Liquid aqueous formulations comprising human anti-tnf alpha antibodies

LIQUID AQUEOUS FORMULATIONS COMPRISING HUMAN ANTI-TNFa ANTIBODIES

Background of the Invention

Tumor necrosis factor (TNF ) is a cytokine produced by numerous cell types, including monocytes and macrophages, that was originally identified based on its capacity to induce the necrosis of certain mouse tumors (see e.g., Old, L. (1985) Science 230:630-632). Subsequently, a factor termed cachectin, associated with cachexia, was shown to be the same molecule as TNFa. TNFa has been implicated in mediating shock (see e.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518; Beutler, B. and Cerami, A. (1989)/bww. Rev. Immunol. 7:625-655). Furthermore, TNFa has been implicated in the pathophysiology of a variety of other human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection and graft- versus-host disease (see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Patent No. 5,231,024 to Moeller et al; European Patent Publication No. 260 610 BI by Moeller, A., et at, Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, K.J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).

Because of the harmful role of human TNFa (hTNFa) in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract hTNFa activity. In particular, antibodies that bind to, and neutralize, hTNFa have been sought as a means to inhibit hTNFa activity. Some of the earliest of such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNFa (see e.g., Hahn T; et al, (1985) ProcNatl Acad Sci USA 82: 3814-3818; Liang, C-M., et al. (1986) Biochem. Biophys. Res.

Commun. 137:847-854: Hirai, M., et al. (1987) J. Immunol. Methods 96:57-62: Fendly, B.M., et al (1987) Hybridoma 6:359-370; Moeller, A., et al, (1990) Cytokine 2:162-169; U.S. Patent No. 5,231,024 to Moeller et al; European Patent Publication No. 186 833 BI by Wallach, D.; European Patent Application Publication No. 218 868 Al by Old et al; European Patent Publication No. 260 610 BI by Moeller, A., et al). While these mouse anti-hTNFa antibodies often displayed high affinity for hTNFa (e.g., Kd < 10 9M) and were able to neutralize hTNFa activity, their use in vivo may be limited by problems associated with administration of mouse antibodies to humans, such as short serum half life, an inability to trigger certain human effector functions and elicitation of an unwanted immune response against the mouse antibody in a human (the "human anti-mouse antibody" (HAM A) reaction). hi an attempt to overcome the problems associated with use of fully-murine antibodies in humans, murine anti-hTNEa antibodies have been genetically engineered to be more ’'human-like.” Bor example, chimeric antibodies, in which the variable regions of the antibody chains are murine-derived and the constant regions of the 5 antibody chains are human-derived, have been prepared (Knight, D.M, et at (1993)

Mol. Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 by Daddona, P.E., et al.). Additionally, humanized antibodies, in which the hypervariable domains of the antibody variable regions are murine-derived but the remainder of the variable regions and the antibody constant regions are human-derived, have also been prepared (PCT 10 Publication No. WO 92/11383 by Adair, J.R., et at). However, because these chimeric and humanized antibodies still retain some murine sequences, they still may elicit an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction, especially when administered for prolonged periods, for chronic indications, such as rheumatoid arthritis (see e.g., Elliott, MJ,, et at (1994) Lancet 344:1125-1127: Elliot, 15 MJ.,efaZ. (1994) Lancet 344:1105-1110). A preferred hTNEa inhibitory agent to murine mAbs or derivatives thereof (e.g., chimeric or humanized antibodies) would be an entirely human anti-hTNEa antibody, since such an agent should not elicit the HAMA reaction, even if used for prolonged periods. Human monoclonal autoantibodies against hTNEa have been prepared using 20 human hybridoma techniques (Boyle, P., et at (1993) Celt Immunol. 152:556-568: Boyle, P., et at (1993) Celt Immunol. 152:569-581: European Patent Application Publication No. 614 984 A2 by Boyle, et at). However, these hybridoma-derived monoclonal autoantibodies were reported to have an affinity for hTNEa that was too low to calculate by conventional methods, were unable to bind soluble hTNEa and were 25 unable to neutralize hTNEa-induced cytotoxicity (see Boyle, et at; supra). Moreover, the success of the human hybridoma technique depends upon the natural presence in human peripheral blood of lymphocytes producing autoantibodies specific for hTNEa. Certain studies have detected serum autoantibodies against hTNEa in human subjects (Fomsgaard, A., et at (1989) Scand. J. Immunol. 30:219-223: Bendtzen, K., et at 30 (1990) Prog. Leukocyte Biot 10B :447-452), whereas others have not (Leusch, H-G,, et at (1991)7. Immunol. Methods 139:145-147).

Alternative to naturally-occurring human anti-hTNEa antibodies would be a recombinant hTNEa antibody. Recombinant human antibodies that bind hTNEa with relatively low affinity (he., I<j ¾) and a fast off rate (he., Koff ~ 10“2 sec~l) have 35 been described (Griffiths, A.D., et al, (1993) EMBO L 12:725-734). However, because of their relatively fast dissociation kinetics, these antibodies may not be suitable for therapeutic use. Additionally, a recombinant human anti-hTNEa has been described 3 166809/2 that does not neutralize hTNFa activity, but rather enhances binding of hTNFa to the surface of cells and enhances internalization of hTNFa (Lidbury, A., et al (1994) Biotechnol. Ther. 5:27-45; PCT Publication No. WO 92/03145 by Aston, R. et al.)

Recombinant human antibodies that bind soluble hTNFa with high affinity and slow dissociation kinetics and that have the capacity to neutralize hTNFa activity, including hTNFa-induced cytotoxicity (in vitro and in vivo) and hTNFa-induced cell activation, have also been described (see U.S. Patent No. 6,090,382).

Summary of the Invention

In one of its aspects, the invention provides a stable liquid aqueous pharmaceutical formulation comprising a human anti-Tumor Necrosis Factor alpha (TNFa) antibody, or antigen-binding fragment thereof, at a concentration of between about 20 and about 150 mg/ml, a polyol, a surfactant, and a buffer system comprising citrate and phosphate, wherein said formulation has a pH of about 4 to about 8, and wherein the antibody, or antigen-binding portion thereof, comprises a light chain variable region comprising a complementary determining region (CDR) 1 domain comprising the amino acid sequence set forth in SEQ ID NO:7; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:5; and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:3, or modified from SEQ ID NO:3 by a single alanine substitution at position 1, 4, 5, 7, or 8, or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8, and/or 9; and comprises a heavy chain variable region comprising a CDR 1 domain comprising the amino acid sequence set forth in SEQ ID NO:8; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:6; and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:4, or modified from SEQ ID NO:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10, or 11, or by one to five conservative amino acid substitutions at positions 2, 3,4, 5, 6, 8, 9, 10, 11, and/or 12.

In some embodiments, the antibody, or antigen-binding portion thereof, is the antibody D2E7, or antigen binding portion thereof.

In other embodiments, the antibody, or antigen binding portion thereof, comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID 3a 166809/1 NQ:1, and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ IDNO:2.

In some other embodiments, the liquid aqueous pharmaceutical formulation comprises about 35-115 mg/ml of the antibody, or antigen-binding portion thereof.

In additional embodiments, the liquid aqueous pharmaceutical formulation comprises about 45-105 mg/ml of the antibody, or antigen-binding portion thereof.

In further embodiments, the pH is between about 4.5 to about 6.0.

In yet further embodiments, the polyol is a sugar alcohol. In such embodiments, the sugar alcohol is mannitol.

In other embodiments, the surfactant is a polysorbate. In such embodiments, the poly sorbate is poly sorbate 80.

In another aspect of the invention there is provided a liquid aqueous pharmaceutical formulation comprising (a) about 20 to about 150 mg/ml of a human anti-Tumor Necrosis Factor alpha (TNFa) antibody, or antigen-binding portion thereof; (b) 5-20 mg/ml of mannitol, (c) 0.1-10 mg/ml of polysorbate-80, and (d) a buffer system comprising citrate and phosphate, with a pH of 4 to 8, wherein the antibody, or antigen-binding portion thereof, comprises a light chain variable region comprising a complementary determining region (CDR) 1 domain comprising the amino acid sequence set forth in SEQ ID NO:7; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:5; and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:3, or modified from SEQ ID NO:3 by a single alanine substitution at position 1, 4, 5, 7, or 8, or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8, and/or 9; and comprises a heavy chain variable region comprising a CDR 1 domain comprising the amino acid sequence set forth in SEQ ID NO:8; a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:6; and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:4, or modified from SEQ ID NO:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10, or 11, or by one to five conservative amino acid substitutions at positions 2, 3,4,5,6, 8, 9, 10, 11, and/or 12. 3b 166809/1

In some embodiments, the liquid aqueous pharmaceutical formulation comprises (a) about 40 to about 100 mg/ml of antibody, or antigen-binding portion thereof, (b) about 7.5 to about 15 mg/ml of mannitol, and (c) about 0.5 to about 5 mg/ml of polysorbate-80.

In other embodiments, the antibody, or antigen-binding portion thereof, is the antibody D2E7 or antigen binding portion thereof.

In some other embodiments, the antibody, or antigen binding portion thereof, comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1, and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:2

In additional embodiments, the liquid aqueous pharmaceutical formulation comprises about 35-115 mg/ml of the antibody, or antigen-binding portion thereof.

In some other additional embodiments, the liquid aqueous pharmaceutical formulation comprises about 45-105 mg/ml of the antibody, or antigen-binding portion thereof.

