-Ig ³ Material prep 2</td><td> 10</td>

<td> 46 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 47</td><td> System Suitability (Mono Std prep 1)</td><td> 10</td>

<td> 48 (Rinse)</td><td> Repeat 1-4</td><td> 10</td>

<td> 49</td><td> System Suitabi lity (Mono S td prep 1)</td><td> 10</td>

<td> 50</td><td> Repeat 1-4</td><td> 10</td>

<td> 51</td><td> System Suitability (Mono Std prep 2)</td><td> 10</td>

<td> 52</td><td> Repeat 1-4</td><td> 10</td>

<td> 53</td><td> System Suitability (Mono Std prep 2)</td><td> 10</td>

[00897] System Suitability

Note: System suitability values are determined using the first injection of system suitability standard unless otherwise specified.

The electropherogram of the first system suitability should be similar to that shown in Figure 81, where peak 1 is GalNAc; peak 2 is ManNAc; peak 3 is GlcNAc.

Note: When CE instruments other than Beckman PACEMDO are to be used, the length of the capillary might be different from that specified in this method due to various configurations of cartridges holding the separation capillary. This would cause variations in analyte migration time, as well as peak intensity.

Resolution between two neighbor peaks is calculated for the first System Suitability standard by the instrument according to the following equation:

p Xt₂-t.) (w,+w₃)

Where:

R - resolution t₂ , t! - migration times of the two neighbor peaks respectively

W יו W2 - peak widths at baseline of the two neighbor peaks respectively

[00898] The R value must be > 1.0. If R < 1.0, rinse the capillary with the washing/rinse sequences; if the problem persists, replace old buffer with freshly prepared

Running Buffer or replace the capillary. For the last System Suitability injection, the last peak (GlcNAc) must have a tailing factor &lt;1.4 using the following formula:

T-W״._n5Z2f

Where:

T - tailing factor

W(1.1)5 - width of peak at 5% of height f - width of the peak front, at peak maximum

If T > 1.4, rinse the capillary with the washing/rinse sequences; if the problem persists, replace old buffer with freshly prepared run buffer or replace the capillary.

The replicate injections show the following exemplary values:

Peak Area Ratio of GlcNAc vs. MaNAc: RSD &lt; 10% (calculated in step 7.1)

Migration time of GlcNAc should be &lt;10.0 minutes

Profile should be equivalent to Figure 81 where the three peaks are observed and the Internal Standard (ManNAc) is the number 2 peak.

If any of the above exemplary values are not reached prior to testing samples, first increase the voltage if the migration time of GlcNAc is greater than 10.0 minutes. Next, if the peak area ratio is > 10%, prepare fresh CE buffer making certain of its pH or replace the capillary. After adjustment to the instrument, repeat System Suitability injections. When analyzing the peak profile, if a significant decrease in the peak height of ManNac occurs, check to make certain the fiber optic cable into the LIF module is not misaligned.

Determine monosaccharide standard percent RSD by comparing peak area ratios of internal standard and monosaccharide standard components. Divide the peak area for each monosaccharide component by the peak area of the internal standard for each monosaccharide standard injection. Calculate the percent RSD for GalNAc and GlcNAc for the two bracketed standards. The RSD should be &lt; 10%. If this averaging exemplary value is not met, then the capillary should be rinsed or replaced as above.

[00899] Calculations

Calculating Peak Area Ratio of GalNAc and GlcNAc relative to the Internal Standard (ManNAc). Used on replicate injections of first four System Suitability Standards so as to meet above exemplary values and performing same calculations on all of the bracketed, System Suitability Standards injected before and after sample(s).

Peak Area Ratio = Divide the peak area for each monosaccharide component (GlcNAc, GalNAc) by the peak area of the internal standard (ManNAc) for each System Suitability Standard injection.

״ . . ״ . monosaccharide peak area Peak Area Ratio =--------------z------MaNAc peak area

Calculate a mean of the Peak Area Ratios for GlcNAc and GalNAc in the System

Suitability Standards. Also calculate a Standard Deviation (S.D.) and percent relative standard deviation (%RSD)

Exemplary Values: RSD for the Peak Area

Ratio of GlcNAc &lt; 10%.

Two, bracketed, System Suitability Standards injected before and after sample(s): Percent RSD for the Peak Area Ratio of GlcNAc and GalNAc < 10%. If this averaging exemplary value is not met (RSD > 10%), then the capillary needs to be rerinsed with the rinse procedures and those samples and bracketed monosaccharide standards need to be run again. If the averaging exemplary value is still not met, replace the capillary and rinse. Run the samples and bracketed monosaccharide standards again.

Standard Deviation = .1”^.

Y «(«-I)

Where:

n — number of measurements in the sample x - individual measurements ״״״ ,״ Standard Deviation % RSD ----------------------X100

Average Measured Peak Area

[00900] Calculate the molar ratio of GalNAc/Protein:

AqUNA&amp;X AwnnN.VD X VoalNAcO X Cg-jINAcO X Rcial NAe. ----------------------------------------------------------------------AManNAc X AOilNAcO X Vp X Cp X MW_flld)AC

Where:

RgbINAc = molar ratio of GalNAc vs. protein

Agsinac =peak area (pVsec) of GalNAc in sample

AmhiiNAc =peak area (pV-sec) oEManNAc in sample

AwnnNAco = peak area (μ V-sec) average of ManNAc in monosaccharide standard

Aguinac□ - peak area (μV-sec) average- of GalNAc in monosaccharide standard

V'ciaiNAco = volume of GalNAc contained in monosaccharide working solution used for hydrolysis (in pL)

Cgu1nac0 = concentration of GalNAc contained in monosaccharide working solution used lor hydrolysis (in mg/mL)

Vp = volume, of protein sample used for hydrolysis (in pL)

Cp = concentration □f protein sample used for hydrolysis (in mg/mL)

MWAbaiacepl — Molecular weight of Abatacept Reference Material as per Certificate of Analysis (COA)

M WgicNAc = Molecular weight of GalNAc (221.2 daltons)

[00901] Standards Bracketing

[00902] When calculating molar ratios of CTLA4-Ig material and samples, use all eight of the bracketed System Suitability Standards. Average the peak areas for inclusion in this equation. This is to be used for the first three samples. For all other samples, always use the average peak area of the next four bracketed monosaccharide standards and the previous four bracketed monosaccharide standards for molar ratio calculations.

[00903] Calculate the molar ratio of GIcNAc/Protein _ AaicNAe X AMnnNAcO X VGkNAoO X CoicNAcO X M W; Rg>IKAc —

CTLA4-lg

Αμλπνλ» X AotaNAu-o X Vp X Cp X MWgicNju

Where:

Rgicnac - molar ratio of GlcNAc vs. protein

Agicnac = peak area (p V-sec) of G1 cNAc in sample.

AmuhNAc = peak area (pV*sec) of MariNAc in sample

A_M״nNAc0 = peak area (pV^sec) average of ManNAc in monosaccharide standard

Agicnaco = peak area (pV-sec) average of GlcNAc in monosaccharide standard

Vgicnaco = volume of GlcNAc contained in monosaccharide working solution used for hydrolysis (in p.L)

CgicNAcO = concentration of GlcNAc contained in monosaccharide working solution used for hydrolysis (in mg/mL)

Vp = volume of protein sample used for hydrolysis (in pL)

Cp = concentration of protein sample used for hydrolysis (in mg/mL)

[00904] MWctla4-i_b = Molecular Weight of CTLA4-Ig Reference Material

[00905] MWqicnac ⁼ Molecular Weigh of GlcNAc (221.2 daltons)

[00906] Exemplary values. The percent RSD for the two, bracketed, amino System Suitability Standard peak area ratios should not exceed 10%. The average molar ratios for amino monosaccharides in the reference material should be within the ranges specified in the Table directly below. For each component, the % RSD for the four results (duplicate injection of duplicate preparations) must be &lt;/= 25%.

Table - Molar Ratio range of CTLA4-Ig Reference Material

<td> Monosaccharide</td><td> -:Range</td>

<td> GalNAc</td><td> 2.0-3.2</td>

<td> GlcNAc</td><td> 18-32</td>

[00907]

Example 17: Determination of molar ratio of aminomonosaccharides (GalNAc and GlcNAc) by capillary electrophoresis (CE)

[00908] In one embodiment, the CTLA4-Ig composition has the characteristic of having from about 15-35 moles GlcNAc / mole of protein and from about 1.7-3.6 moles GalNac / moles protein. The following example describes a method of determining these molar ratios.

