Affinity purification of polypeptide on protein a matrix

BACKGROUND OF THE INVENTION

Field of the invention

0001This invention relates generally to protein purification. In particular, the invention relates to a method for purifying C_H2/C_H3 region-containing proteins, by Protein A affinity chromatography.

Description of Related Art

0002The large-scale, economic purification of proteins is increasingly an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Separation of the desired protein from the mixture of compounds fed to the cells and from the byproducts of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.

0003Procedures for purification of proteins from cell debris initially depend on the site of expression of the protein. Some proteins can be caused to be secreted directly from the cell into the surrounding growth media; others are made intracellularly. For the latter proteins, the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins in the course of the protein production run.

0004Once a clarified solution containing the protein of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of different chromatography techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, or size. Several different chromatography resins are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively to the separation medium, being then differentially eluted by different solvents. In some cases, the desired protein is separated from impurities when the impurities specifically adhere to the column, and the protein of interest does not, that is, the protein of interest is present in the "flow-through."

0005Affinity chromatography, which exploits a specific interaction between the protein to be purified and an immobilized capture agent, may also be an option for some proteins. Protein A is a useful adsorbent for affinity chromatography of proteins, such as antibodies, which contain an Fc region. Protein A is a 41 kD cell wall protein from <i>Staphylococcus aureas</i> which binds with a high affinity (about 10^-8M to human IgG) to the Fc region of antibodies.

SUMMARY OF THE INVENTION

0006A problem associated with Protein A chromatography of contaminated protein preparations has been identified herein. In particular, it has been observed that in Protein A chromatography using a glass or silica surface for adsorbing the Protein A (<i>e.g.</i> where the Protein A is immobilized on a controlled pore glass column or a silicic acid column), contaminants in the protein preparation (such as Chinese Hamster Ovary proteins (CHOP), where the protein preparation is derived from a CHO cell) adhere to the glass or silica surface of the solid phase. This was found to occur even when the solid phase is coated with a reagent (such as glycerol) in an attempt to prevent nonspecific adherence thereto. An intermediate wash step has been devised herein which addresses this problem. This wash step serves to remove the contaminants, but not the immobilized Protein A or the protein of interest bound to the Protein A, from the solid phase. In particular, it has been found that the hydrophobic electrolytes, tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC) tetrapropylammonium chloride and tetrabutylammonium chloride can be used in this intermediate wash step.

0007Accordingly, the invention provides a method for purifying a protein from a contaminated solution thereof by Protein A chromatography, wherein said protein to be purified comprises a CH2/CH3 region fused to, or conjugated with a polypeptide selected from the group of renin; a growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; a clotting factor; an anti-clotting factor; atrial natriuretic factor; lung surfactant; a plasminogen activator; bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); a serum albumin; Muellerianinhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA); inhibin; activin; vascular endothelial growth factor (VEGF); a receptor for a hormone or growth factor; or D; a rheumatoid factor; a neurotrophic factor; platelet-derived growth factor (PDGF); fibroblast growth factor; epidermal growth factor (EGF); a transforming growth factor (TGF); insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF I (brainIGF-I), insulin-like growth factor binding proteins; a CD protein; erythropoietin; an osteoinductive factor; an immunotoxin; a bone morphogenetic protein (BMP); an interferon; a colony stimulating factor (CSF); an interleukin; superoxide dismutase; a T-cell receptor; a surface membrane protein; a decay accelerating factor; a viral antigen; a transport protein; a homing receptor; an addressin; a regulatory protein; an integrin; a tumor associated antigen; or a fragment of any of the above-listed polypeptides; the method comprising the following steps performed sequentially: (a) adsorbing the protein to Protein A immobilized on a solid phase comprising silica or glass; (b) removing contaminants bound to the solid phase by washing the solid phase with a hydrophobic electrolyte solvent, wherein the hydrophobic electrolyte solvent comprises tetrapropylammonium chloride, tetrabutylammonium chloride, tetramethylammonium chloride (TMAC) or tetraethylammonium chloride (TEAC); and (c) recovering the protein from the solid phase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions:

0008When used herein, the term "Protein A" encompasses Protein A recovered from a native source thereof, Protein A produced synthetically (<i>e.g.</i> by peptide synthesis or by recombinant techniques), and variants thereof which retain the ability to bind proteins which have a C_H2/C_H3 region. Protein A can be purchased commercially from Repligen, Pharmacia and Fermatech.