In further embodiments, the pH is between about 4.5 to about 6.0.

In other embodiments, the liquid aqueous pharmaceutical formulation comprises 10 to 14 mg/ml of the mannitol.

In yet further embodiments, the liquid aqueous pharmaceutical formulation comprises 0.5 to 5 mg/ml of the polysorbate 80. 3c 166809/1

Detailed Description of the Invention

There is a need for a stable aqueous pharmaceutical formulation with an extended shelf life, comprising an antibody which is suitable for therapeutic use to inhibit or counteract detrimental hTNFa activity. There is also a need for a stable aqueous pharmaceutical formulation with an extended shelf life, comprising an antibody suitable for therapeutic use which is easily administered and contains a high protein concentration.

This invention provides a liquid aqueous pharmaceutical formulation consisting of a therapeutically effective amount of an antibody in a buffered solution forming a formulation having a pH between about 4 and about 8 and having a shelf life of at least 18 months. The invention also includes an aqueous pharmaceutical formulation comprising a therapeutically effective amount of an antibody in a buffered solution forming a formulation having a pH between about 4 and 8 and having a shelf life of at least 18 months in the liquid state. In one embodiment, the pharmaceutical formulation has enhanced stability. In a further embodiment, the formulation of the invention is stable following at least 3 freeze/thaw cycles of the formulation. In another embodiment, the antibody is directed to TNFa. In yet another embodiment, the antibody is directed to human TNFa. In still another embodiment, the antibody is D2E7.

This invention also provides a liquid aqueous pharmaceutical formulation comprising a therapeutically effective amount of an antibody in a buffered solution forming a formulation having a pH between 4 and 8 and having enhanced stability of at least 12 months at a temperature of 2 - 8°C. In one embodiment, the formulation has enhanced stability of at least 18 months. In another embodiment, the antibody is directed to TNFa. In yet another embodiment, the antibody is directed to human TNFa. In a further embodiment, the antibody is D2E7,

The invention further provides a liquid aqueous pharmaceutical formulation comprising a therapeutically effective amount of an antibody in a buffered solution _ 4 - forming a formulation having a pH between about 4 and about 8 which is easily administratable. In one embodiment, the antibody is directed to TNFa. In yet another embodiment, the antibody is directed to human TNFa. In a further embodiment, the antibody is D2E7. 5 In one embodiment of the invention, the liquid aqueous pharmaceutical formulation is suitable for injection. In a further embodiment, the formulation is suitable for single use sc injection. In another embodiment, the concentration of the antibody in the liquid aqueous pharmaceutical formulation is about 1-150 mg/ml. In yet another embodiment, the concentration of the antibody in the formulation is about 50 10 mg/ml. In still another embodiment, the formulation has a high protein concentration.

In yet another embodiment of the invention, the formulation is not light sensitive.

In one embodiment of the invention, the liquid aqueous pharmaceutical formulation contains an antibody, or an antigen-binding portion thereof, that dissociates from human TNFa with a Kd of 1 x I0"8 M or less and a Koff rate constant of 1 x 10"3 s’ 15 1 or less, both determined by surface plasmon resonance, and neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC5Q of 1 x 10~7 M or less. In another embodiment, the formulation of the invention contains an antibody, or antigenbinding portion thereof, which dissociates from human TNFa with a rate constant of 5 x IO"4 s’1 or less. In a further embodiment, the formulation contains an antibody, 20 or antigen-binding portion thereof, which dissociates from human TNFa with a Koff rate constant of 1 x 10"4 s"1 or less. In still a further embodiment, the formulation of the invention contains an antibody, or antigen-binding portion thereof, which neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an of 1 x 10-8 M or less. In yet another embodiment of the invention, the claimed formulation includes 25 an antibody, or antigen-binding portion thereof, which neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC50 of 1 x IO’9 M or less.

Another embodiment of the invention, includes a formulation where the antibody, or antigen-binding portion thereof, neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC5Q of 1 x I0~w M or less. 30 In another embodiment of the invention, the liquid aqueous pharmaceutical formulation contains of an antibody, or antigen-binding portion thereof, which is a recombinant antibody, or antigen-binding portion thereof. In another embodiment, the formulation contains an antibody, or antigen-binding portion thereof, which inhibits human TNFa-induced expression of ELAM-1 on human umbilical vein endothelial 35 cells. In still another embodiment, the claimed formulation includes the D2E7 antibody. In another embodiment of the invention, the liquid aqueous pharmaceutical formulation includes an antibody, or antigen-binding portion, thereof which dissociates from human TNEa with a Koff rate constant of 1 x 10“3 s*1 or less, as determined by surface plasmon resonance; b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position I, 5 4, 5,7 or 8 or by one to five conservative amino acid substitutions at positions 1,3,4, 6, 7, 8 and/or 9; c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3,4,5, 6, 8, 9,10 or 11 or by one to five conservative amino acid substitutions at 10 positions 2, 3,4, 5, 6, 8, 9,10,11 and/or 12. In another embodiment, the formulation of the invention includes an antibody, or an antigen-binding portion thereof, which dissociates from human TNEa with a Koff rate constant of 5 x 10"4 s"1 or less. In yet another embodiment of the invention, the formulation includes an antibody, or an antigen-binding portion thereof, which dissociates from human TNEa with a Koff rate 15 constant of 1 x lCH s“l or less.

In yet another embodiment of the invention, the liquid aqueous pharmaceutical formulation, contains of an antibody, or antigen-binding portion thereof, which has a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3-by a single alanine 20 substitution at position 1,4,5,7 or 8, and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alanine substitution at position 2, 3,4, 5, 6, 8, 9,10 or 11. In a further embodiment, the formulation of the invention contains an antibody, wherein the LCVR of the antibody, or an antigen-binding portion thereof, 25 further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5 and the HCVR of the antibody, or an antigen-binding portion thereof, further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6. In yet another embodiment, the formulation of the invention contains an antibody, wherein the LCVR of the antibody, or an antigen-binding portion thereof, further has CDRI domain 30 comprising the amino acid sequence of SEQ ID NO: 7 and the HCVR has a CDRI domain comprising the amino acid sequence of SEQ ID NO: 8. '

In yet another embodiment of the invention, the antibody or antigen-binding portion thereof, contained in the liquid aqueous pharmaceutical formulation has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NOf 1 35 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2. In another embodiment, the antibody, or antigen-binding portion thereof, has an IgGl heavy chain constant region. In still another embodiment, the antibody, or antigen-binding portion thereof, has an IgG4 heavy chain constant region. In another embodiment, the antibody, or antigen-binding portion thereof, is a Fab fragment. In still a further embodiment, the antibody, or antigen-binding portion thereof, is a single chain Fv fragment. 5 In one embodiment of the invention, the liquid aqueous pharmaceutical

formulation, contains an antibody, or antigen-binding portion thereof, which has a light chain variable region (LCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ED NO: 15, SEQ ED NO: 16, SEQ ID

10 NO: 17, SEQ ED NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ED NO: 25, SEQ ID NO: 26 or with a heavy chain variable region (HCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ED NO: 27, SEQ ED NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ED NO: 31, SEQ ED NO: 32, SEQ ED 15 NO: 33 and SEQ ID NO: 34. In still another embodiment, the antibody, or antigenbinding portion thereof, neutralizes the activity of human TNFa, chimpanzee TNFa and at least one additional primate TNFa selected from the group consisting of baboon TNFa, marmoset TNFa, cynomolgus TNFa and rhesus TNFa. In a further embodiment, the formulation of the invention includes an antibody, or an antigen- 20 binding portion thereof, which also neutralizes the activity of mouse TNFa. The formulation of the invention also an antibody, or an antigen-binding portion thereof, which neutralizes the activity of pig TNFa.

In a further embodiment, the invention provides a liquid aqueous pharmaceutical formulation which contains an antibody, or antigen-binding portion thereof, which binds 25 to human TNFa and comprises: a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine substitution at position 1,4,5,7 or 8 or by one to five conservative amino acid substitutions at positions 1,3,4,6,7, 8 and/or 9, and

30 a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQ ED NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9,10 or 11 or by one to rive conservative amino acid substitutions at positions 2, 3,4, 5,6, 8, 9,10,11 and/or 12. In one embodiment, the liquid aqueous pharmaceutical formulation includes an antibody which bind human TNFa and 35 comprises a light chain variable region (LCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ED NO: Π, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ED NO: 15, SEQ ID NO: 16, SEQ ED NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ED NO: 20, SEQ ED NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ED NO: 26 or a heavy chain variable region (HCVR) having a CDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ED NO: 5 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ED NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ED NO: 34. In a further embodiment of the invention, the antibody, or antigen-binding portion thereof, binds human TNFa and is the antibody D2E7 or an antigen binding portion thereof.

The invention also provides an aqueous pharmaceutical composition comprising 10 a polyol, a surfactant, and a buffer system comprising citrate and/or phosphate with a pH of about 4 to 8, in amounts sufficient to formulate an antibody for therapeutic use at a concentration of greater than about 45 mg/ml. In one embodiment, the polyol is mannitol and the surfactant is polysorbate 80. In another embodiment, the composition includes 5-20 mg/ml of mannitol and 0.1-10 mg/ml of polysorbate 80. In a further 15 embodiment, the composition includes the antibody D2E7.