[00909] Reagents: Hydrolysis solution (4N HCI); Derivatization solution I (0.1M 8-amino-

1,3,6, trisulfonic acic, trisodium salt (APTS) aqueous solution); Derivatization solution II (0.25M NaBH3CN in IM acetic acid); Re-acetylation buffer (25mM sodium bicarbonate, pH9.5); Running buffer (60 ± 5 mM sodium tetraborate, pH9.25); Capillary rinsing solutions (INNaOH; IN HCI; 80% methanol); Monosaccharide standard stock solutions of GalNAc, GlcNAc, and ManNAc at concentration of 5mg/ml; Monosaccharide working solution I: Internal standard working solution is lOOfold dilution of ManNAc stock solution; Monosaccharide working solution II: Amino mix standard working solutions, lOOfold dilution of GalNAc and GlcNAc stock solutions.

[00910] Instrumentation: CE system is Beckman P/ACE MDQ CE sytem; Detector: Beckman laser induced (L1F) detection system coupled with P/ACE MDQ); Uncoated capillary (i.d. 25pm, o.d. 360pm) 27-31 cm total length to accommodate P/ACE MDQ.

Capillary Electrophoresis running conditions: Running buffer (60mM sodium tetraborate, pH 9.25); Capillary cartridge temperature: 25°C; Voltage: 25-30 kV, positive mode; Detector condition: LIF detector, excitation at 488nm, emission at 520m; Sample injection: pressure injection mode, 20s at 0.5PSI; Run time: 10 min; Sample storage: 10°C.

[00911] Hydrolysis: 1 OpL of ManNAc working solution and 200pJL of 4N HCI were mixed to make the system blank. 1 OpL of ManNAc working solution and 1 OpL of Amino mix standard solution were mixed with 200pL of 4N HC1 to make the monosaccharide standard. 10pL of ManNAc working solution and ΙΟμΕ of CTLA4-Ig dimer (approximately 1 mg/ml) were mixed with 200μΕ of 4N HC1 to make the test sample. All tubes were vortexed for lOsec, and centrifuge for lOsec, followed by incubation at 95°C for 6 hours. After the hydrolysis step the samples were places at -20°C for 10 min to cool down. Samples were spun down for 1 Osec and evaporated to dryness in SpeedVac.

[00912] Re-acetylation: Hydrolyzed and dried samples were reconstituted with 100μΕ of HPLC grade water. Reconstituted samples were re-acetylated by addition ofΙΟμΕ of M6 reN- acetylation buffer (Glyko) and 4μΕ of M3 re-acetylation reagent (Glyko), followed by mixing and with incubation on ice (30min). Samples were spun down for 1 Osec and evaporated to dryness in SpeedVac.

[00913] Derivatization: Reconstituted samples (100μΕ of HPLC grade water) were equilibrated 55 °C, followed by addition of 1 ΟμΕ of Derivatization solution I, a brief mix, and addition of 5μΕ of Derivatization solution II. Samples were loaded in a pre-warmed centrifuge and incubated for 3 hours at 55°C while centrifuging at 2000rpm.

[00914] CE injection: The final volume of the samples after derivatization was brought to ΙΟΟμΕ by addition of HPLC grade water, and 10μΕ of samples were transferred to a CE micro vial with 190μΕ HPLC grade water. Before sample injections the CE cartridge was rinsed extensively with HPLC grade water (1-3 min run time), followed by an equilibrating rinse with running buffer (5min run time). Following the initial rinse, monosaccharide standards and samples for analysis were injected in the CE cartridge (10 min run time).

Following the injection run of each standard or test sample, the CE cartridge was rinsed and. equilibrated with HPLC grade water and running buffer. The electopherograpm of the system suitability should be similar to FIG. 30.

[00915] Calculations: Calculating peak area ratio of GalNAc and GLCNAc relative to internal standard ManNAc.

Peak area ratio=monosaccharide peak area (GalNAc or GlcNAc)/ManNAc peak area, wherein the relative standard deviation (RSD) for the peak area ratio is equal or less that

10%.

Calculating ratio of monosaccharide (for example GalNAc) to CTLA4-Ig protein:

Rati0GalNAc⁼ (AoalNAc x AftlanNAcO X VGalNAcO X CoalNAcO X MW CTLA4-Ig dimer) / (AfrianNAc X

A_GalNAcO X Vp X Cp X MW_GalNAc)

Ratio_Ga1NAc= molar ratio of GalNAc versus protein

Agsinac = peak area (^V.sec) in GalNAc sample

A_ManNAc = peak area (gV.sec) in ManNAc sample

A_Ma״NAco = peak area (pV.sec) average of ManNAc in monosaccharide standard

A_Ga1NAco = peak area (μν.βεο) average of GalNAc in monosaccharide standard

V_Ga1NAco = volume of GalNAc contained in monosacchride working solution used for hydrolysis (in μΙΤ)

C_Ga1NAcO = concentration of GalNAc contained in monosacchride working solution used for hydrolysis (in mg/ml)

Vp = volume of protein sample used for hydrolysis (in μΕ)

Cp = concentration of protein sample used for hydrolysis (in mg/ml)

MW c־TLA4-1g = Molecular weight of CTLA4-Ig dimer

MW_Ga!NAc =221.2 daltons.

Table 18: Average Molar Ratio of Monosaccharide to CTLA4-Ig molecules or dimer

<td> MONOSACCHARIDE</td><td> RANGE</td>

<td> GalNAc</td><td> 2.0-3.2</td>

<td> GlcNAc</td><td> 18-32</td>

Example 18: Determination of molar ratio of neutral monosaccharides (mannose, fucose and galactose) by capillary electrophoresis (CE)

[00916] Reagents: Hydrolysis solution (2M trifluoroacetic acid (TFA)); Derivatization solution I (0.1M 8-amino-1,3,6, trisulfonic acic, trisodium salt (APTS) aqueous solution);

Derivatization solution II (0.25M NaBH₃CN in IM acetic acid); Running buffer (60 ± 5 mM sodium tetraborate, pH9.25); Capillary rinsing solutions (IN NaOH; IN HCI; 80% methanol); Monosaccharide standard stock solutions of mannose (Man), fucose (Fuc), galactose (Gal), and xylose (Xyl) at concentration of 5mg/ml; Monosaccharide working solution I: Internal standard working solution is 100 fold dilution of Xyl stock solution; Monosaccharide working solution II: Neutral mix standard working solutions, 100 fold dilution of Man, Fuc and Gal stock solutions.

[00917] Instrumentation: CE system is Beckman P/ACE MDQ CE sytem; Detector: Beckman laser induced (LIF) detection system coupled with P/ACE MDQ); Uncoated capillary (i.d. 25gm, o.d. 360gm) 27-31 cm total length to accommodate P/ACE MDQ.

[00918] Capillary Electrophoresis running conditions: Running buffer (60mM sodium tetraborate, pH 9.25); Capillary cartridge temperature: 25°C; Voltage: 25-30 kV, positive mode; Detector condition: LIF detector, excitation at 488nm, emission at 520m; Sample injection: pressure injection mode, 20s at 0.5PSI; Run time: 10 min; Sample storage: 10°C.

[00919] Hydrolysis: 1 OgL of Xylose working solution and 200gL of 2M TFA were mixed to make the system blank. lOgL of Xylose working solution and lOgL of Neutral mix standard solution were mixed with 200gL of 2M TFA to make the monosaccharide standard. lOgL of Xylose working solution and lOgL of CTLA4-Ig dimer (approximately 1 mg/ml) were mixed with 200gL of 2M TFA to make the test sample. All tubes were vortexed for lOsec, and centrifuge for lOsec, followed by incubation at 95°C for 6 hours. After the hydrolysis step the samples were places at -20°C for 10 min to cool down. Samples were spun down for lOsec and evaporated to dryness in SpeedVac.

[00920] Derivatization: Samples were reconstituted with 100pL of HPLC grade water and were equilibrated 55°C, followed by addition of 1 OpL of Derivatization solution I, a brief mix, and addition of 5pL of Derivatization solution II. Samples were loaded in a pre-warmed centrifuge and incubated for 3 hours at 55°C while centrifuging at 2000rpm.

