WO2009058769A1 - Purification of antibodies containing hydrophobic variants - Google Patents

Abstract

The present invention encompasses methods of purifying samples containing antibodies. In particular the present invention provides methods of reducing hydrophobic variants in these samples using a series of column chromatography steps, comprising protein A affinity chromatography, ion exchange chromatography and hydrophobic interaction chromatography.

Description

PURIFICATION OF ANTIBODIES CONTAINING HYDROPHOBIC VARIANTS

FJELD OF THE INVENTION

[0001 J The present invention provides a method of purifying proteins containing hydrophobic variants. In particular, methods of purifying Ig-containing proteins are provided.

BACKGROUND OF THE INVENTION

[0002] The immune system is composed of many interdependent cell types that collectively protect the body from bacterial, parasitic, fungal, viral infections and from the growth of turnout cells. The guards of the immune system are macrophages that continually roam the bloodstream of their host. When challenged by infection or immunization, macrophages respond by engulfing invaders marked with foreign molecules known as anti s gene. This event, mediated by helper T cells, sets forth a complicated chain of responses that result in the stimulation of B-cells. These B-cells, in turn, produce proteins called antibodies, which bind to the foreign invader. The binding event between antibody and antigen marks the foreign invader for destruction via phagocytosis or activation of the complement system, A number of different classes of antibodies, or immunoglobulins, so exist, such as IgA, IgD. IgE. IgG. and IgM. They differ not only in their physiological roles but also in their structures. From a structural point of view, IgG antibodies are a particular class of immunoglobulins that have been extensively studied, perhaps because of the dominant role they play in a mature immune response.

|0003| The biological activity , which the immunoglobulins possess, is today exploited in a range of different applications in the human and veterinary diagnostic, health care and therapeutic sector, in fact, in the last few years, monoclonal antibodies and recombinant antibody constructs have become the largest class of proteins currently investigated in clinical trials and receiving FDA approval as therapeutics and diagnostics. Complementary to expression systems and production strategies, purification protocols are designed to obtain highly pure antibodies in a simple and cost-effcieπt manner. [0004] A commonly utilized method for isolation of immunoglobulins is chromatography, which embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions many times before the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile to phase moving through a column containing the stationary phase. Separated components emerge in the order of increasing interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column. Efforts are continuously being made to design the optimal stationary phase for each specific separation purpose.

[0005] Reversed phase high pressure liquid chromatography (RP-HPLC) separates on the basis of hydrophobicity. As with other HPLC techniques there is a stationary phase, either silica or polymeric based, for example polystyrene/divinylbenzene. The mobile phase is usually a combination of a weak aqueous buffer or a dilute acid and a water miscible organic solvent. For effective separation of proteins the mobile phase is generally a gradient system, required to achieve separation and is preferably linear for convenience. [0006] Protein A and Protein G affinity chromatography are popular and widespread methods for isolation and purification of immunoglobulins, particularly for isolation of mono-clonal antibodies, mainly due to the ease of use and the high purity obtained. Used in combination with ion exchange, hydrophobic interaction, hydroxyapatite and/or gel fil-tration steps, especially protein A-based methods have become the antibody purification method of choice for many biopharmaceutical companies, see e. g. WO 8400773 and US 5,151,350. [0007] However, antibody preparation often include various contaminants and product related impurities, e.g., hydrophobic variants, that affect purity and yield. The present invention provides methods of purification using analytical liquid chromatography and column chromatography. SUMMARY OF THE INVENTION

[0008] The present invention is based, in part, upon the discovery that antibody purity levels were adversely affected by the presence of hydrophobic variants. The present invention provides a method of purifying a protein in a sample comprising: a) loading the sample on a Protein A column; b) eluting the sample from the Protein A column; c) loading the sample on an ion exchange column; d) eluting the sample from the ion exchange column; e) determining if the sample contains hydrophobic variants of the protein; f) if the sample contains hydrophobic variants of the protein, loading the sample on a hydrophobic interaction chromatography (HIC) column, wherein the HIC column is in a flow through mode; and g) collecting flow through from the HIC column. In further embodiments, the protein is an antibody or antibody fragment thereof; the antibody or antibody fragment is humanized; or the antibody or antibody fragment is binds to a human cytokine, including IL-IO or IL- 17. In other embodiments the ion exchange column is a cation exchange column; or the HIC column is a Butyl HIC column.

[0009] The sample can be subjected to viral inactivation between steps b and c; and/or can be subjected to ultrafiltration/diafiltration after step g. In another embodiment, the sample is loaded on and eluted from a second ion exchange column after ultrafiltration/diafiltration. The second ion exchange column can be an anion exchange column. In a further step, the sample is further subjected to virus filtration and ultrafiltration/diafiltration after the virus filtration after elution from the anion exchange column.

[0010] In one embodiment the sample is from a cell culture and reverse phase high pressure liquid chromatography (RP-HPLC) is used to determine if the sample contains hydrophobic variants.

[0011] The present invention provides A method of purifying a protein in a sample comprising: a) loading the sample on a Protein A column; b) eluting the sample from the Protein A column; c) determining if the sample contains hydrophobic variants of the protein; d) loading the sample on an ion exchange column; e) eluting the sample from the ion exchange column; f) loading the sample on a hydrophobic interaction chromatography (HIC) column, wherein the HIC column is in a flow through mode; and g) collecting flow through from the HIC column. In further embodiments, the protein is an antibody or antibody fragment thereof; the antibody or antibody fragment is humanized; or the antibody or antibody fragment is binds to a human cytokine, including IL-IO or IL-17. In other embodiments the ion exchange column is a cation exchange column; or the HIC column is a Butyl HIC column.