The invention also provides a liquid aqueous pharmaceutical formulation consisting of 1-150 mg/ml of antibody, 5-20 mg/ml of mannitol, 0.1-10 mg/ml of Tween-80, and a buffer system comprising citrate and/or phosphate, with a pH of 4 to 8. In one embodiment, the antibody is directed to hTNFa. In another embodiment, the 20 formulation contains about 40 mg of antibody. The invention further provides a liquid aqueous pharmaceutical formulation comprising about 50 mg/ml of antibody, about 12 mg/ml of mannitol, about 1 mg/ml of Tween-80, and a buffer system comprising citrate and/or phosphate, with a pH of about 4 to about 8. In one embodiment, the pH of the formulation is between about 4.5 to about 6.0. In another embodiment, the pH is 25 between about 4.8 to about 5.5. In yet another embodiment, the pH of the invention is between about 5.0 to about 5.2.

In one embodiment of the invention, the liquid aqueous pharmaceutical formulation also includes about 1,305 mg/ml of citric acid, about 0.305 mg/ml of sodium citrate, about 1.53 mg/ml of disodium phosphate dihydrate, about 0.86 mg/ml of 30 sodium dihydrogen phosphate dihydrate, and about 6.165 mg/ml of sodium chloride. In another embodiment, the formulation of the invention includes an antibody which is directed to hTNEa. In yet another embodiment, the formulation of the invention includes the antibody D2E7. In yet a further embodiment, the formulation of the invention is administered to a subject suffering from a disorder in which TNEa activity 35 is detrimental such that TNFa activity in the subject is inhibited 8 166809/2

Detailed Description of Embodiments

This invention pertains to a liquid aqueous pharmaceutical formulation with a pH of about 4 to about 8 which contains a high protein concentration, including an antibody concentration ranging from about 1 to about 150 mg/ml, and has enhanced stability. This invention also pertains to a liquid aqueous pharmaceutical formulation for therapeutic use in a subject suffering from a condition characterized by detrimental TNFa activity. The formulation of the invention comprises the following constituents: an antibody which binds to human TNFa with high affinity, a low off rate and high neutralizing capacity; a buffer, which includes citric acid, sodium citrate, disodium phosphate dihydrate, and sodium dihydrogen phosphate dihydrate; tonicity agents, which include mannitol and sodium chloride; a detergent, including polysorbate 80; and sodium hydroxide, for pH adjustment.

Definitions

In order that the present invention may be more readily understood, certain terms are first defined.

The term "subject" is intended to include living organisms, e.g., prokaryotes and eukaryotes. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals, in specific embodiments of the invention, the subject is a human.

The term "pharmaceutical formulation" refers to preparations which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective, and which contain no additional components which are significantly toxic to the subjects to which the formulation would be administered. "Pharmaceutically acceptable" excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed. A "stable" formulation is one in which the antibody therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage.

Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. Stability can be measured at a selected temperature for a selected time period. Preferably, the formulation is stable at room temperature (about 30°C) or at 40°C for at least 1 month and or stable at about 2-8°C. for at least 1 year for at least 2 years. Furthermore, the formulation Is preferably stable following freezing (to, e.g., -70°C) and thawing of the formulation, hereinafter referred to as a "freeze/thaw cycle?’

An antibody "retains its physical stability" in a pharmaceutical formulation if it 5 shows substantially no signs of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering or by size exclusion chromatography.

An antibody "retains its chemical stability" in a pharmaceutical formulation, if the chemical stability at a given time is such that the antibody is considered to still retain 10 its biological activity as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the antibody. Chemical alteration may involve size modification (e.g. clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example. Other types of chemical 15 alteration include charge alteration (e.g. occurring as a result of deamidation) which can be evaluated by ion-exchange chromatography, for example.

An antibody "retains its biological activity” in a pharmaceutical formulation, if the antibody in a pharmaceutical formulation is biologically active for its intended purpose. For example, biological activity is retained if the biological activity of the 20 antibody in the pharmaceutical formulation is within about 30%, about 20%, or about 10% (within the errors of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared (e.g., as determined in an antigen binding assay). “Isotonic” is a term recognized in the art. Isotonic can mean, for example, that 25 the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example. A "tonicity agent" is a compound which renders the formulation isotonic. 30 A "polyol" is a substance with multiple hydroxyl groups, and includes sugars (reducing and nonreducing sugars), sugar alcohols and sugar acids. Preferred polyols herein have a molecular weight which is less than about 600 kD (e.g. in the range from about 120 to about 400 kD), A "reducing sugar” is one which contains a hemiacetal group that can reduce metal ions or react covalently with lysine and other amino groups 35 in proteins and a "nonreducing sugar" is one which does not have these properties of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Nonreducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol and glycerol are examples of sugar alcohols. As to sugar acids, these include L-gluconate and metallic salts thereof. Where it desired that the formulation is freeze-thaw stable, the polyol is preferably one which does not crystallize at freezing 5 temperatures (e.g. -20°C) such that it destabilizes the antibody in the formulation. The polyl may also act as a tonicity agent. In one embodiment of the invention, one ingredient of the formulation is mannitol in a concentration of 5 to 20 mg/ml. In a preferred embodiment of the invention, the concentration of mannitol is 7.5 to 15 mg/ml. In a more preferred embodiment of the invention, the concentration of mannitol 10 is 10-14 mg/ml.

As used herein, "buffer" refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. The buffer of this invention has a pH in the range from about 4 to about 8; preferably from about 4.5 to about 7; and most preferably has a pH in the range from about 5.0 to about 6.5. Examples of buffers that 15 will control the pH in this range include acetate (e.g. sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.

In a pharmacological sense, in the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody refers to an amount effective in the prevention or treatment of a disorder for the treatment of which 20 the antibody is effective. A "disorder" is any condition that would benefit from treatment with the antibody. This includes chronic and acute disorders or diseases including those pathological conditions which predisposes the subject to the disorder in question. A "preservative" is a compound which can be included in the formulation to 25 essentially reduce bacterial action therein, thus facilitating the production of a multi-use formulation, for example. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of 30 preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, andm-cresol. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as 35 those in which the disorder is to be prevented.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, -π- usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. 5 The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. 10 The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administration to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each 15 carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

The term "human TNFa" (abbreviated herein as hTNFa, or simply hTNF), as used herein, is intended to refer to a human cytokine that exists as a 17 Id) secreted form and a 26 kD membrane associated form, the biologically active form of which is 20 composed of a trimer of noncovalently bound 17 kD molecules. The structure of hTNEa is described further in, for example, Pennica, D., et at (1984) Nature 312:724-729; Davis, J.M., et at (1987) Biochemistry 26:1322-1326; and Jones, E.Y., et at (1989) Nature 338:225-228. The term human TNFa is intended to include recombinant human TNFa (rhTNFa), which can be prepared by standard recombinant expression 25 methods or purchased commercially (R & D Systems, Catalog No. 210-TA, Minneapolis, MN).

The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (E) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy 30 chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into 35 regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).

Each VH and VL is composed of three CDRs and four FRs, arranged from amino- 12 166809/2 terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one embodiment of the invention, the formulation contains an antibody with CDR1, CDR2, and CDR3 sequences like those described in U.S. Patent Nos. 6,090,382 and 6,258,562.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hTNFa). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546 ), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al (1988) Science 242:423-426: and Huston et al. (1988) Proc, Natl Acad. Sci, USA 85:5879-5883) . Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating twa antigen binding sites (see e.g., Holliger, P., et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et ah (1994) Structure 2:1121-1123). In one embodiment of the invention, the formulation contains an antigen-binding portions described in U.S. Patent Nos. 6,090,382 and 6,258,562. Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Tab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, 5 antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not 10 encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto 15 human framework sequences.

The term "recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section Π, below), antibodies isolated from a 20 recombinant, combinatorial human antibody library (described further in Section ΪΠ, below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. 25 Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VT regions of the recombinant antibodies are

30 sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an 35 isolated antibody that specifically binds hTNFa is substantially free of antibodies that specifically_bind antigens other than hTNFa). An isolated antibody that specifically binds hTNFa may, however, have cross-reactivity to other antigens, such as TNFa 14 166809/2 molecules from other species. Moreover, an isolated antibody may be substantially free of . other cellular material and/or chemicals. A "neutralizing antibody", as used herein (or an "antibody that neutralized hTNFa activity"), is intended to refer to an antibody whose binding to hTNFa results in inhibition of the biological activity of hTNFa. This inhibition of the biological activity of hTNFa can be assessed by measuring one or more indicators of hTNFa biological activity, such as hTNFa-induced cytotoxicity (either in vitro or in vivo), hTNFa-induced cellular activation and hTNFa binding to hTNFa receptors. These indicators of hTNFa biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art, and described in U.S. Patent Nos. 6,090,382 and 6,258,562. Preferably, the ability of an antibody to neutralize hTNFa activity is assessed by inhibition of hTNFa-induced cytotoxicity of L929 cells. As an additional or alternative parameter of hTNFa activity, the ability of an antibody to inhibit hTNFa-induced expression of ELAM-1 on HUVEC, as a measure of hTNFa-induced cellular activation, can be assessed.

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26: Jonsson, U., et al (1991) Biotechniques LL620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term "KOfA as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.