[00921] CE injection: The final volume of the samples after derivatization was brought to lOOpL by addition of HPLC grade water, and lOpL of samples were transferred to a CE micro vial with 190pL HPLC grade water. Before sample injections the CE cartridge was rinsed extensively with HPLC grade water (1-3 min run time), followed by an equilibrating rinse with running buffer (5min run time). Following the initial rinse, monosaccharide standards and samples for analysis were injected in the CE cartridge (15 min run time). Following the injection run of each standard or test sample, the CE cartridge was rinsed and equilibrated with HPLC grade water and running buffer. The electopherograpm of the system suitability should be similar to FIG. 31.

[00922] Calculations: Calculating peak area ratio of Man, Gal and Fuc relative to internal standard Xylose.

Peak area ratio=monosaccharide peak area (Gal, Fuc or Man)/Xylose peak area, wherein the relative standard deviation (RSD) for the peak area ratio is equal or less that 10%.

Calculating ratio of monosaccharide (for example Man) to CTLA4-Ig protein:

RatiOMan⁼ (Αμππ X Axyio X VjvianO X CManO X MW CTLA4-Ig dimer) / (Axy! X A!v1anO X Vp X Cp X

MW Man)

RatioMan = molar ratio of Man versus protein

AMan = peak area (pV.sec) in Man in sample

Axy! = peak area (pV.sec) in Xyl in sample

<td> AxylO</td><td> = peak area (μν-sec) average of Xyl in monosaccharide standard ־</td>

<td> AtvIanO</td><td> - peak area (frV.sec) average of Man in monosaccharide standard</td>

<td> VlVtanO</td><td> = volume of Mannose contained in monosacchride working solution used for</td>

hydrolysis (in μΕ)

CMano = concentration of Mannose contained in monosacchride working solution used for hydrolysis (in mg/ml)

<td> • Vp</td><td> = volume of protein sample used for hydrolysis (in μΕ)</td>

<td> Cp</td><td> = concentration of protein sample used for hydrolysis (in mg/ml)</td>

<td> MW CTLA4-lg</td><td> = Molecular weight of CTLA4-Ig dimer</td>

<td> MW_Man</td><td> = 180.2 daltons.</td>

Table 19: Average Molar Ratio of Monosaccharide to CTLA4-Ig molecules or dimer

<td> MONOSACCHARIDE</td><td> RANGE</td>

<td> Mannose</td><td> 10-20</td>

<td> Fucose</td><td> 4.2 -7.0</td>

<td> Galactose</td><td> 9.2-17</td>

EXAMPLE 19: PRODUCTION OF CTLA4^A29YL104E־Ig

[00923] CTLA4^A29YU04E-lg is a genetically engineered fusion protein, which consists of the functional binding domain of modified human CTLA-4 and the Fc domain of human immunoglobulin of the IgGl class. Two amino acid substitutions were made in the

B7 binding region of the CTLA-4 domain (L104E and A29Y) to generate this molecule. It is is comprised of two glycosylated polypeptide chains of 357 amino acids each. It exists as covalent dimer linked through an inter-chain disulfide bond. CTLA4^A29YL104E-Ig has an average mass of approximately 91,800 Da as determined by matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass spectrometry.

[00924] CTLA4^A29YL104E-Ig is a modified form of CTLA4-Ig. The modification consists of point mutations that result in two amino acid substitutions (L104E and A29Y). Relative to CTLA4-Ig, CTLA4^A29YL104E-Ig binds CD80 (B7-1) with ~2-fold increased avidity, and binds CD86 (B7-2) with ~4-fold increased avidity. CTLA4^A29YLI04E-Ig is approximately 10-fold more effective than abatacept at inhibiting T cell proliferation, cytokine production, and CD28-dependent killing of target cells by natural killer cells. CTLA4^A29YL104E-Ig causes modest inhibition of B7-1 mediated T cell proliferation but is markedly more potent at blocking B7-2 mediated T cell proliferation. This Example describes the production of CTLA4^A29YL104E־Ig molecules comprising SEQ ID NO:4. The methods described in this Example can be adapted and extended for the production of other recombinant proteins, including but not limited to, secreted proteins such as cytokines and other hormones, secreted proteins that are members of the Ig superfamily or comprise a portion of an Ig superfamily protein, and generally any protein expressed in CHO cells.

[00925] A process flow diagram for the CTLA4^A29YL104E-Ig culturing steps is shown in FIG. 24. CTLA4^A29YL104E-Ig is produced in 5000-L production bioreactors with an approximate working volume of 4000 L. One batch of drug substance is produced from a single production bioreactor derived from a single vial from a cell bank. The production process involves three-stages consisting of inoculum expansion, production cell culture and downstream purification. The inoculum expansion stage is conducted using animal component-free medium. The production cell culture stage is also performed in animal component-free medium with the exception ofthe use of D-galactose.

[00926] Cell Culture Media. All media are prepared in clean medium vessels of the appropriate size and sterilized by filtration. The composition of the medium utilized for inoculum expansion is presented in the Table below.

Inoculum Cell Growth Basal Medium

<td> Component</td><td> Concentration</td>

<td> CD-CHO, 25x Concentrate Acid Solubles I</td><td> 40 mL/L</td>

<td> CD-CHO, 25 x Concentrate Acid Solubles II</td><td> 40 mL/L</td>

<td> CD-CHO, 25x Concentrate Salts I</td><td> 40 mL/L</td>

<td> CD-CHO, 25x Concentrate Salts II</td><td> 40 mL/L</td>

Inoculum.Cell Growth Basal Medium

<td> Component</td><td> Concentration</td>

<td> L-glutamine</td><td> 0.88 g/L</td>

<td> Sodium Bicarbonate</td><td> 2.22 g/L</td>

<td> Recombinant Human Insulin (10 mg/mL)</td><td> 0.1 mL/L</td>

<td> Methotrexate (20 mM)</td><td> 0.05 mL/L</td>

[00927]

<td> Seed and Production Bioreactor Cell Growth Basal</td><td> Medium</td>

<td> Component CD-CHO, 25x Concentrate Acid Solubles 1</td><td> Concentration 40 mL/L</td>

<td> CD-CHO, 25x Concentrate Acid Solubles II CD-CHO, 25x Concentrate Salts I CD-CHO, 25x Concentrate Salts II L-glutamine Sodium Bicarbonate Recombinant Human Insulin (10 mg/mL)</td><td> 40 mL/L 40 mL/L 40 mL/L 1.32 g/L 2.22 g/L 0.1 mL/L</td>

Production Bioreactor Feed Medium

<td> Component</td><td> Concentration</td>

<td> eRDF powder³</td><td> 25.2 g/L</td>

<td> Dextrose</td><td> 30.9 g/L</td>

<td> D-galactose</td><td> 12.5 g/L</td>

<td> L-glutamine</td><td> 4.1 g/L</td>

<td> Recombinant Human Insulin (10 mg/mL)</td><td> 1.0 mL/L</td>

<td> Dextran Sulfate (added as bolus feed)</td><td> 50 mg/L</td>

[00928] Inoculum Expansion

[00929] A frozen vial from the cell bank is thawed at a controlled temperature and centrifuged to remove the cryoprotectant media. The cells are resuspended in inoculum medium and recovered in a T-flask. A minimum cell viability after thaw of 80% is an exemplary value. Temperature and carbon dioxide are controlled during the T-flask

7׳ incubation step. The T-flask is incubated until a viable cell number of 1.0 x 10 cells is obtained, and the contents are transferred into a shake flask. The culture is expanded through a series of shake flasks to achieve the required inoculum volume. The seeding density range for the shake flask passages is 1.0 to 3.0 x 10⁵ viable cells/mL. Temperature, carbon dioxide, and shaker speed are controlled during the shake flask incubation steps. The shake flask cultures are pooled into a sterile inoculum transfer vessel upon reaching a viable cell density range of 1.5 to 3.0 x 10⁶ cells/mL. Approximately 20 liters from the final shake flask inoculum expansion step is transferred to the 140-L seed bioreactor to achieve an initial cell density range of 0.2 to 1.0 x 10⁶ viable cells/mL.

[00930] Seed Bioreactor Operation

[00931] A 140-L seed bioreactor with a working volume of approximately 90 liters is operated in batch mode. The temperature, pH, pressure, and dissolved oxygen concentration in the 140-L seed bioreactor are monitored and controlled using a distributed control system (DCS). Samples are obtained daily from the 140-L seed bioreactor to monitor cell growth. The seeding density range of the 140-L seed bioreactor is 0.2 to 1.0 x 10⁶ viable cells/mL.