0009The Protein A is immobilized on a solid phase. By "solid phase" is meant a non-aqueous matrix to which the Protein A can adhere. The solid phase of interest herein is one which comprises a glass or silica surface. The solid phase may be a purification column or a discontinuous phase of discrete particles. In preferred embodiments, the solid phase is a controlled pore glass column or a silicic acid column. In certain embodiments, the solid phase is coated with a reagent (such as glycerol) which is intended to prevent nonspecifc adherence of contaminants to the solid phase.

0010The protein of interest herein is one which comprises a C_H2/C_H3 region and therefore is amenable to purification by Protein A chromatography. The term "C_H2/C_H3 region" when used herein refers to those amino acid residues in the Fc region of an immunoglobulin molecule which interact with Protein A. In preferred embodiments, the C_H2/C_H3 region comprises an intact C_H2 region followed by an intact C_H3 region, and most preferably comprises a Fc region of an immunoglobulin. Examples of C_H2/C_H3 region-containing proteins include antibodies, immunoadhesins and fusion proteins comprising a protein of interest fused to, or conjugated with, a C_H2/C_H3 region.

0011The "intermediate wash step" is a step performed after the protein of interest is loaded on the solid phase and adsorbed to the Protein A, but before the protein is recovered from the column. The intermediate wash step serves to remove contaminants nonspecifically bound to the solid phase, without significantly eluting the protein of interest from the solid phase. In the intermediate wash step, the solid phase is washed with a hydrophobic electrolyte solvent (<i>e.g.</i> the hydrophobic electrolyte solvent is passed through the Protein A column, where the solid phase is a column).

0012The "hydrophobic electrolyte solvent" in the intermediate wash step is that which is able to elute contaminants bound to the solid phase, without significantly eluting the immobilized Protein A or the protein of interest adsorbed thereto. Preferably the hydrophobic electrolyte solvent is an aqueous carrier (<i>e.g</i>. a buffer) comprising one or more hydrophobic electrolytes. The hydrophobic electrolytes are the alkylamines tetramethylammonium chloride (TEMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride and tetrabutylammonium chloride.

0013A "buffer" is a buffered solution that resists changes in pH by the action of its acid-base conjugate components. The "equilibration buffer" herein is that used to prepare the solid phase (with immobilized Protein A) for loading the protein of interest. The equilibration buffer is preferably isotonic and commonly has a pH in the range from about 6 to about 8. The equilibration buffer of the example was 25mM Tris, 25mM NaCl, 5mM EDTA, pH 7.1. The "loading buffer" is that which is used to load the mixture of the C_H2/C_H3 region-containing protein and contaminants onto the solid phase to which the Protein A is immobilized. Often, the equilibration and loading buffers are the same. The "elution buffer" is used to elute the C_H2/C_H3 region-containing protein from the immobilized Protein A. Preferably the elution buffer has a low pH and thereby disrupts interactions between Protein A and the protein of interest. Preferably, the low pH elution buffer has a pH in the range from about 2 to about 5, most preferably in the range from about 3 to about 4. Examples of buffers that will control the pH within this range include phosphate, acetate, citrate and ammonium buffers, as well as combinations of these. The preferred such buffers are citrate and acetate buffers, most preferably sodium citrate or sodium acetate buffers. Other elution buffers are contemplated including high pH buffers <i>(e.g.</i> those having a pH of 9 or more) or buffers comprising a compound or composition such as MgCl₂ (2mM) for eluting the protein of interest.

0014The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (<i>e</i>.<i>g</i>., bispecific antibodies), and antibody fragments so long as they retain, or are modified to comprise, a C_H2/C_H3 region as herein defined.

0015"Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; single-chain antibody molecules; diabodies; linear antibodies; and multispecific antibodies formed from antibody fragment.

0016The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, <i>i</i>.<i>e</i>., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by <nplcit id="ncit0001" npl-type="s"><text>Kohler et al., Nature 256:495 (1975</text></nplcit>), or may be made by recombinant DNA methods (see, <i>e</i>.<i>g</i>., <patcit id="pcit0001" dnum="US4816567A"><text>U.S. Patent No. 4,816,567</text></patcit>). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in <nplcit id="ncit0002" npl-type="s"><text>Clackson et al., Nature 352:624-628 (1991</text></nplcit>) and <nplcit id="ncit0003" npl-type="s"><text>Marks et al., J. Mol. Biol. 222:581-597 (1991</text></nplcit>), for example.