The term "Ka", as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction. Π. Antibodies of the Formulation

The invention is directed to a liquid aqueous pharmaceutical formulation comprising a therapeutically effective amount of an antibody in a buffered solution forming a formulation having a pH between about 4 and about 8 and having an extended shelf life, preferably of at least about 18 months. In another embodiment, the liquid aqueous pharmaceutical formulation of the invention has enhanced stability. In a further embodiment of the invention, the formulation is not light sensitive. In yet another embodiment of the invention, the claimed formulation remains stable following at least 166809/2 3 freeze/thaw cycles. In still another embodiment, the pharmaceutical formulation of the invention is suitable for single use sc injection.

Antibodies that can be used in the formulation include polyclonal, monoclonal, recombinant antibodies, single chain antibodies, hybrid antibodies, chimeric antibodies, 5 humanized antibodies, or fragments thereof. Antibody-like molecules containing one or two binding sites for an antigen and a Fc-part of an immunoglobulin can also be used.

An example of an antibody-like molecule is the active ingredient etanercept or infliximab. Preferred antibodies used in the formulation are human antibodies which are cloned from human cells or from gene-archives representing the human antibody- 10 reservoir. Especially preferred among the human antibodies are antibodies directed against the antigen TNFa, including human TNFa (or hTNFa).

In one embodiment, the formulation of the invention includes a combination of antibodies (two or more), or a ’’cocktail*’ of antibodies. For example, the formulation can include the antibody D2E7 and one or more additional antibodies. 15 In a preferred embodiment of the invention, the formulation contains an antibody, or antigen-binding portion thereof, dissociates from human TNFa with a Kd of 1 x 10"8 M or less and a Karate constant of 1 x 10"3 s_1 or less, both determined by surface plasmon resonance, and neutralizes human TNFa cytotoxicity in a standard in vitro E929 assay with an IC5Q of 1 x 10“7 M or less. In another preferred embodiment, 20 the formulation of the invention contains an antibody, or antigen-binding portion thereof, like those described in U.S. Patent Nos. 6,090,382 and 6,258,562,

In one aspect, the formulation of the invention contains D2E7 antibodies and antibody portions, D2E7-related antibodies and antibody portions, and other human 25 antibodies and antibody portions with equivalent properties to D2E7, such as high affinity binding to hTNFa with low dissociation kinetics and high neutralizing capacity. In another one embodiment, the formulation of the invention contains an isolated human antibody, or an antigen-binding portion thereof, that dissociates from human TNFa with a Kd of lx 10~8 M or less and a Koff rate constant of 1 x 10’3 s_1 or less, both 30 determined by surface plasmon resonance, and neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC50 of 1 x 10"7 M or less. More preferably, the isolated human antibody, or antigen-binding portion thereof, dissociates from human TNFa with aKoffOf 5 x 10-4 S"1 or less, or even more preferably, with aKoff of l x 10"4 s”1 or less. More preferably, the isolated human antibody, or antigen-binding portion 35 thereof, neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC50 of 1 x IO"8 M or less, even more preferably with an IC50 of 1 x ΙΌ'9 M or less and still more preferably with an IC50 of 5 x 10-10 M or less. In a preferred embodiment, the 166809/2 - 16 - formulation contains an antibody which is an isolated human recombinant antibody, or an antigen-binding portion thereof. In another preferred embodiment, the formulation contains an antibody which also neutralizes ΤΝΕ'α-induced cellular activation, as assessed using a standard in vitro assay for TNFci-induced ELAM-1 expression on 5 human umbilical vein endothelial cells (HUVEC). ΙΏ. Preparation of Formulation 10 The present invention features formulations (e.g·., protein formulations and/or antibody formulations) having improved properties as compared to art-recognized formulations. For example, the formulations of the invention have an improved shelf life and/or stability as compared to art recognized formulations. In a preferred aspect, the formulations of the invention comprise a high protein concentration, including, for 15 example, a protein concentration greater than about 45 mg/ml, a protein concentration greater than about 50 mg/ml, a protein concentration greater than about 100 mg/ml, or a protein concentration greater than about 150 mg/ml. In a preferred embodiment of the invention, the protein is an antibody. In another preferred embodiment, the antibody is D2E7. The invention also provides an aqueous pharmaceutical composition comprising. 20 a polyol, a surfactant, and a buffer system comprising citrate and/or phosphate with a pH of about 4 to 8, in amounts sufficient to formulate an antibody for therapeutic use at a concentration of greater than about, for example, 45 mg/ml.

Preparation of the antibody of interest is performed according to standard methods known in the art. In a preferred embodiment of the invention, the antibody 25 used in the formulation is expressed in CHO cells and purified by a standard series of chromatography steps. In a further preferred embodiment, the antibody is directed to hTNEa, and is prepared according to the methods described in U.S. Patent Nos. 6,090,382 and 6,258,562,

After preparation of the antibody of interest, the pharmaceutical formulation 30 comprising the antibody is prepared. The therapeutically effective amount of antibody present in the formulation is determined, for example, by taking into account the desired dose volumes and mode(s) of administration. In one embodiment of the invention, the concentration of the antibody in the formulation is between about 1 to about 150 mg of antibody per ml of liquid formulation. In a preferred embodiment, the concentration of 35 the antibody in the formulation is between about 5 to about 80 mg per ml. In another preferred embodiment, the concentration of the antibody in the formulation is between about 25 to about 50 mg/ml. The formulation is especially suitable for large antibody dosages of more than 15 mg/ml. In a preferred embodiment, the concentration of the antibody is 50 mg/ml.

In another embodiment of the invention, the concentration of the antibody in the formulation is about 1-150 mg/ml, about 5-145 mg/ml, about 10-140 mg/ml, about 15- 5 135 mg/ml, about 20-130 mg/ml, about 25-125 mg/ml, about 30-120 mg/ml, about 35- 115 mg/ml, about 40-110 mg/ml, about 45-105 mg/ml, about 50-100 mg/ml, about 55-95 mg/ml, about 60-90 mg/ml, about 65-85 mg/ml, about 70-80 mg/ml, or about 75 mg/ml. Ranges intermediate to the above recited concentrations, e.g., about 6-144 mg/ml, are also intended to be part of this invention. For example, ranges of values 10 using a combination of any of the above recited values as upper and/or lower limits are intended to be included.

In one embodiment, the invention provides a formulation with an extended shelf life comprising of an active ingredient, preferably an antibody, in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, 15 sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide. In a further embodiment, the formulation of the invention has an extended shelf life of at least about 18 months in the liquid state. Freezing the formulation of the invention can also he used to further extend its shelf life.

An aqueous formulation is prepared comprising the antibody in a pH-buffered 20 solution. The buffer of this invention has a pH ranging Hom about 4 to about 8, preferably from about 4.5 to about 6.0, more preferably from about 4.8 to about 5.5, and most preferably has a pH of about 5.0 to about 5.2. Ranges intermediate to the above recited pH's are also intended to be part of this invention. For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are 25 intended to be included. Examples of buffers that will control the pH within this range include acetate (e.g. sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.

In a preferred embodiment of the invention, the formulation comprises a buffer system which contains citrate and phosphate to maintain the pH in a range of about 4 to 30 about 8. hi a further preferred embodiment the pH range is from about 4.5 to about 6.0, more preferably from about pH 4.8 to about 5.5, and most preferably in a pH range of about 5.0 to about 5.2. In another preferred embodiment, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In a further preferred embodiment, the buffer system 35 includes about 1,3 mg/ml of citric acid (e.g., 1.305 mg/ml), about 0.3 mg/ml of sodium citrate (e.g., 0.305 mg/ml), about 1.5 mg/ml of disodium phosphate dihydrate (e.g. 1.53 mg/ml), about 0.9 mg/ml of sodium dihydrogen phosphate dihydrate (e.g., 0.86), and about 6.2 mg/ral of sodium chloride (e.g., 6.165 mg/ml). In additional preferred embodiments, the buffer system includes 1-1.5 mg/ml of citric acid, 0.25 to 0.5 mg/ml of sodium citrate, 1.25 to 1.75 mg/ml of of disodium phosphate dihydrate, 0.7 to 1.1 mg/ml of sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg/ml of sodium 5 chloride. In a further embodiment, the pH of the formulation is adjusted with sodium hydroxide. A polyol, which acts as a tonicifier and may stabilize the antibody, is also included in the formulation. The polyol is added to the formulation in an amount which may vary with respect to the desired isotonicity of the formulation. Preferably the 10 aqueous formulation is isotonic. The amount of polyol added may also alter with respect to the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g. mannitol) may be added, compared to a disaccharide (such as trehalose). In a preferred embodiment of the invention, the polyol which is used in the formulation as a tonicity agent is mannitol. In a preferred embodiment of the invention, 15 the mannitol concentration is about 5 to 20 mg/ml. In another preferred embodiment of the invention, the concentration of mannitol is about 7.5 to 15 mg/ml. In a more preferred embodiment of the formulation of the invention, the concentration of mannitol is about 10-14 mg/ml. In the most preferred embodiment, the concentration of mannitol is about 12 mg/ml. In another embodiment of the invention, the polyol sorbitol is 20 included in the formulation. A detergent or surfactant is also added to the antibody formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g. polysorbates 20, 80 etc) or poloxamers (e.g. poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and/or minimizes the formation of 25 particulates in the formulation and/or reduces adsorption. In a preferred embodiment of the invention, the formulation includes a surfactant which is a polysorbate. In another preferred embodiment of the invention, the formulation contains the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag 30 Aulendorf, 4th edi., 1996). In one preferred embodiment, the formulation contains between about 0.1 and about 10 mg/ml of polysorbate 80, more preferably between about 0.5 and about 5 mg/ml. In another preferred embodiment, about 0.1% polysorbate 80 is found in the formulation of the invention.