. The 140-L seed bioreactor culture is used to inoculate a 1100-L seed bioreactor when a viable cell density of >1.5 x 10⁶ cells/mL is achieved. The duration of the 140-L seed bioreactor step is approximately 3 days. The initial target viable cell density in the 1100-L seed bioreactor is 0.4 to 1.5 x 10⁶ viable cells/mL.

[00932] The 1100-L seed bioreactor contains an initial culture volume of260 liters. The 1100-L seed bioreactor is operated in batch mode. The temperature, pH, pressure, and dissolved oxygen concentration in the 1100-L seed bioreactor are monitored and controlled using a DCS. The volume of the culture is increased to 900 liters with basal medium when the viable cell density has reached >1.5 x 10⁶ cells/mL. Samples are obtained daily from the 1100-L seed bioreactor to monitor cell growth. The 1100-L seed bioreactor culture is used to inoculate a 5000-L production bioreactor when a viable cell density of >2.0 x 10⁶ cells/mL is achieved. The duration of the 1100-L seed bioreactor step is approximately 4 days. The initial target viable cell density in the 5000-L production bioreactor is 0.4 to 1.5 x 10⁶ viable cells/mL.

[00933] Production Bioreactor Operation

[00934] The 5000-L production bioreactor contains an initial culture volume of 3000 I liters. The 5000-L production bioreactor is operated in fed-batch mode with temperature, pH, pressure, and dissolved oxygen concentration monitored and controlled using a DCS. A bolus of dextran sulfate is added to the culture at approximately 72 hours. During the operation of the production bioreactor, the culture temperature setpoint is shifted from 37° to 34°C at 144 ± 8 hours. The temperature shift and the dextran sulfate addition are performed to prolong the duration of high cell viability in the 5000-L production bioreactor step. Samples are obtained from the bioreactor to monitor cell growth and viability, glucose, lactate and ammonia concentration. The samples are also tested for CTLA4^A29YL104E-Ig concentration and sialic acid to CTLA4^A29YL104E-Ig protein molar ratio. The feed medium is added to the bioreactor to maintain a desired glucose concentration. The primary harvest criterion for the production bioreactor is the sialic acid to CTLA4^A29YLI04E-Ig protein molar ratio. The production bioreactor is harvested at a target sialic acid to CTLA4^A29YL104E-Ig protein molar ratio of >6. The duration of the 5000-L production bioreactor step is approximately 14 days. The harvest volume of the 5000-L production bioreactor is approximately 4000 liters.

[00935] Cell Removal and Product Concentration

[00936] Cells are removed from the culture broth by tangential flow microfiltration using 0.65 pm membranes. The microfiltration permeate is concentrated by tangential flow ultrafiltration using 30 kDa nominal molecular weight cutoff (NMWCO) membranes. Transmembrane pressure and flow rates are controlled during the microfiltration and ultrafiltration steps. The concentrate is then passed through a series of membrane filters, with a final filtration through a 0.2 pm single-use filter. The pH of the concentrate is adjusted to 8.0 by the addition of a 0.5 M Tris solution. The microfiltration and ultrafiltration filters are multi-use. The microfiltration filters are cleaned with sodium hypochlorite and Triton X-100 and stored in phosphoric acid. The ultrafiltration filters are cleaned with sodium hypochlorite and sodium hydroxide and then stored in sodium hydroxide.

EXAMPLE 20: PURIFICATION OF RECOMBINANT CTLA4^A29YLI04E־Ig

[00937] Example 20-A: An example of a purification process of CTLA4^A29YLI04E-Ig is shown in the following flow diagram and a description of a purification process is provided by this example.

<img file="IL192098A_D0002.tif" />

[00938] Viral Inactivation

[00939] The pH of the clarified concentrated harvest material is adjusted to S.O by the addition of a 0.5 M Tris solution. Potential adventitious viral agents are inactivated by the addition of 20% Triton X-100 to a final concentration of 0.5% (v/v). The Triton X-100treated protein solution is mixed for > 2 hours.

[00940] Affinity Chromatography

[00941] Affinity chromatography using a column of MabSelect Protein A resin (GE Healthcare, formerly known as Amersham Biosciences) is used to capture the

CTLA4^A29YL104E-Ig protein from the in-process material from the viral inactivation step and to separate the belatacept protein from the majority of impurities.

[00942] The MabSelect Protein A column is equilibrated with a 25 mM NaH2PO4, 150 mM NaCI, pH 7.5 buffer. The dynamic binding capacity of the affinity resin is 25 g of CTLA4^A29YLI04E-Ig protein per liter of resin at a linear velocity of 350 cm/hour. The 157-L column bed is capable of binding approximately 3.9 kg of CTLA4^A29YLI04E-Ig protein.

[00943] The Triton X-100-treated in-process material is applied to the MabSelect Protein A column, and the column is washed with a minimum of 3 column volumes (CV) of equilibration buffer to remove weakly retained impurities. These impurities include the cytokine monocyte chemotactic protein-1 (MCP-1) and Triton X-100. The CTLA4^a29yl104eIg protein is then eluted from the column with a 250 mM glycine, pH 3.0 buffer. ^,The CTLA4^A29YLI04E-Ig protein elutes as a narrow peak in approximately 2 to 3 CV of elution buffer and is collected into a tank containing 2 M HEPES, pH 7.5 buffer in order to increase the pH rapidly and thereby minimize the formation of belatacept high molecular weight (HMW) species.

[00944] Anion Exchange Chromatography

[00945] Anion exchange chromatography using Q-Sepharose Fast Flow (QFF) resin (GE Healthcare) is used primarily to enrich the amount of more highly sialylated species of the CTLA4^A29YL104E-Ig protein. The pH-adjusted belatacept product pool from the MabSelect Protein A column is diluted approximately two-fold with water for injection (WFI) prior to application to the QFF column.

[00946] The QFF column is equilibrated with a 50 mM HEPES, 50 mM NaCI, pH 7.0 buffer. The pH- and conductivity-adjusted MabSelect Protein A step product pool is applied to the QFF column, and the column is washed with a minimum of 3 CV of equilibration buffer to remove weakly bound impurities. The column is then washed with 50 mM HEPES, 140 mM NaCI, pH 7.0 buffer, to remove CTLA4^A29YL104E-Ig protein species with low sialic acid content. The more highly sialylated species of the CTLA4^A29YLI04E-Ig protein are subsequently eluted from the column using &lt; 5 CV of 50 mM HEPES, 200 mM NaCI, pH 7.0 buffer.

[00947] Hydrophobic Interaction Chromatography

[00948] Hydrophobic interaction chromatography (HIC) using Toyopearl Phenyl 650M resin (Tosoh Biosciences) is used primarily to reduce the amount of ctLA4^a29yl104eIg HMW species in the product pool from the QFF chromatography step. Prior to application to the HIC column, the QFF chromatography step product pool is diluted using 50 mM HEPES, pH 7.0 buffer and 50 mM HEPES, 3.6 M ammonium sulfate, pH 7.0 buffer to achieve a conductivity of approximately 135 mS/cm and a CTLA4^A29YL,04E-Ig concentration of &lt; 1 g/L in the QFF product pool.

[00949] The HIC column is equilibrated with a 50 mM HEPES, 1.2 M ammonium sulfate, pH 7.0 buffer. The concentration- and conductivity-adjusted CTLA4^A29YL104E-Ig QFF chromatography step product pool is applied to the column. The column is then washed with a 50 mM HEPES, 1.2 M ammonium sulfate, pH 7.0 buffer to remove weakly bound impurities. The CTLA4^A29YLI04E-Ig protein is eluted from the HIC column using a 50 mM HEPES, 0.55 M ammonium sulfate, pH 7.0 buffer.

[00950] Viral Filtration

[00951] Concentration and diafiltration of the CTLA4^A29YLI04E-Ig product pool from the HIC step is achieved by ultrafiltration (UF). The UF step utilizes a 30-kDa NMWCO membrane and a 25 mM NaH₂PO₄, 10 mM NaCl, pH 7.5 buffer. The UF step is followed by a viral filtration step using a 15-nm Planova membrane (Asahi Kasei). The CTLA4^a29yli04eIg protein product pool is then adjusted to a protein concentration of 25 g/L by UF using a 30-kDa NMWCO membrane.