0017The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class o: subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (<patcit id="pcit0002" dnum="US4816567A"><text>U.S. Patent No. 4,816,567</text></patcit>; and <nplcit id="ncit0004" npl-type="s"><text>Morrison et al., Proc. Natl. Acad Sci. USA 81:6851-6855 (1984</text></nplcit>)).

0018The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (<i>i.e</i> residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; <nplcit id="ncit0005" npl-type="b"><text>Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991</text></nplcit>)) and/or those residues from a "hypervariable loop" (<i>i</i>.<i>e</i>. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; <nplcit id="ncit0006" npl-type="s"><text>Chothia and Lesk J. Mol. Biol. 196:901-917 (1987</text></nplcit>)). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

0019"Humanized" forms of non-human (<i>e</i>.<i>g</i>., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see <nplcit id="ncit0007" npl-type="s"><text>Jones et al., Nature 321:522-525 (1986</text></nplcit>); <nplcit id="ncit0008" npl-type="s"><text>Riechmann et al., Nature 332:323-329 (1988</text></nplcit>); and <nplcit id="ncit0009" npl-type="s"><text>Presta, Curr. Op. Struct. Biol. 2:593-596 (1992</text></nplcit>).

0020As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the "binding domain" of a heterologous "adhesin" protein (<i>e.g.</i> a receptor, ligand or enzyme) with the effector functions of an immunoglobulin constant domain. Structurally, the immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (<i>i.e</i>. is "heterologous") and an immunoglobulin constant domain sequence. The immunoglobulin constant domain sequence in the immunoadhesin is preferably derived from γ1, γ2, or γ4 heavy chains since immunoadhesins comprising these regions can be purified by Protein A chromatography (<nplcit id="ncit0010" npl-type="s"><text>Lindmark et al., J. Immunol. Meth. 62:1-13 (1983</text></nplcit>)).

0021The term "ligand binding domain" as used herein refers to any native cell-surface receptor or any region or derivative thereof retaining at least a qualitative ligand binding of a corresponding native receptor. In a specific embodiment, the receptor is from a cell-surface polypeptide having an extracellular domain which is homologous to a member of the immunoglobulin supergenefamily. Other receptors, which are not members of the immunoglobulin supergenefamily but are nonetheless specifically covered by this definition, are receptors for cytokines, and in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules, <i>e</i>.<i>g</i>. (E-, L- and P-) selectins.

0022The term "receptor binding domain" is used to designate any native ligand for a receptor, including cell adhesion molecules, or any region or derivative of such native ligand retaining at least a qualitative receptor binding ability of a corresponding native ligand. This definition, among others, specifically includes binding sequences from ligands for the above-mentioned receptors.

0023An "antibody-immunoadhesin chimera" comprises a molecule which combines at least one binding domain of an antibody (as herein defined) with at least one immunoadhesin (as defined in this application). Exemplary antibody-immunoadhesin chimeras are the bispecific CD4-IgG chimeras described in <nplcit id="ncit0011" npl-type="s"><text>Berg et al., PNAS (USA) 88:4723-4727 (1991</text></nplcit>) and <nplcit id="ncit0012" npl-type="s"><text>Chamow et al., J. Immunol. 153:4268 (1994</text></nplcit>).

Modes for Carrying Out the Invention

0024The process herein involves purifying a C_H2/C_H3 region-containing protein from contaminants by Protein A chromatography. The protein to be purified using Protein A chromatography is a protein fused to, or conjugated with, a C_H2/C_H3 region according to claim 1. Techniques for generating such molecules will be discussed below.

1.

C

H

2/C

H

3 region-containing proteins

0025The protein to be purified is one which is fused to, or conjugated with, a C_H2/C_H3 region. Such fusion proteins may be produced so as to increase the serum half-life of the protein and/or to facilitate purification of the protein by Protein A chromatography.

0026Examples of biologically important proteins which can be conjugated this way include renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor, lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); a serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; Protein D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derived growth factor(PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), <i>e</i>.<i>g</i>., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), <i>e</i>.<i>g</i>., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the HIV envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 or HER4 receptor; and fragments of any of the above-listed polypeptides.

2.