In a preferred embodiment of the invention, the formulation is a 0.8 mL solution 35 in a vial containing the ingredients shown below in Table 1.

Table 1 1 vial with 0.8 ml, solution for injection^ contains;

Name of ingredient Quantity Function Active substance: Antibody (D2E7)2) 40.0 mg Active substance Excipients: Mannitol 9.6 mg Tonicity agent Citric acid monohydrate Citric acid 1.044 mg Buffer Sodium citrate Sodium citrate 0.244 mg Buffer Disodium phosphate dihydrate Dibasic sodium phosphate dihydrate 1.224 mg Buffer Sodium dihydrogen phosphate dihydrate Monobasic sodium phosphate dihydrate 0.688 mg Buffer Sodium chloride 4.932 mg Tonicity agent Polysorbate 80 0.8 mg Detergent Water for injections Water for injection 759.028 - 759.048 mg Solvent Sodium hydroxide3'1 0.02-0.04 mg pH adjustment Total 817.6 mg 5 l> Density of the solution: 1.022 g/mL Is used as concentrate 3) Addition as 1M solution

In one embodiment, the formulation contains the above-identified agents (i.e. j0 antibody, buffer, polyol and detergent) and is essentially free of one or more preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol and benzethonium Cl. In another embodiment, a preservative may be included in the formulation, particularly where the formulation is a multidose formulation. One or more other pharmaceutically acceptable carriers, excipients or stabilizers such as those 0 described in Remington’s Pharmaceutical Sciences 16th edition, Osol, A, Ed. (19S0) may be included in the formulation provided that they do not significantly adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include; additional buffering agents; co-solvents; antioxidants including ascorbic acid 2θ and methionine; chelating agents such as EDTA; metal complexes (e.g. Zn-protein 166809/2 complexes); biodegradable polymers such as polyesters; and/or salt-forming counterions such as sodium.

The formulation herein may also be combined with one or more other therapeutic agents as necessary for the particular indication being treated, preferably 5 those with complementary activities that do not adversely affect the antibody of the formulation. Such therapeutic agents are suitably present in combination in amounts that are effective for the purpose intended. Additional therapeutic agents which can be combined with the formulation of the invention are further described in U.S. Patent Nos. 6,090,382 and 6,258,562. 10 The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to, or following, preparation of the formulation. IV. Administration of Formulation 15

The formulation of the invention can be used in similar indications as those described in U.S. Patent Nos. 6,090,382 and 6,258,562,

The language “effective amount” of the formulation is that amount necessary or 20 sufficient to inhibit TNFa activity, e.g., prevent the various morphological and somatic symptoms of a detrimental TNFa activity-associated state. In another embodiment, the effective amount of the formulation is the amount necessary to achieve the desired result. In one example, an effective amount of the formulation is the amount sufficient to inhibit detrimental TNFa activity. In another example, an effective amount of the 25 formulation is 0.8 mL of the formulation containing 40 mg of antibody, as described in table 1. The effective amount can vary depending on such factors as the size and weight of the subject, or the type of illness. For example, the choice of a TNFa activity-inhibiting formulation can affect what constitutes an "effective amount". One of ordinary skill in the art would be able to study the aforementioned factors and make the 30 determination regarding the effective amount of the TNFa activity inhibiting formulation without undue experimentation.

The regimen of administration can affect what constitutes an effective amount. The TNFa activity-inhibiting formulation can be administered to the subject either prior to or after the onset of detrimental TNFa activity. Further, several divided dosages, as 35 well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the TNFa activity-inhibiting formulation can be proportionally increased or decreased as indicated WO 2004/016286 FCT/IB2003/004502 by the exigencies of the therapeutic or prophylactic situation.

The term “treated,” “treating” or “treatment” includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. For example, treatment can be diminishment of one or several symptoms 5 of a disorder or complete eradication of a disorder.

Actual dosage levels of the active ingredients (antibody) in the pharmaceutical formulation of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. 10 The selected dosage level will depend upon a variety of factors including the activity of the antibody found in the formulation, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health 15 and prior medical history of the patient being treated, and like factors well known in the medical arts, A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition of the present invention required. For example, the physician or veterinarian could start doses of the 20 compounds of the invention employed in the pharmaceutical formulation at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a formulation of the invention will be that amount of the formulation that is the lowest dose effective to produce a therapeutic 25 effect. Such an effective dose will generally depend upon the factors described above. An effective amount of the formulation of the present invention is an amount that inhibits TNFa activity in a subject suffering from a disorder in which TNFa activity is detrimental. In a preferred embodiment, the formulation provides an effective dose of 40 mg per injection of the active ingredient, the antibody. In another embodiment, the 30 formulation provides an effective dose which ranges from about 1 to 150 mg of antibody. If desired, the effective daily dose of the pharmaceutical formulation may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

In one embodiment of the invention, the dosage of the antibody in the 35 formulation is between about 5 to about 80 mg. In another embodiment, the dosage of the antibody in the formulation is between about 25 to about 50 mg. The formulation is especially suitable for large antibody dosages of more than 15 mg. In a preferred - 22 - · embodiment of the invention, the formulation provides an antibody at a dose of about 40 mg. -In another preferred embodiment, the antibody is directed to TNFa. In the most preferred embodiment, the antibody is D2E7.

In one embodiment of the invention, the dosage of the antibody in the 5 formulation is between about 1-150 mg, about 5-145 mg, about 10-140 mg, about 15-135 mg, about 20-130 mg, about 25-125 mg, about 30-120 mg, about 35-115 mg, about 40-110 mg, about 45-105 mg, about 50-100 mg, about 55-95 mg, about 60-90 mg, about 65-85 mg, about 70-80 mg, or about 75 mg. In a preferred embodiment, the dosage of the antibody is 40 mg. In a further preferred embodiment, the antibody is directed to 10 TNFa. In the most preferred embodiment, the antibody is D2E7, Ranges intermediate to the above recited dosages, e.g., about 2-149 mg, are also intended to be part of tills invention. For example, ranges of values using a combination of any of the above l recited values as upper and/or lower limits are intended to be included.

It is to be noted that dosage values may vary with the severity of the condition to 15 be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. 20 The invention provides a pharmaceutical formulation with an extended shelf life, which, in one embodiment, is used to inhibit TNFa activity in a subject suffering from a disorder in which TNFa activity is detrimental, comprising administering to the subject

I an antibody or antibody portion of the invention such that TNFa activity in the subject is inhibited. Preferably, the TNFa is human TNFa and the subject is a human subject. 25 Alternatively, the subject can be a mammal expressing a TNFa with which an antibody of the invention cross-reacts. Still further the subject can be a mammal into which has been introduced hTNFa (e.g., by administration of hTNFa or by expression of an hTNFa transgene). A formulation of the invention can be administered to a human subject for therapeutic purposes (discussed further below). In one embodiment of the 30 invention, the liquid pharmaceutical formulation is easily administratable, which includes, for example, a formulation which is self-administered by the patient. In a preferred embodiment, the formulation of the invention is administered through sc injection, preferably single use. Moreover, a formulation of the invention can be administered to a non-human mammal expressing a TNFa with which the antibody 35 cross-reacts (e.g., a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (<?.g., testing of dosages 166809/2 and time courses of administration).

As used herein, the term "a disorder in which TNFa activity is detrimental" is intended to include diseases and other disorders in which the presence of TNFa in a subject suffering from the disorder has been shown to be or is suspected of being either 5 responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which TNFa activity is detrimental is a disorder in which inhibition of TNFa activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of TNFa in a biological fluid of a subject 10 , suffering from the disorder (e.g., an increase in the concentration of TNFa in serum, plasma, synovial fluid, etc. of the subject), which can be detected, for example, using an anti-TNFa antibody as described above.

There are numerous examples of disorders in which TNFa activity is detrimental. Examples of disorders in which TNFa activity is detrimental are described 15 in U.S. Application No. 60/397275, Examples in which TNFa activity is detrimental are also described in U.S. Patent Nos. 6,015,557, 6,177,077,6,379,666, 6,419,934,6,419,944, 6,423,321, and 6,428,787; U.S. Patent Application Nos. US2001/0016195, US2001/0004456, and US2001/026801; WO 00/50079 and WO 01/49321, 20 The use of the antibodies and antibody portions of the invention in the treatment of specific disorders is discussed further below: A. Sepsis

Tumor necrosis factor has an established role in the pathophysiology of sepsis, 25 with biological effects that include hypotension, myocardial suppression, vascular leakage syndrome, organ necrosis, stimulation of the release of toxic secondary mediators and activation of the clotting cascade (see e.g., Tracey, KJ. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503; Russell, D and Thompson, R.C. (1993) Curr. Opin. Biotech. 4:714-721). Accordingly, the formulation of the invention can be used to treat 30 sepsis in any of its clinical settings, including septic shock, endotoxic shock, gram negative sepsis and toxic shock syndrome.