[00952] Column Sanitization and Storage

[00953] The MabSelect Protein A chromatography column is sanitized using 0.1 N NaOH solution, washed with 25 mM NaH₂PO₄, 150 mM NaCl, pH 7.5 buffer to lower the pH, and then stored in 20% ethanol at 2° to 8 °C. The QFF chromatography column is sanitized with 1 N NaOH solution and stored in 0.1 N NaOH solution at room temperature. The HIC column is sanitized with 0.1 N NaOH solution, washed with 20% ethanol, and stored in 20% ethanol at room temperature.

Example 20-B: A further example of such purification method follows:

[00954] Viral Inactivation

[00955] The pH of the clarified concentrated harvest material is adjusted to 8.0 by the addition of a 0.5 M Tris solution. Potential adventitious viral agents are inactivated by the addition of 20% Triton X-100 to a final concentration of 0.5% (v/v). The Triton X-100treated protein solution is mixed for > 2 hours.

[00956] Protein A Affinity Chromatography for CTLA4^A29YLI04E-Ig Purification: Affinity chromatography using a column of MabSelect Protein A resin (GE Healthcare, formerly known as Amersham Biosciences) is used to capture CTLA4^A29YL104E-Ig from the in-process material from the viral inactivation step and to separate CTLA4^A29YLI04E-Ig from the majority of impurities.

[00957] A 140 cm inner diameter column is packed with MabSelect PrA resin to a height of 18 to 25 cm, representing a volume of about 339 to 372 L. The column is qualified for use by determining HETP and A_s of the packed column. A HETP of 0.02 to 0.08 cm and an A_sof 0.8 to 1.2 are employed for qualification of the column.

[00958] The MabSelect PrA column operation is carried out at ambient temperature. The viral inactivation product pool is loaded onto the equilibrated MabSelect PrA column. The MabSelect PrA step is operated at a maximum flow rate of 26.7 L/min and an operating pressure of &lt; 13 psig. The maximum CTLA4^A29YL104E-Ig protein load applied to the MabSelect PrA column is 25 g of CTLA4^A29YL104E-Ig protein per liter of resin at a linear velocity of 350 cm/hour. The column bed is capable of binding approximately 3.9 kg of CTLA4^A29YL104E-Ig protein.

[00959] The MabSelect PrA column is equilibrated with a 25 mM NaH₂PO4, 150 mM NaCl, pH 7.5 buffer. Equilibration is complete when a minimum of 3 CV of equilibration buffer have been passed through the column and the pH and conductivity values of the effluent are between 7.3 to 7.7 and 14.5 to 17.5 mS/cm, respectively.

[00960] The Triton X-100-treated in-process material is applied to the equilibrated MabSelect PrA column. The column is washed with a minimum of 3 CV of a 25 mM NaH₂PO4, 150 mM NaCl, 0.5 % Triton X-100, pH 7.5 buffer to remove weakly retained impurities from the MabSelect PrA column. These impurities include the cytokine monocyte chemotactic protein-1 (MCP-1) and Triton X-100. Subsequent wash steps are performed using a 25 mM NaH2PO4, 150 mM NaCI, pH 7.5 buffer to remove the residual Triton X-100 from the MabSelect PrA column.

[00961] The CTLA4^A29YL104E-Ig is eluted from the MabSelect PrA chromatography column with a 250 mM glycine, pH 3.0 buffer. The eluate is diverted into a collection vessel when the A280 increases to > 0.2 AU above the baseline. The column effluent is filtered through a 0.2 pm cellulose acetate filter into a collection vessel equipped with an agitator. The eluate is collected until the A280 °f the trailing edge of the elution peak decreases to a value of < 0.2 AU. The CTLA4^A29YL104E-Ig elutes as a narrow peak in approximately 2 to 3 CV of elution buffer. The pH of the eluate pool is adjusted to pH 7.5 ± 0.2 with a 2 M HEPES, pH 7.5 buffer in order to increase the pH rapidly and thereby minimize the formation of CTLA4^A29YL104E-Ig high molecular weight (HMW) species. The MabSelect PrA chromatography step product pool is held at ambient temperature for a maximum of 5 days. The product pool may be cooled for storage; the stability profile of the CTLA4^A29YL,04E-Ig was the same at 5 °C and 22°C. The product may be stored for up to 5 days.

[00962] A CTLA4^A29YL104E-Ig dimer product with a molar ratio of moles sialic acid to moles CTLA4^A29YLI04E-Ig protein that is about 6, or from about 5.2 to about 7.6, is collected.

[00963] QFF Anion Exchange Chromatography for CTLA4^A29YU04E-Ig Purification:

Anion exchange chromatography using Q-Sepharose Fast Flow (QFF) resin (GE Healthcare) is used primarily to enrich the amount of more highly sialylated species of the CTLA4^A29YL104E-Ig as well as reduce the residual Protein A levels. The pH-adjusted CTLA4^A29YLI04E-Ig product pool from the MabSelect Protein A column is diluted, approximately two-fold with water for injection (WFI) prior to application to the QFF column.

[00964] A 80 cm inner diameter column is packed with QFF resin to a height of 27 to 35 cm, representing a volume of about 136 to 176 L. The column is qualified for use by determining the HETP and A_s of the packed column. A HETP of 0.02 to 0.08 cm and an asymmetry (A_s) of 0.8 to 1.2 are employed for qualification of the column.

[00965] The QFF column operation is carried out at ambient temperature. The QFF column is equilibrated with a 50 mM HEPES, 50 mM NaCI, pH 7.0 buffer. The pH- and conductivity-adjusted MabSelect Protein A step product pool is applied to the QFF column.

The QFF step is operated at a maximum flow rate of 16.4 L/min (196 cm/h) and a maximum operating pressure of 35 psi.

[00966] The column is sanitized both prior to and following use with a 1 N NaOH solution. A minimum of 2 column volumes of the sodium hydroxide solution is passed over the column. The column is then held static for 60 to 120 minutes. The acceptable conductivity range for the solution and the column effluent is 136 to 202 mS/cm.

[00967] The column is equilibrated with a minimum of 5 column volumes of a 50 mM HEPES, 50 mM sodium chloride, pH 7.0 buffer. The pH and conductivity ranges for this buffer are 6.8 to 7.2 and 5.0 to 7.0 mS/cm, respectively. ׳These ranges are also used to determine whether the column is equilibrated.

[00968] The pH- and conductivity-adjusted MabSelect Protein A step product pool is applied to the QFF column, and the column is washed with a minimum of 3 CV of equilibration buffer to remove weakly bound impurities. The column is then washed with 50 mM HEPES, 135 mM NaCI, pH 7.0 buffer, to remove CTLA4^A29YLi04E-Ig species with low sialic acid content.

[00969] The more highly sialylated species of the CTLA4^A29YL104E-Ig are eluted from the QFF chromatography column using a 50 mM HEPES, 200 mM NaCI, pH 7.0 buffer. The eluate collection is initiated when the elution buffer is first applied to the column. During elution, the column effluent is filtered through a 0.2 pm filter into the collection vessel. The eluate is collected until the absorbance of the trailing edge of the elution peak decreases to — 0.2 AU above the baseline. The CTLA4^A29YL104E-Ig elutes from the column using &lt; 5 CV of 50 mM HEPES, 200 mM NaCI, pH 7.0 buffer. The collection vessel is then cooled to 2° to 8°C. The maximum hold time for the QFF chromatography step product pool at 2° to 8°C is 3 days.

[00970] A CTLA4^A29YL104E-Ig dimer product with a molar ratio of moles sialic acid to moles CTLA4^A29YLI04E-Ig protein that is about 6, or from about 5.2 to about 7.6 is collected.

[00971] Phenyl Sepharose FF HIC for CTLA4^A2nL,04E-Ig Purification: Hydrophobic interaction chromatography (HIC) using Toyopearl Phenyl 650M resin (Tosoh Biosciences) is used primarily to reduce the amount of CTLA4^A29YL104E-Ig HMW species in the product pool from the QFF chromatography step.

[00972] A 100 cm inner diameter column is packed with Phenyl Sepharose Phenyl 650M resin to a height of 18 to 22 cm, representing a volume of about 141 to 173 L. The column is qualified for use by determining the HETP and A_s of the packed column. A HETP of 0.02 to 0.08 cm and an A_s of .0.8 to 1.2 are employed for qualification of the HIC column.