Protein Purification

0027The protein to be purified using the method described herein is generally produced using recombinant techniques. Methods for producing recombinant proteins are described, <i>e</i>.<i>g</i>., in <patcit id="pcit0003" dnum="US5534615A"><text>US Pat No's 5,534,615</text></patcit> and <patcit id="pcit0004" dnum="US4816567A"><text>4,816,567</text></patcit> . In preferred embodiments, the protein of interest is produced in a CHO cell (see, <i>e</i>.<i>g</i>. <patcit id="pcit0005" dnum="WO9411026A"><text>WO 94/11026</text></patcit>).

0028US Patent <patcit id="pcit0006" dnum="US5429746A"><text>US5429746</text></patcit> discloses the purification of antibodies on immobilised Protein A on controlled pore glass beads. Therein the wash step is performed at pH 7 using PBS/glycine buffer.

0029When using recombinant techniques, the protein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the protein is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Where the protein is secreted into the medium, the recombinant host cells may be separated from the cell culture medium by tangential flow filtration, for example.

0030Protein A immobilized on a solid phase is used to purify the C_H2/C_H3 region-containing protein. The solid phase is preferably a column comprising a glass or silica surface for immobilizing the Protein A. Preferably, the solid phase is a controlled pore glass column or a silicic acid column. Sometimes, the column has been coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adherence to the column. The PROSEP A™ column, commercially available from Bioprocessing Limited, is an example of a Protein A controlled pore glass column which is coated with glycerol.

0031The solid phase for the Protein A chromatography is equilibrated with a suitable buffer. For example, the equilibration buffer may be 25mM Tris, 25mM NaCl, 5mM EDTA, pH 7.1.

0032The contaminated preparation derived from the recombinant host cells is loaded on the equilibrated solid phase using a loading buffer which may be the same as the equilibration buffer. As the contaminated preparation flows through the solid phase, the protein is adsorbed to the immobilized Protein A and, as discovered herein, other contaminants (such as Chinese Hamster Ovary Proteins, CHOP, where the protein is produced in a CHO cell) bind nonspecifically to the solid phase.

0033The next step performed sequentially entails removing the contaminants bound to the solid phase by washing the solid phase with a hydrophobic electrolyte solvent in an intermediate wash step. In preferred embodiments, the hydrophobic electrolyte in this wash solvent is TEMAC and/or TEAC. While a single hydrophobic electrolyte may be present in the wash solvent, in certain embodiments, two or more such electrolytes may be used. The hydrophobic electrolyte is preferably added to a pH buffered solution having a pH in the range from about 4 to about 8, and preferably in the range from about 5 to about 7. Suitable buffers for this purpose include Tris, phosphate, MES, and MOPSO buffers. The preferred final concentration for the hydrophobic electrolyte in the wash solvent is in the range from about 0.1 to about 1.0M, and preferably in the range from about 0.25 to about 0.5M.

0034Following the intermediate wash step of the preceding paragraph, !he protein of interest is recovered from the column. This is normally achieved using a suitable elution buffer. The protein may, for example, be eluted from the column using an elution buffer having a low pH, <i>e</i>.<i>g</i>. in the range from about 2 to about 5, and preferably in the range from about 2.5 to about 3.5. Examples of elution buffers for this purpose include citrate or acetate buffers.

0035The eluted protein preparation may be subjected to additional purification steps either prior to, or after, the Protein A chromatography step. Exemplary further purification steps include hydroxylapatite chromatography; dialysis; affinity chromatography using an antibody to capture the protein; hydrophobic interaction chromatography (HIC); ammonium sulphate precipitation; anion or cation exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on silica; chromatofocusing; and gel filtration.

0036The protein thus recovered may be formulated in a pharmaceutically acceptable carrier and is used for various diagnostic, therapeutic or other uses known for such molecules.

EXAMPLE 1

0037The PROSEP A™ column (Bioprocessing, Ltd) has Protein A immobilized on a glycerol coated-controlled pore glass column. The glycerol coating reduces the glass surface available for non-specific interactions with contaminants but, as demonstrated herein, contaminants can still adhere to the column.