Furthermore, to treat sepsis, the formulation of the invention can be coadministered with one or more additional therapeutic agents that may further alleviate sepsis, such as an interleukin-1 inhibitor (such as those described in PCT Publication 35 Nos. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see e.g., PCT Publication No. WO 93/11793) or an antagonist of platelet activating factor (see e.g.i European Patent Application Publication No. EP 374 510).

Additionally, in a preferred embodiment, the formulation of the invention is administered to a human subject within a subgroup of sepsis patients having a serum or plasma concentration of 30L-6 above 500 pg/ml, and more preferably 1000 pg/ml, at the time of treatment (see PCT Publication No. WO 95/20978 by Daum, L., et at). 5 B. Autoimmune Diseases

Tumor necrosis factor has been implicated in playing a role in the pathophysiology of a variety of autoimmune diseases. For example, TNFa has been implicated in activating tissue inflammation and causing joint destruction in rheumatoid 10 arthritis (see e.g.t Tracey and Cerami, supra; Arend, W.P. and Dayer, J-M. (1995) Arth. Rheum. 38:151-160: Fava, R.A., et at (1993) Clin. Exp. Immunot 94:261-266). TNFa also has been implicated in promoting the death of islet cells and in mediating insulin resistance in diabetes (see e.g., Tracey and Cerami, supra; PCT Publication No. WO 94/08609). TNFa also has been implicated in mediating cytotoxicity to 15 ; oligodendrocytes and induction of inflammatory plaques in multiple sclerosis (see e.g.,

Tracey and Cerami, supra}. Chimeric and humanized murine anti-hTNFa antibodies have undergone clinical testing for treatment of rheumatoid arthritis (see e.g., Elliott, M.J., et at (1994) Lancet 344:1125-1127; Elliot, MJ., et at (1994) Lancet 344:1105-1110; Rankin, E.C., et at (1995) Br. 3. Rheumatol. 34:334-342). 20 The formulation of the invention can be used to treat autoimmune diseases, in particular those associated with inflammation, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis and nephrotic syndrome. Typically, the formulation is administered systemically, although for certain disorders, local 25 administration of the antibody or antibody portion at a site of inflammation may be beneficial (e.g., local administration in the joints in rheumatoid arthritis or topical application to diabetic ulcers, alone or in combination with a cyclohexane-ylidene derivative as described in PCT Publication No. WO 93/19751). 30 C. Infectious Diseases

Tumor necrosis factor has been implicated in mediating biological effects observed in a variety of infectious diseases. For example, TNFa has been implicated in mediating brain inflammation and capillary thrombosis and infarction in malaria (see e.g., Tracey and Cerami, supra). TNFa also has been implicated in mediating brain 35 inflammation, inducing breakdown of the blood-brain barrier, triggering septic shock syndrome and activating venous infarction in meningitis (see e.g., Tracey and Cerami, supra). TNFa also has been implicated in inducing cachexia, stimulating viral proliferation and mediating central nervous system injury in acquired immune deficiency syndrome (AIDS) (see e.g., Tracey and Cerami, supra). Accordingly, the antibodies, and antibody portions, of die invention, can be used in the treatment of infectious diseases, including bacterial meningitis (see e.g., European Patent Application 5 Publication No. EP 585 705), cerebral malaria, AIDS and AIDS-related complex (ARC) (see e.g., European Patent Application Publication No. EP 230 574), as well as cytomegalovirus infection secondary to transplantation (see e.g., Fietze, E„ et al. (1994) Transplantation 58:675-680). The formulation of the invention, also can be used to alleviate symptoms associated with infectious diseases, including fever and myalgias 10 due to infection (such as influenza) and cachexia secondary to infection (e.g., secondary to AIDS or ARC). D. Transplantation

Tumor necrosis factor has been implicated as a key mediator of allograft 15 rejection and graft versus host disease (GVHD) and in mediating an adverse reaction that has been observed when the rat antibody OKT3, directed against the T cell receptor CD3 complex, is used to inhibit rejection of renal transplants (see e.g., Tracey and Cerami, supra', Eason, J.D., et al. (1995) Transplantation 59:300-305; Suthanthiran, M. and Strom, T.B. (1994) New Engl. J. Med. 331:365-375). Accordingly, the formulation 20 of the invention, can be used to inhibit transplant rejection, including rejections of allografts and xenografts and to inhibit GVHD. Although the antibody or antibody portion may be used alone, more preferably it is used in combination with one or more other agents that inhibit the immune response against the allograft or inhibit GVHD. Eor example, in one embodiment, the formulation of the invention is used in 25 combination with OKT3 to inhibit OKT3-induced reactions. In another embodiment, the formulation of the invention is used in combination with one or more antibodies directed at other targets involved in regulating immune responses, such as the cell surface molecules CD25 (interleukin-2 receptor-α), CDlla (EFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet another 30 embodiment, the formulation of the invention is used in combination with one or more general immunosuppressive agents, such as cyclosporin A orEK506. E. Malignancy

Tumor necrosis factor has been implicated in inducing cachexia, stimulating 35 tumor growth, enhancing metastatic potential and mediating cytotoxicity in malignancies (see e.g., Tracey and Cerami, supra). Accordingly, the formulation of the invention, can be used in the treatment of malignancies, to inhibit tumor growth or metastasis and/or to alleviate cachexia secondary to malignancy. The formulation may be administered systemically or locally to the tumor site. F. Pulmonary Disorders 5 Tumor necrosis factor has been implicated in the pathophysiology of adult respiratory distress syndrome, including stimulating leukocyte-endothelial activation, directing cytotoxicity to pneumocytes and inducing vascular leakage syndrome (see e.g., Tracey and Cerami, supra). Accordingly, the formulation of the invention, can be used to treat various pulmonary disorders, including adult respiratory distress syndrome (see 10 e.g., PCT Publication No. WO 91/04054), shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. The formulation may be administered systemically or locally to the lung surface, for example as an aerosol. · G. Intestinal Disorders 15 Tumor necrosis factor has been implicated in the pathophysiology of inflammatory bowel disorders (see e.g., Tracy, K.J., et al. (1986) Science 234:470-474; Sun, X-M., et al. (1988)/. Clin. Invest. 81:1328-1331; MacDonald, T.T., et al. (1990) Clin. Exp. Immunol. 81:301-305). Chimeric murine anti-hTNFa antibodies have undergone clinical testing for treatment of Crohn's disease (van Dullemen, H.M., et al. 20 (1995) Gastroenterology 109:129-135). The formulation of the invention, also can be used to treat intestinal disorders, such as idiopathic inflammatory bowel disease, which includes two syndromes, Crohn's disease and ulcerative colitis, H. Cardiac Disorders 25 The formulation of the invention, also can be used to treat various cardiac disorders, including ischemia of the heart (see e.g., European Patent Application Publication No. EP 453 898) and heart insufficiency (weakness of the heart muscle)(see e.g., PCT Publication No. WO 94/20139). 30 I. Others

The pharmaceutical formulation of the invention, also can be used to treat various other disorders in which TNEa activity is detrimental. Examples of other diseases and disorders in which TNFa activity has been implicated in the pathophysiology, and thus which can be treated using the formulation of the invention, 35 include inflammatory bone disorders and bone resorption disease (see e.g., Bertolini, D.R., et al. (1986) Nature 319:516-518: Konig, A., et al. (1988) /. Bone Miner. Res. 3:621-627; Lerner, U.H. and Ohlin, A. (1993) /. Bone Miner. Res. 8:147-155; and

Shankar, G. and Stem, P.H. (1993) Bone 14:871-876), hepatitis, including alcoholic hepatitis (see e.g., McClain, CJ. and Cohen, D.A. (1989) Hepatology 9:349-351; Felver, ME., et al (1990) Alcohol. Clin. Exp. Res. 14:255-259; and Hansen, J., et al. (1994) Hepatology 20:461-474) and viral hepatitis (Sheron, N., et al. (1991) J. Hepatol. 5 12:241-245; and Hussain, MJ., et al (1994) J. Clin. Pathol 47:1112-1115), coagulation disturbances (see e.g., van der Poll, T., et al (1990) N. Engl. J. Med. 322:1622-1627: and van der Poll, T., et al (1991) Prog. Clin. Biol Res. 367:55-60), bums (see e.g, Giroir, B.P., et al (1994) Am, J. Physiol. 267:H118-124; and Liu, X.S., et al. (1994) Bums 20:40-44). reperfusion injury (see e.g., Scales, W.E., et al. (1994) Am. J. Physiol 10 267:G1122-1127; Serrick, C., et al (1994) Transplantation 58:1158-Π62; and Yao, Y.M., et al (1995) Resuscitation 29:157-168), keloid formation (see e.g., McCauley, R.L., et al (1992) 7. Clin. Immunol 12:300-308), scar tissue formation; pyrexia; periodontal disease; obesity and radiation toxicity.