[00973] The HIC column operation is carried out at ambient temperature. Prior to application to the HIC column, the QFF chromatography step product pool is diluted using 50 mM HEPES, pH 7.0 buffer and 50 mM HEPES, 3.6 M ammonium sulfate, pH 7.0 buffer to achieve a conductivity of approximately 135 mS/cm and a CTLA4^A29YL104E-Ig concentration of &lt; 1 g/L in the QFF product pool. The HIC step is operated at a maximum flow rate of 22.7 L/min (173 cm/h) and at a maximum operating pressure of 45 psi. Multiple cycles of the HIC step can be employed based on the amount of CTLA4^A29YL104E-Ig present in the QXL eluate pool.

[00974] The HIC column is first sanitized with a 1 N sodium hydroxide solution. The sanitization is complete when 2 to 4 CV of the 1 N sodium hydroxide solution have been passed through the column. The column is then held for 60 to 120 minutes to ensure sanitization.

[00975] After the sanitization step, the'HIC column is equilibrated with a 50 mM HEPES,

1.2 M ammonium sulfate, pH 7.0 buffer. The equilibration is complete when a minimum of 3 CV of equilibration buffer have been passed through the column and the pH of the effluent is 7.0 ± 0.3 and the conductivity of approximately 135 mS/cm.

[00976] The concentration- and conductivity-adjusted CTLA4^A29YL104E-Ig QFF chromatography step product pool is applied to the column. The column is then washed with a 50 mM HEPES, 1.2 M ammonium sulfate, pH 7.0 buffer to remove weakly bound impurities. The CTLA4^A29YLI04E-Ig is eluted from the HIC column using a 50 mM HEPES, 0.55 M ammonium sulfate, pH 7.0 buffer. This HIC product pool is held in the collection vessel at 2° to 8°C. The maximum hold time in the collection vessel is 3 days.

[00977] A CTLA4^A29YL104E-Ig dimer product with a molar ratio of moles sialic acid to moles CTLA4^A29YL,04E-Ig protein that is about 6, or from about 5.2 to about 7.6; a pool of

CTLA4^A29YLI04E-Ig high molecular weight material is present at ^2.5%; a pool of

CTLA4^A29YLI04E-Ig low molecular weight material (for example CTLA4^A29YL104E-Ig monomer) is present at ^).5%; and a pool of MCP-1 &lt;9.5 ng/mL is present.

[00978] Viral Filtration. Concentration and diafiltration of the CTLA4^A29YL104E-Ig product pool from the HIC step is achieved by ultrafiltration (UF). The UF step utilizes a 30-kDa NMWCO membrane and a 25 mM NaH₂PO₄,10 mM NaCl, pH 7.5 buffer. The UF step is followed by a viral filtration step using a 15-nm Planova membrane (Asahi Kasei). The CTLA4^A29YL,04E-Ig product pool is then adjusted to a protein concentration of 25 g/L by UF using a 30-kDa NMWCO membrane.

[00979] The Pall Filtron TFF system is used in the concentration and diafiltration step of the downstream CTLA4^A29YLI04E-Ig production process. The objective of this step is to concentrate the HIC chromatography step product pool to 45 to 55 g/L and to exchange the elution buffer used in the HIC chromatography step with the final buffer used for CTLA4^A29YL,04E-Ig compositions. The concentrated CTLA4^A29YL104E-Ig product pool is transferred through a 0.2 pm polyvinylidene fluoride filter and into a 50-L bioprocess bag.

[00980] EXAMPLE 21: BIOLOGICAL ACTIVITY - DETERMINATION OF BTOSPECIFIC BINDING OF CTLA4^A29YL104E-Ig TO THE B-7IG CO-RECEPTOR BY

SURFACE PLASMON RESONANCE

[00981] Surface Plasmon Resonance (B7 Binding)

[00982] ^,This method measures the binding of CTLA4^A29YLI04E-Ig to a representative B7 co-receptor by surface plasmon resonance. B7Ig is immobilized at high density via primary amino groups to the surface of an activated CM5 sensorchip. CTLA4^A29YL104E-Ig material, Quality Controls, and samples are diluted to concentrations between 0.125 and 8 ng/mL and injected over the B7Ig surface to generate binding sensorgrams. The initial rate (slope) of CTLA4^A29YL104E-Ig binding to immobilized B7lg is measured under mass transfer (diffusion) limited conditions on this B7Ig surface. The initial binding rate in resonance units per second (RU/s) correlates directly with the active concentration. The binding rates of samples are converted into an active concentration using the reference standard curve where the binding rate of a CTLA4^A29YLI04E-Ig material is plotted against concentration. The final results are either expressed as percent binding of sample relative to CTLA4^A29YL104E-Ig material.

[00983] The presence of the human IgGl Fc region in CTLA4^A29YL104E-Ig was detected using surface plasmon resonance (SPR). SPR enables measurement of bio specific interactions in real time. An antibody fragment specific for the Fc region of human IgG (goat F(_ab’)2 anti-human IgG Fc) was covalently immobilized on the surface of a sensorchip.

Binding of CTLA4^A29YL,04E-Ig samples was detected by measuring the response obtained on this surface, compared to an unmodified sensor chip surface. The results in resonance units bound for the Process B, Process C and the Co-mixture are comparable as shown in FIG. 6 and Table 23.

Table 23. Detection of Human IgG Fc in CTLA4^A29YL104E-Ig Drug Substance Lots Using SPR

<td> Lot No</td><td> RU bound to anti-Fc Antibody</td><td> RU bound to unmodified surface</td>

<td> Lot A</td><td> 1295 .</td><td> 1</td>

<td> LotB</td><td> 1309</td><td> 1</td>

<td> Co-mixture</td><td> 1268</td><td> 1</td>

[00984] Human Cell IL 2 Inhibition Assay

[00985] The method is based on the inhibition of IL-2 production from T cells by CTLA4^A29YLI04E-Ig when stimulated with anti-CD3 and B cells. Jurkat T cells, transfected with the luciferase gene under the. control of the IL-2 promoter, are co-stimulated with Daudi B cells and anti-CD3 in the presence of various concentrations of CTLA4^A29YL104E-Ig. The co-stimulation activates the IL-2 promoter, which in turn produces luciferase protein. The resulting luminescent signal is measured using a luciferase assay system. In this system, CTLA4^A29YL104E-Ig produces a dose-dependent decrease in luciferase activity.

[00986] The results for the Process B Lot 000929-278, Process C Lot 224818-2004-007 and the co-mixture Lot 55128-162 are comparable as shown in Table 24. The EC50 values, slope factors, and upper and lower asymptotes are similar for all three samples, within one standard deviation. This indicates that CTLA4^A29YL104E-Ig from the Process C and from the Process B behave comparably in the in vitro potency assay.

Table 24. Comparison of Human IL-2 Promoter Mediated Luciferase Activity in the In Vitro Potency Bioassay. Dose Response Curve

Parameters for Processes of the Invention

<td> Lot No.</td><td> EC50 (ng/mL)</td><td> Slope Factor</td><td> Upper Asymptote (CPS)</td><td> Lower Asymptote (CPS)</td>

<td> Process A</td><td> 19.1 ±1.9</td><td>.91 ± 0.06</td><td> 85,000 ± 15,000</td><td> 30,000 ±6,000.</td>

<td> Co-mixture</td><td> 21.5 + 2.7</td><td>.93 ± 0.08</td><td> 88,000 ± 16,000</td><td> 29,000 ± 5,000</td>

<td> Process B</td><td> 21.8 ±1.4</td><td>.91 ± 0.09</td><td> 81,000 ± 17,000</td><td> 27,000 ± 7,000</td>

[00987] Materials:

-Sensor Chip CM5, certified grade Biacore (Catalog No. BR-1000-13)

-HBS-EP Buffer BIA Certified 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, .005% v/v

-Surfactant P20 Biacore (Catalog No. BR-1001-88)

-Amine Coupling Kit BIA Certifiedll5 mg N-hydroxysuccinimide (NHS), 750 mg l-ethyl-3(3 dimethylaminopropyl) carbodiimde hydrochloride (EDC), 10.5 mL ethanolamine HC1 Biacore (Catalog No. BR-1000-50)

-Biacore C Instrument with a PC compatible computer Biacore, (Catalog No. BR-1100-51) -Biacore C Control Software Biacore, as provided with Biacore C instrument, version 1.0.1

[00988] Amine Coupling Kit BIA Certified: The kit contains one vial each: 115 mg NHS, 750 mg EDC, and 10.5 mL ethanolamine. Prepare each vial according to manufacturers directions. Aliquot 200 pL volumes of NHS and EDC solutions into individual plastic/glass vials of appropriate size and cap. These solutions are stable for 2 months when stored at -20°C. Aliquot 200 pL of Ethanolamine into individual plastic/glass vials of appropriate size and cap. This solution is stored at 2-8°C and is stable according to manufacturer’s directions.