0038Protein A chromatography was the initial chromatography step in the purification of the C_H2/C_H3 region-containing protein; humanized anti-HER2 antibody (humAb4D5-8) (<nplcit id="ncit0013" npl-type="s"><text>Carter et al. Proc. Natl. Acad Sci. 89: 4285-4289 (1992</text></nplcit>)). This anti-HER2 antibody was produced recombinantly in CHO cells. Following protein production and secretion to the cell culture medium, the CHO cells were separated from the cell culture medium by tangential flow filtration (PROSTACK™). In this expression system, the most prevalent contaminants were found to be Chinese Hamster Ovary Proteins (CHOP).

0039The PROSEP A™ column was equilibrated with 25mM Tris, 25mM NaCl, 5mM EDTA, pH 7.1 (Buffer A). Protein A chromatography was performed by applying the Harvested Cell Culture Fluid (HCCF) from the CHO cells directly to the equilibrated PROSEP A™ column using Buffer A as the loading buffer. The column was then washed with Buffer A to wash out the HCCF and unbound proteins. The anti-HER2 antibody was eluted from the Protein A column by washing the column with Buffer B having a low pH (2.5-3.5). Buffer B was 25mM sodium citrate, pH 2.8. The low pH of Buffer B disrupted the interactions between Protein A and the anti-HER2 antibody. It also disrupted the non-specific interactions between the exposed glass surfaces of the column and non-specifically bound contaminating CHOP. This resulted in a level of CHOP contamination in the eluted anti-HER2 containing protein pool of ∼4000 ppm (Table 1). <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1^a</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="25mm" /><colspec colnum="2" colname="col2" colwidth="33mm" /><colspec colnum="3" colname="col3" colwidth="16mm" /><colspec colnum="4" colname="col4" colwidth="20mm" /><thead><row><entry morerows="1" align="center" valign="top">Sample</entry><entry morerows="1" align="center" valign="top">Conditions</entry><entry namest="col3" nameend="col4" align="center" valign="top">CHOP</entry></row><row><entry align="center" valign="top">(µg/ml)</entry><entry align="center" valign="top">(ppm)</entry></row></thead><tbody><row><entry>HCCF</entry><entry>Load</entry><entry align="right">760</entry><entry align="right">1,461,538</entry></row><row><entry>Antibody Pool</entry><entry>No wash</entry><entry align="right">32</entry><entry align="right">4,270</entry></row><row><entry>Antibody Pool</entry><entry>TMAC, pH 5.0 wash</entry><entry align="right">3</entry><entry align="right">408</entry></row><row><entry>Antibody Pool</entry><entry>TEAC, pH 5.0 wash</entry><entry align="right">2</entry><entry align="right">317</entry></row><row><entry>Antibody Pool</entry><entry>TMAC, pH 7.1 wash</entry><entry align="right">4</entry><entry align="right">537</entry></row><row><entry>Antibody Pool</entry><entry>TEAC, pH 7.1 wash</entry><entry align="right">5</entry><entry align="right">620</entry></row></tbody></tgroup><tgroup cols="4" rowsep="0"><colspec colnum="1" colname="col1" colwidth="25mm" /><colspec colnum="2" colname="col2" colwidth="33mm" /><colspec colnum="3" colname="col3" colwidth="16mm" /><colspec colnum="4" colname="col4" colwidth="20mm" /><tbody><row><entry namest="col1" nameend="col4" align="justify">^a The wash buffer was 25mM Tris, 25mM NaCl, 5mM EDTA including either 0.5M TMAC or TEAC at the indicated pH. Following the wash, the protein was eluted at pH 2.8.</entry></row></tbody></tgroup></table></tables>

0040In order to address this problem concerning contaminants in the eluted protein pool, an "intermediate wash step" was evaluated. Prior to eluting the antibody from the column, the Protein A column was washed with wash buffer (at varying pH's) to which various concentrations of TMAC or TEAC were added (Table 1).

0041As shown in Table 1, the intermediate wash step using a buffer containing either TMAC or TEAC was effective at lowering the level of CHOP in the eluted antibody pool. The concentration of either TMAC or TEAC was preferably at least 0.25M. Accordingly, it was found that preferred TMAC or TEAC concentrations in the wash solvent are from about 0.1 to about 1.0M, and preferably from about 0.25 to about 0.5M.

0042As to variations in the pH of the wash solution, it was found that the lower the pH, the greater removal of CHOP in the wash step. However, at pH's below 7.0, protein may also be partially eluted during the wash step. Therefore, preferred pH's for the intermediate wash step are from about 4 to about 8, and preferably from about 5 to about 7.