Other disorders in which TNFa activity is detrimental include, but are not 15 limited to, adult Still’s disease, Alzheimer’s disease, ankylosing spondylitis, asthma, cancer and cachexia, atherosclerosis, chronic atherosclerosis, chronic fatigue syndrome, liver failure, chronic liver failure, obstructive pulmonary disease, chronic obstructive pulmonary disease, congestive heart failure, dermatopolymyositis, diabetic macro vasculopathy, endometriosis, familial periodic fevers, fibrosis, hemodialysis, 20 Jarisch-Herxheimer reaction, juvenile RA, Kawasaki syndrome, myelo dysplastic syndrome, myocardial infarction, panciaticular vulgaris, periodontal disease, peripheral neuropathy, polyarticular, polymyositis, progressive renal failure, psoriasis, psoriatic arthritis, Reiter’s syndrome, sarcoidosis, scleroderma, spondyloarthropathies, Still’s disease, stroke, therapy associated syndrome, therapy induced inflammatory syndrome, 25 inflammatory syndrome following IL-2 administration, thoracoabdominal aortic aneurysm repair (TAAA), Vasulo-Behcet’s disease, Yellow Pever vaccination, type 1 diabetes mellitus, type 2 diabetes mellitus, neuropathic pain, sciatica, cerebral edema, edema in and/or around the spinal cord, vasculitide, Wegener’s granulomatosis, temporal arteritis, polymyalgia rheumatica, Takayasu’s arteritis, polyarteritis nodosa, 30 microscopic polyangiitis, Churg-Strauss syndrome, Felty’s syndrome, Sjogren’s syndrome, mixed connective tissue disorder, relapsing polychondritis, pseudogout, loosening of prostheses,, autoimmune hepatitis, sclerosing cholangitis, acute pancreatitis, chronic pancreatitis, glomerulonephritides, post-streptococcal glomerulonephritis or IgA nephropathy, rheumatic heart disease, cardiomyopathy, 35 orchitis, pyoderma gangerenosum, multiple myeloma, TNF receptor associated periodic syndrome [TRAPS], atherosclerosis, steroid dependent giant cell arteritismyostitis, uveitis, and drug reactions. 166809/2

Examples 10 EXAMPLE 1: Preparation of the Formulation

The pharmaceutical formulation of the invention was made according to the following protocol. 15 Materials which were used in the formulation include: mannitol, citric acid monohydrate (citric acid), sodium citrate, disodium phosphate dihydrate (dibasic sodium phosphate dihydrate), sodium dihydrogen phosphate dihydrate (monobasic sodium phosphate dihydrate), sodium chloride, polysorbate 80, water for the injections, sodium hydroxide, which was used as a 1M solution to adjust the pH, and protein concentrate 20 (e.g,, antibody concentrate).

Preparation of20L of buffer (equivalent to 20.180kg - density of the solution: 1.009 gM).

Ingredients were weighed out as follows: 240.0 g mannitol, 26,1 g citric acid 25 monohydrate, 6.1 g sodium citrate, 30.6 g disodium phosphate dihydrate, 17.2 g sodium dihydrogen phosphate dihydrate, 123.3 g sodium chloride, 20.0 g polysorbate 80, and 19,715.7 to 19,716.1 g of water. A sodium hydroxide solution was prepared by combining 40.0 g of sodium hydroxide with 1000,8 g of water for injections. 30 Next, a buffer was prepared by dissolving the following pre-weighed ingredients (described above) in about 90% of the water for injections: mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate, sodium chloride, and polysorbate 80. It was determined that the sequence of the addition of the buffer constituents was not important and can, therefore, be chosen at 35 will.

Following addition of all of the buffer constituents, the pH of the solution was adjusted with 1M sodium hydroxide which was prepared as described above. After the addition of the sodium hydroxide, the final weight of the water was added. The buffer solution was then filtered through a sterilized filter (hydrophilic poly vinylidene difluoride, 0.22 pm pore size) into a sterilized receptacle. The filtration medium used was filtration sterilized nitrogen. 5 Preparation of40L of formulation (equivalent to 40.88 kg)

The filtered buffer solution was then added to the thawed and pooled antibody concentrate (the active ingredient of the pharmaceutical formulation), prepared as follows. The antibody (concentrate) was thawed in a water bath prior to the preparation of the pharmaceutical formulation. 34.207 g of antibody concentrate was used, which is 10 equivalent to 2.0 kg of protein with 60 mg protein/mL protein concentrate. The density of the concentrate was 1.0262 g/mL. Any protein concentrate ranging from 25,655 to 37.316, which is equivalent to a protein concentration in the protein concentrate of 55 to 80 mg/mL, can be used. The buffer was added while stirring, until the final weight of the bulk solution was reached. 15 The formulation, with all of its ingredients included, was then sterilized by filtration as described above, except the formulation was filtered through two sterile 0.22 pm membrane filters. Following sterilization, the formulation was packaged for use in either a vial or a pre-filled syringe.

The skilled artisan will also appreciate that the weight quantities and/or weight- 20 to-volume ratios recited herein, can be converted to moles and/or molarities using the art-recognized molecular weights of the recited ingredients. Weight quantities exemplified herein (e.g., g or kg) are for the volumes (e.g., of buffer or pharmaceutical formulation) recited. The skilled artisan will appreciate that the weight quantities can be proportionally adjusted when different formulation volumes are desired. For example, 25 32L, 20L, 10L, 5L, or IL formulations would include 80%, 50%, 25%, 12.5%, or 2.5%, respectively, of the exemplified weight quantities. EXAMPLE 2: Ereeze/Thaw Studies 30

After the formulation buffer for the D2E7 antibody was selected the drug substance was formulated in the same matrix as the finished product.

Freeze thaw behavior of the D2E7 antibody drug substance at a protein concentration of 63 mg/mL was evaluated by cycling drug substance 3 times from the 35 frozen state to the liquid state. Table N shows the results of an experiment evaluating the effect of three fast and slow freeze-thaw cycles in the presence and absence of 0.1 % polysorbate 80 starting from ™80°C or -30°C, respectively.

Table 2 shows that the D2E7 antibody drug substance can be thawed/frozen at least 3 times without any detrimental effect on either chemical (cation exchange HPLC, size exclusion HPLC, colour, pH), physicochemical properties (subvisihle particles, clarity) or biological activity (in vitro TNE neutralization assay). Also table 2 shows that 5 the inclusion of polysorbate 80 improved the physicochemical properties of the D2E7 antibody drug substance as evidenced by the lower number of subvisihle particles regardless whether a slow or fast freeze/thaw cycle was being used (see shaded areas in table 2). 10 Table 2: Effect of freeze thaw on the D2E7 antibody drug substance with/without polysorbate 80

Test criteria Polysorbate (0.1 %)υ No freeze/ thaw Slow thaw -30°C in refrigerator Past thaw -30°C in water bath Siow thaw -80°in refrigerator Fast thaw -80°C in water bath Clarity - 25.0 22.5 25.3 25.8 ' 25.6 + 27.8 28.1 28.2 28.0 28.1 Colour - <B9 <B9 <B9 <B9 <B9 + <B9 <B9 <B9 <B9 <B9 pH - 5.01 5.02 5.02 5.02 5.02 + 5.02 5.02 5.02 5.02 5.02 ‘ SubVisible' . \ -'.42'. -"303 -Ά .1891 ·, particles - ’ 2' ._· 4- -. - A 5 λ'" 8 O' · - 0 , 5 7.. ;’A %' /,·' · 'd '8 ί .0 - .Λ.. , o · N„,’ y-0,A ,.1 .y/ Size exclusion .- 99.8 99.8 99.8 99.8 99.8 HPLC + 99.8 99.8 99.8 99.8 99.8 Cation 87,1 87.0 87.2 86.9 86.9 exchange HPLC 4* 86.8 87.0 87,1 87.3 86.8 In vitro TNF - 118.0 123.8 118.0 103.3 120.5 neutralization test + 111.8 96.2 100.9 96.7 95.8 1)+ = formulation with 0.1 % polysorbate 80; - = formulation without 0.1 % polysorbate 80 15 166809/2 ΕΧΑΜΡΙ,Ε 3: Microbial Studies

Tests were performed to determine if the formulation can support microbial growth. The results from these experiments showed that the formulation does not 5 support microbial growth if stored at 20 to 25 °C for 14 days. This result was determined by directly inoculating the sterile formulation with microorganisms (e.g., Staphylococous aureus, ATCC-No.: 6538P, Candida albicans, ATCC-No.: 10231, Aspergillus niger, ATCCC-No.: 16404, Pseudomonas aeruginosa, ATCC-No.: 9027, an environmental isolate) at low level (NMT 100 cfu/mL). Inoculated formulations were 10 then examined for overall microbial growth, e.g., for changes in turbidity. A lack of turbidity was an indication of no overall growth, and was detected in the inoculated containers after 14 days. Further, no organisms could be reisolated from these containers. Thus it was concluded that the formulation does not support microbial growth under these conditions. 15 20 25

Seqlist

SEQUENCE LISTING <110> Krause, Hans Juergen Baust, Lisa Diekes, Michael

<12 0> FORMULATION OF HUMAN ANTIBODIES FOR TREATING TNF-ALPA ASSOCIATED DISORDERS <130> BBC-166 <140> 10/222140 <141> 2002 16 <160> 34 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 108

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 1

Pro Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp- Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly lie Arg Asn 20 25 30 Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 lie Tyr Ala Ala Ser Thr Leu Gin Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gin 65 70 75 80 Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gin Arg Tyr Asn Arg Ala Pro 85 90 95 Tyr Thr Phe Gly Gin .Gly Thr Lys Val Glu lie Lys 100 105 <210> 2 <211> 122