[00989] To ensure good binding to the flow cell, a flow cell will be used for one week or 286 injections, which ever comes first. A new flow cell will be immobilized at the beginning of each week. Immobilization of B7.1 Ig in Preparation For Sample Testing.

NOTE: Aliquot 200 pL of all solutions into 7 mm Biacore tubes for analysis. Thaw one vial containing B7.1 Ig at ambient temperature. Dilute B7.1 Ig using 10 mM Acetate pH 5.0 buffer (1.7) to achieve a surface mass of between 3000-9000 Resonance Units (RU). Thaw one vial (200pL) each of EDC, and NHS to ambient temperature. Remove Ethanolamine

HCI from the refrigerator and allow warming to room temperature. From the Biacore software: Open the published project “B7 Ig Immobilization” selected from the “Immobilization Wizard.” Open the published file “B7 Immob.blw.” Step through the wizard and confirm or change selection by clicking “Next.” Under “User Information” select flow cell and provide experimental information in the “Notebook” tab. Place reagent and ligand vials in sample rack as outlined. Review instructions. Save the template file as: B7 Immob BIOQC# Date Initials Chip # Flow cell #.blw. Start immobilization by clicking on “Start.” Save result file as: B7 Immob BIOQC# Date Initial chip # flow cell # .blr. When the assay is finished, print the Wizard results and sensorgram.

EXAMPLE 22: CARBOHYDRATE CONTENT ANALYSIS OF A CTLA4^A29YL104E-Ig COMPOSITION, TRYPTIC PEPTIDE MAPPING AND IEF

[00990] Tryptic digest peptide mapping

[00991] In this trypsin digest method, CTLA4^A29YLI04E-Ig samples are denatured using guanidine-HCl, and reduced and alkylated using DTT and IAA. Samples are desalted using an NAP-5 column and digested with trypsin. The digestion mixture is separated by reversed phase (Cl 8) chromatography and peaks are detected by UV absorbance at 215 nm.

[00992] REAGENTS: Mobile Phase A solution (0.02 % Trifluoroacetic Acid (TFA) in Water (v/v)); Mobile Phase B solution (0.02 % TFA in 95 % ACN (Acetonitrile) and 5 % Water (v/v)); Alkylating Agent (200 mM lodoacetamide (IAA)); Dilution Buffer (100 mM Tris, 25 mM NaCl, pH 8.0); Denaturing Buffer (8 M Guanidine, 50 mM TRIS, pH 8.0); Digestion Buffer (50 mM TRIS, 10 mM CaCl₂, pH 8.0); Reducing Agent (100 mM DTT).

[00993] IN STRUMENT ATI ON: (equivalent instrumentation may be used) NAP-5 columns (Amersham, cat. # 17-0853-02); HPLC Column Heater; Water’s Alliance HPLC system with column heater and UV detector.

[00994] Overview of Procedure

CTLA4^A29YU04E-Ig

Denatured/Reduced/Alkylated Protein

Desalted Protein I

Digested Protein

Map Profile and MS information

[00995] Reduction and Alkylation: Samples (for example, CTLA4^A29YL104E-Ig, etc.) were diluted to 10 mg/ml by adding water to a final volume of 100 pL (1 mg). 560 pl of denaturing buffer and 35 pL of Reducing Agent (100 mM DTT) were added to the 100 pl samples, were mixed, and spun down in a microcentrifuge for 3 seconds. Samples were then incubated at 50° C for 20 minutes ± 2 minutes. 35 pL of Alkylating Agent (200 mM IAA) was then added to each sample, and again were mixed, and spun down in a microcentrifuge for 3 seconds. Samples were then covered with aluminum foil and incubated at 50° C for 20 min. + 2 minutes. After the NAP-5 columns were equilibrated by pouring 3 columns volumes (about 7-8 mL) of digestion buffer, 500 pl of the reduced and alkylated mixtures were poured over the NAP-5 columns, allowing the liquid to drain through column. Samples were then collected from the NAP-5 columns via eluting sample off of the column with 1 mL of digestion buffer.

[00996] Digestion: Samples were digested with 20 pL of trypsin (0.5 pg/pL) in 38° C water bath for 4 hours (+ 0.5 hr). Upon completion of digest, samples were acidified with 2.5 pL of TFA. Samples were then placed into autosampler vials for subsequent analysis.

[00997] Instrument Method: The instrument method is shown below:

<td> Time (min)</td><td> Flow (mL/min)</td><td> Mobile Phase A</td><td> Mobile Phase B</td>

<td> 0</td><td> 0.7</td><td> 100</td><td> 0</td>

<td> 17</td><td> 0.7</td><td> 83</td><td> 17</td>

<td> 27</td><td> 0.7</td><td> 78</td><td> 22</td>

<td> 42</td><td> 0.7</td><td> 73</td><td> 27</td>

<td> 58</td><td> 0.7</td><td> 65</td><td> 35</td>

<td> 74</td><td> 0.7</td><td> 52</td><td> 48</td>

<td> 79</td><td> 0.7</td><td> 0</td><td> 100</td>

<td> 84</td><td> 0.7</td><td> 100</td><td> 0</td>

Time (min)

Flow (mL/min)

0.7

Mobile Phase A

Mobile Phase B 0

[00998] The column was equilibrated with 100% Mobile Phase A buffer for 25 minutes prior to the first injection. UV absorbance was monitored at 215 nm while column temperature was manintained at 37°C and the autosampler temperature at 4°C. A mobile phase A buffer blank was run before the first system suitability standard, thereafter followed by a single 50 gL injection of each sample. A reference material injection should bracket every six sample injections.

[00999] Number of Theoretical Plates: Column efficiency, evaluated as the number of theoretical plates, can be measured quantitatively using the retention time and the width of peak according to the Equation:

( t N = \6 — ,2

Where:

w is the peak width at the baseline measured by extrapolating the relatively straight sides to the baseline, t is the retention time of the peak measured from time of injection to time of elution of peak maximum.

[001000] If the N &lt;50000, re-equilibrate the column.

[001001] Resolution: Determine The resolution (R) between 2 peaks, for example peak T2 and peak T12 as indicated in FIG. 32, can be determined using the following equation:

₌ ^(*2 ) (w, +w₂)

Where:

t!, t₂ = retention times of fragments peak T2 and peak T12, respectively w!, w₂ = tangent-defined peak width at baseline of the peaks with retention times t! and t₂, respectively.

[001002] If R &lt; 1.5, the column should be re-equilibrate and if the problem persists, the column should be replaced.

[001003] FIG. 32 and Table 25 show the peptide fragments obtained from a trypsin digestion of CTLA4^A29YL104E-Ig- The region showing peptides T7 and T9 at -50 minutes sometimes reflects incomplete sample digestion and peaks can show different qualities from day to day; however, within a run all samples show comparability.

Table 25: Tryptic Peptide Fragments of CTLA4^A29Y04״E-Ig

<td> Fragme nt No.</td><td> Residue No.</td><td> Theoretic al Mass</td><td> Observed Mass</td><td> Peptide Sequence</td>

<td> T1</td><td> 1-14</td><td> 1465.8</td><td> 1464.8</td><td> MHVAQPAVVLASSR</td>

<td> T2</td><td> 15-28</td><td> 1485.7</td><td> 1484.8</td><td> GIASFVCEYASPGK</td>

<td> T3</td><td> 29-33</td><td> 666.3</td><td> 666.4</td><td> YTEVR</td>

<td> T4</td><td> 34-38</td><td> 586.7</td><td> 586.4</td><td> VTVLR</td>

<td> T5^a</td><td> 39-83⁰</td><td> 4900.4</td><td> ___d</td><td> QADSQVTEVCAATYMMGNELTFLDDSI CTGTSSGNQVNLTIQGLR</td>

<td> T6</td><td> 84-93</td><td> 1171.4</td><td> 1170.5</td><td> AMDTGLYICK</td>

<td> T7^b</td><td> 94-128°</td><td> 3983.5</td><td> ___d</td><td> VELMYPPPYYEGIGNGTQIYVIDPEPCPD SDQEPK</td>

<td> T8^b</td><td> 129-132</td><td> 435.4</td><td> ND</td><td> SSDK</td>

<td> T9^b</td><td> 133-158⁰</td><td> 3345.7</td><td> ___d</td><td> THTSPPSPAPELLGGSSVFLFPPKPK</td>

<td> T10</td><td> 159-165</td><td> 834.9</td><td> 834.5</td><td> DTLMISR</td>

<td> TH</td><td> 166-184</td><td> 2140.3</td><td> 2138.5</td><td> TPEVTCWVDVSHEDPEVK</td>

<td> T12</td><td> 185-198</td><td> 1677.8</td><td> 1677.8</td><td> FNWYVDGVEVHNAK</td>

<td> T13</td><td> 199-202</td><td> 500.6</td><td> 500.3</td><td> TKPR</td>

<td> T14^a</td><td> 203-211⁰</td><td> 1189.2</td><td> ___d</td><td> EEQYNSTYR</td>

<td> T15</td><td> 212-227</td><td> 1808.1</td><td> 1808</td><td> WSVLTVLHQDWLNGK</td>

<td> T16</td><td> 228-230</td><td> 438.5</td><td> 438.2</td><td> EYK</td>

<td> T17</td><td> 231-232</td><td> 307.4</td><td> ND</td><td> CK</td>

<td> T18</td><td> 233-236</td><td> 446.5</td><td> ND</td><td> VSNK</td>

<td> T19</td><td> 237-244</td><td> 838.0</td><td> 837.4</td><td> ALPAPIEK</td>

<td> T20</td><td> 245-248</td><td> 447.5</td><td> 447.2</td><td> TISK</td>

<td> T21</td><td> 249-250</td><td> 217.2</td><td> ND</td><td> AK</td>

<td> T22</td><td> 251-254</td><td> 456.5</td><td> 456.3</td><td> GQPR</td>

<td> T23</td><td> 255-265</td><td> 1286.4</td><td> 1285.6</td><td> EPQVYTLPPSR</td>

Table 25: Tryptic Peptide Fragments of CTLA4^A29YL104E-Ig

<td> Fragme nt No.</td><td> Residue No.</td><td> Theoretic al Mass</td><td> Observed Mass</td><td> Peptide Sequence</td>

<td> T24</td><td> 266-270</td><td> 604.7</td><td> 604.3</td><td> DELTK</td>

<td> T25</td><td> 271-280</td><td> 1161.4</td><td> 1160.7</td><td> NQVSLTCLVK</td>

<td> T26</td><td> 281-302</td><td> 2544.7</td><td> 2544</td><td> GFYPSDIAVEWESNGQPENNYK</td>

<td> T27</td><td> 303-319</td><td> 1874.1</td><td> 1873.9</td><td> TTPPVLDSDGSFFLYSK</td>

<td> T28</td><td> 320-324</td><td> 574.7</td><td> 574.3</td><td> LTVDK</td>

<td> T29</td><td> 325-326</td><td> 261.28</td><td> ND</td><td> SR</td>

<td> T30</td><td> 327-349</td><td> 2803.09</td><td> 2802.1</td><td> WQQGNVFSCSVMHEALHNHYTQK</td>

<td> T31</td><td> 350-356</td><td> 659.7</td><td> 659.2</td><td> SLSLSPG</td>

<td> T6</td><td> 84-93</td><td> 1171.4</td><td> 1170.5</td><td> AMDTGLYICK</td>

<td> T7^b</td><td> 94-128“</td><td> 3983.5</td><td> _ _d</td><td> VELMYPPPYYEGIGNGTQIYVIDPEPCPD SDQEPK</td>

<td> T8^b</td><td> 129-132</td><td> 435.4</td><td> ND</td><td> SSDK</td>

<td> T9^b</td><td> 133-158'</td><td> 3345.7</td><td> d</td><td> THTSPPSPAPELLGGSSVFLFPPKPK</td>

<td> T10</td><td> 159-165</td><td> 834.9</td><td> .834.5</td><td> DTLMISR</td>

<td> Til</td><td> 166-184</td><td> 2140.3</td><td> 2138.5</td><td> TPEVTCVWDVSHEDPEVK</td>

<td> T12</td><td> 185-198</td><td> 1677.8</td><td> 1677.8</td><td> FNWYVDGVEVHNAK</td>

<td> T13</td><td> 199-202</td><td> 500.6</td><td> 500.3</td><td> TKPR</td>

<td> T14^a</td><td> 203-211'</td><td> 1189.2</td><td> ___d</td><td> EEQYNSTYR</td>

<td> T15</td><td> 212-227</td><td> 1808.1</td><td> 1808</td><td> WSVLTVLHQDWLNGK</td>

<td> T16</td><td> 228-230</td><td> 438.5</td><td> 438.2</td><td> EYK</td>

<td> T17</td><td> 231-232</td><td> 307.4</td><td> ND</td><td> CK</td>

<td> T18</td><td> 233-236</td><td> 446.5</td><td> ND</td><td> VSNK</td>

<td> T19</td><td> 237-244</td><td> 838.0</td><td> 837.4</td><td> ALPAPIEK</td>

<td> T20</td><td> 245-248</td><td> 447.5</td><td> 447.2</td><td> TISK</td>

<td> T21</td><td> 249-250</td><td> 217.2</td><td> ND</td><td> AK</td>

<td> T22</td><td> 251-254</td><td> 456.5</td><td> 456.3</td><td> GQPR</td>

<td> T23</td><td> 255-265</td><td> 1286.4</td><td> 1285.6</td><td> EPQVYTLPPSR</td>

Table 25: Tryptic Peptide Fragments of CTLA4^A29YL104E-Ig

<td> Fragme nt No.</td><td> Residue No.</td><td> Theoretic al Mass</td><td> Observed Mass</td><td> Peptide Sequence</td>

<td> T24</td><td> 266-270</td><td> 604.7</td><td> 604.3</td><td> DELTK</td>

<td> T25</td><td> 271-280</td><td> 1161.4</td><td> 1160.7</td><td> NQVSLTCLVK</td>

<td> T26</td><td> 281-302</td><td> 2544.7</td><td> 2544</td><td> GFYPSDIAVEWESNGQPENNYK</td>

<td> T27</td><td> 303-319</td><td> 1874.1</td><td> 1873.9</td><td> TTPPVLDSDGSFFLYSK</td>

<td> T28</td><td> 320-324</td><td> 574.7</td><td> 574.3</td><td> LTVDK</td>

<td> T29</td><td> 325-326</td><td> 261.28</td><td> ND</td><td> SR</td>

<td> T30</td><td> 327-349</td><td> 2803.09</td><td> 2802.1</td><td> WQQGNVFSCSVMHEALHNHYTQK</td>

<td> T31</td><td> 350-356</td><td> 659.7</td><td> 659.2</td><td> SLSLSPG</td>

^a Peptides with N-linked carbohydrate ^b Peptides with O-linked carbohydrate ^c Masses for T5, T7, T9 and T14 are masses without the carbohydrate moieties ^d A number of masses corresponding to glycosylated peptides were observed

[001004] Isoelectric focusing

[001005] Isoelectric focusing (IEF) is used to evaluate the isoelectric points (pl) of the various isoforms of CTLA4^A29YL,04E-Ig in both drug substance and drug product. This method uses Pharmacia Biotech Ampholine® PAGplates at pH gradient of 4.0-6.5 and a Multiphore II Flatbed Electrophoresis System. Samples (for example, CTLA4^A29YU04E-Ig, etc.) are diluted in Milli-Q water and loaded directly onto the gel using sample application strips. The gel is focused for 2.5 hours under increasing voltage using a 100 mM β-alanine soaked cathode strip and a 100 mM glutamic acid/500 mM phosphoric acid soaked anode strip. After focusing, the gel is fixed using sulfosalicylic acid/trichloroacetic acid and then stained using a Coomassie blue staining system. After staining, the wet gel is scanned into a digital image file using a laser-based densitometer at a 50 or 100 μιη spatial resolution with up to 4096 levels of optical density resolution. CTLA4^A29YL104E-Ig focuses into 10 to 15 bands ranging from a pl of 4.5 to 5.5.