<212> PRT <213> Artificial Sequence WO 2004/016286 FCT/3B2003/004502

Seqlist <220> <223> mutated human antibody <400> 2

Pro Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly 1 5 10 15 Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp 20 25 30 Tyr Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Ala He Thr Trp Asn Ser Gly His Xie Asp Tyr Ala Asp Ser 50 55 60 Val Glu Gly Arg Phe Thr Xie Ser Arg Asp Asn Ala Lys Asn Ser Leu 65 70 75 ’ 80 Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala hys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp 100 105 110 Gly Gin Gly Thr Leu Val Thr Val Ser Ser .115 120 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <220> <221> VARIANT <222> 10 <223> Xaa = Thr or Ala <400> 3

Pro Gin Arg Tyr Asn Arg Ala Pro Tyr Xaa 1 5 10 <210> 4 <211> 13

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody

Seqlist <220>

<221> VARIANT <222> 13 <223> Xaa - Tyr or Asn <400> 4

Pro Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa 15 10 <210> 5 <211> 8

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 5

Pro Ala Ala Ser Thr Leu Gin Ser 1 5 <210> 6 <211> 18

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 6

Pro Ala lie Thr Trp Asn Ser Gly His He Asp Tyr Ala Asp Ser Val 1 5 10 15

Glu Gly <210> 7 <211> 12

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 7

Pro Arg Ala Ser Gin Gly lie Arg Asn Tyr Leu Ala

Seqlist 15 10 <210> 8 <211> 6

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 8

Pro Asp Tyr Ala Met His 1 5 <210> 9 <400> 9 000 <210> 10 <400> 10 000 . <210> 11 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 11

Pro Gin Lys Tyr Asn Ser Ala Pro Tyr Ala 1 . 5 . 10 <210> 12 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 12

Seqlist

Pro Gin Lys Tyr Asn Arg Ala Pro Tyr Ala 1 5 10 <210 > 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 13 Pro Gin Lys Tyr Gin Arg Ala Pro Tyr Thr 1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <2 2 3 > mutated human antibody <400> 14 Pro Gin Lys Tyr Ser Ser Ala Pro Tyr Thr 1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 15 Pro Gin Lys Tyr Asn Ser Ala Pro Tyr Thr 1 . 5 10 <210> 16 <211> 10 <212> PRT <213> Artificial . Sequence

Seqlist <220> <223> mutated human antibody <400> 16

Pro Gin Lys Tyr Asn Arg Ala. Pro Tyr Thr 1 5 10 <210> 17 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 17

Pro Gin Lys Tyr Asn Ser Ala Pro Tyr Tyr 15 10 <210> 18 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 18

Pro Gin Lys Tyr Asn Ser Ala Pro Tyr Asn 15 10 <210> 19 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 19

Pro Gin Lys Tyr Thr Ser Ala Pro Tyr Thr 15 10 <210> 20

Seqlist <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 20

Pro Gin Lys Tyr Asn Arg Ala Pro Tyr Asn 15 10 <210> 21 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 21

Pro Gin Lys Tyr Asn Ser Ala Ala Tyr Ser 15 10 <210> 22 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 22

Pro Gin Gin Tyr Asn Ser Ala Pro Asp Thr 15 10 <210> 23 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 23

Pro Gin Lys Tyr Asn Ser Asp Pro Tyr Thr

Seqlist 1 5 10 <210> 24 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 24

Pro Gin Lys Tyr He Ser Ala Pro Tyr Thr 1 5 10 <210> 25 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 25

Pro Gin Lys Tyr Asn Arg Pro Pro Tyr Thr .1 5 10 <210> 26 <211> 10

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 26

Pro Gin Arg Tyr Asn Arg Ala Pro Tyr Ala 1 5 10 <210> 27 <211> 13

<212> PRT <213> Artificial Sequence <220> 40 166809/2

SEQUENCE LISTING <110> Krause, Hans Juergen

Baust, Lisa

Dickes, Michael

<120> FORMULATION OF HUMAN ANTIBODIES FOR TREATING TNF-ALPA ASSOCIATED DISORDERS <130> BBC-166 <140> 10/222140 <141> 2002 16 <160> 34 <170> FastSEQ for Windows Version 4.0 <210> 1 <2H> 107

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 1 Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Gin Gly He Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu He 35 40 45 Tyr Ala Ala Ser Thr Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gin Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gin Gly Thr Lys Val Glu He Lys 100 105 <210> 2 <2H> 121

<212> PRT 01596188X129-01 41 166809/2 <213> Artificial Sequence <220> <223> mutated human antibody <400> 2

Glu Val Gin Leu Val Glu Ser Gly 1 5

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ala Met His Trp Val Arg Gin Ala 35 40

Ser Ala lie Thr Trp Asn Ser Gly 50 55

Glu Gly Arg Phe Thr lie Ser Arg 65 70

Leu Gin Met Asn Ser Leu Arg Ala 85

Ala Lys Val Ser Tyr Leu Ser Thr 100

Gin Gly Thr Leu Val Thr Val Ser 115 120 <210> 3 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <220>

<221> VARIANT <222> 9 <223> Xaa = Thr or Ala <400> 3

Gin Arg Tyr Asn Arg Ala Pro Tyr 1 5 <210> 4 <211> 12

Gly Gly Leu Val Gin Pro Gly Arg 10 15 Ser Gly Phe Thr Phe Asp Asp Tyr 25 30 Pro Gly Lys Gly Leu Glu Trp Val 45 His He Asp Tyr Ala Asp Ser Val 60 Asp Asn Ala Lys Asn Ser Leu Tyr 75 80 Glu Asp Thr Ala Val Tyr Tyr Cys 90 95 Ala Ser Ser Leu Asp Tyr Trp Gly 105 110

Ser

Xaa 01596188M29-01 42 166809/5

<212> FRT <213> Artificial Sequence <220> <223> mutated human antibody <220>

<221> VARIANT <222> 12 <223> Xaa = Tyr or Asn <400> 4

Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa 15 10 <210> 5 <211> 7

<212> FRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 5

Ala Ala Ser Thr Leu Gin Ser 1 5 <210> 6 <211> 17

<212> FRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 6

Ala lie Thr Trp Asn Ser Gly His He Asp Tyr Ala Asp Ser Val Glu 15 10 15

Gly <210> 7 <211> 11

<212> FRT <213> Artificial Sequence <220> 01596188M 29-01 43 166809/5 <223> mutated human antibody <400> 7

Arg Ala Ser Gin Gly Xie Arg Asn Tyr Leu Ala 15 10 <210> 8 <211> 5

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 8

Asp Tyr Ala Met His 1 5 <210> 9 <400> 9 000 <210> 10 <400> 10 <210> 11 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 11

Gin Lys Tyr Asn Ser Ala Pro Tyr Ala 1 5 <210> 12 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 12

Gin Lys Tyr Asn Arg Ala Pro Tyr Ala 01596188U29-01 44 166809/5 1 5 <210> 13 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 13

Gin Lys Tyr Gin Arg Ala Pro Tyr Thr 1 5 <210> 14 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 14

Gin Lys Tyr Ser Ser Ala Pro Tyr Thr 1 5 <210> 15 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 15

Gin Lys Tyr Asn Ser Ala Pro Tyr Thr 1 5 <210> 16 <211> 9

<212> PRT <213> Artificial Sequence <220> 01596188M29-01 45 166809/5 <223> mutated human antibody <4Q0> 16

Gin Lys Tyr Asn Arg Ala Pro Tyr Thr 1 5 <210> 17 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 17

Gin Lys Tyr Asn Ser Ala Pro Tyr Tyr 1 5 <210> 18 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 18

Gin Lys Tyr Asn Ser Ala Pro Tyr Asn 1 5 <210> 19 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 19

Gin Lys Tyr Thr Ser Ala Pro Tyr Thr 1 5 <210> 20 <211> 9

<212> PRT <213> Artificial Sequence 01596188X129-01 46 166809/2 <220> <223> mutated human, antibody <400> 20

Gin Lys Tyr Asn Arg Ala Pro Tyr Asn 1 5 <210> 21 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 21

Gin Lys Tyr Asn Ser Ala Ala Tyr Ser 1 5 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 22

Gin Gin Tyr Asn Ser Ala Pro Asp Thr 1 5 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 23

Gin Lys Tyr Asn Ser Asp Pro Tyr Thr 1 5 <210> 24 <211> 9

<212> PRT 015961S8\129-01 47 166809/1 <213> Artificial Sequence <220> <223> mutated human antibody <400> 24

Gin Lys Tyr lie Ser Ala Pro Tyr Thr 1 5 <210> 25 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 25

Gin Lys Tyr Asn Arg Pro Pro Tyr Thr 1 5 <210> 26 <211> 9

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 26

Gin Arg Tyr Asn Arg Ala Pro Tyr Ala 1 5 <210> 27 <211> 12

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 27

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn 15 10 <210> 28 <211> 12 01596188X129-01 48 166809/1 <212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 28 Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Lys 1 5 10 <210> 29 <211> 12 <212> IT 1\ X <213> Artificial Sequence <220> <223> mutated human antibody <400> 29 Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Tyr 1 5 10 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 30 Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp 1 5 10 <210> 31 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 31 Ala Ser Tyr Leu Ser Thr Ser Phe Ser Leu Asp Tyr 1 5 10 <210> 32 01596188U29-01 49 166809/1 <211> 12

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 32

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu His Tyr 15 10 <210> 33 <211> 12

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 33

Ala Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr 15 10 <210> 34 <211> 12

<212> PRT <213> Artificial Sequence <220> <223> mutated human antibody <400> 34

Ala Ser Tyr Leu Ser Thr Ala Ser Ser Leu Glu Tyr 15 10 01596188\129-01