[0012] The sample can be subjected to viral inactivation between steps c and d; and/or can be subjected to ultrafiltration/diafiltration after step g. In another embodiment, the sample is loaded on and eluted from a second ion exchange column after ultrafiltration/diafiltration. The second ion exchange column can be an anion exchange column. In a further step, the sample is further subjected to virus filtration and ultrafiltration/diafiltration after the virus filtration after elution from the anion exchange column.

[0013] In one embodiment the sample is from a cell culture and reverse phase high pressure liquid chromatography (RP-HPLC) is used to determine if the sample contains hydrophobic variants.

DETAILED DESCRIPTION

[0014] As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the," include their corresponding plural references unless the context clearly dictates otherwise.

[0015] All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent, was specifically and individually indicated to be incorporated by reference.

I. Definitions.

[0016] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecifϊc antibodies), and antibody fragments so long as they retain, or are modified to comprise, a ligand-specific binding domain. The antibody herein is directed against an "antigen" of interest. Preferably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal. However, antibodies directed against nonpolypeptide antigens (such as tumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) are also contemplated. Where the antigen is a polypeptide, it may be a transmembrane molecule (e.g. receptor) or ligand such as a growth factor. Exemplary antigens include those polypeptides.

[0017] "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 fragments. [0018] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., 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 Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al, J. MoI. Biol. 222:581-597 (1991), for example.

[0019] The 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 or 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 (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984) ). [0020] The 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.e. residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (i.e. residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. MoI. Biol. 196:901-917 (1987)). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined. [0021] "Humanized" forms of non-human (e.g., 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 Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). [0022] As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the "binding domain" of a heterologous "adhesin" protein (e.g. 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.e. is "heterologous") and an immunoglobulin constant domain sequence. The immunoglobulin constant domain sequence in the immunoadhesin is preferably derived from γl, γ2, or γ4 heavy chains since immunoadhesins comprising these regions can be purified by Protein A chromatography (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983) ).

[0023] The 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 super gene family. Other receptors, which are not members of the immunoglobulin super gene family 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, e.g. (E-, L-and P-) selectins. [0024] The 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.

[0025] An "impurity" is a material that is different from the desired polypeptide product or protein of interest. The impurity includes, but is not limited to, a host cell protein (HCP, such as CHOP), a polypeptide other than the target polypeptide, nucleic acid, endotoxin etc.

[0026] The terms "Protein A" and "ProA" are used interchangeably herein and encompasses Protein A recovered from a native source thereof, Protein A produced synthetically (e.g. 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, such as an Fc region. Protein A can be purchased commercially from Repligen, Pharmacia and Fermatech. Protein A is generally immobilized on a solid phase support material. The term "ProA" also refers to an affinity chromatography resin or column containing chromatographic solid support matrix to which is covalently attached Protein A.

[0027] The term "chromatography" refers to the process by which a solute of interest in a mixture is separated from other solutes in a mixture as a result of differences in rates at which the individual solutes of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes.

[0028] The term "ion-exchange" and "ion-exchange chromatography" refers to the chromatographic process in which a solute of interest (such as a protein) in a mixture interacts with a charged compound linked (such as by covalent attachment) to a solid phase ion exchange material such that the solute of interest interacts non-specifically with the charged compound more or less than solute impurities or contaminants in the mixture. The contaminating solutes in the mixture elute from a column of the ion exchange material faster or slower than the solute of interest or are bound to or excluded from the resin relative to the solute of interest. "Ion-exchange chromatography" specifically includes cation exchange, anion exchange, and mixed mode chromatography.

[0029] The phrase "ion exchange material" refers to a solid phase that is negatively charged (i.e. a cation exchange resin) or positively charged (i.e. an anion exchange resin). The charge may be provided by attaching one or more charged ligands to the solid phase, e.g. by covalent linking. Alternatively, or in addition, the charge may be an inherent property of the solid phase (e.g. as is the case for silica, which has an overall negative charge). [0030] By "solid phase" is meant a non-aqueous matrix to which one or more charged ligands can adhere. The solid phase may be a purification column, a discontinuous phase of discrete particles, a membrane, or filter etc. Examples of materials for forming the solid phase include polysaccharides (such as agarose and cellulose); and other mechanically stable matrices such as silica (e.g. controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles and derivatives of any of the above. [0031] A "cation exchange resin" refers to a solid phase which is negatively charged, and which thus has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. A negatively charged ligand attached to the solid phase to form the cation exchange resin may, e.g., be a carboxylate or sulfonate. Commercially available cation exchange resins include carboxy-methyl-cellulose, sulphopropyl (SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOW® or SP-SEPHAROSE HIGH PERFORMANCE®, from Pharmacia) and sulphonyl immobilized on agarose (e.g. S- SEPHAROSE FAST FLOW® from Pharmacia). A "mixed mode ion exchange resin" refers to a solid phase which is covalently modified with cationic, anionic, and hydrophobic moieties. A commercially available mixed mode ion exchange resin is BAKERBOND ABX® (J.T. Baker, Phillipsburg, N.J.) containing weak cation exchange groups, a low concentration of anion exchange groups, and hydrophobic ligands attached to a silica gel solid phase support matrix.

[0032] The term "anion exchange resin" is used herein to refer to a solid phase which is positively charged, e.g. having one or more positively charged ligands, such as quaternary amino groups, attached thereto. Commercially available anion exchange resins include DEAE cellulose, QAE SEPHADEX® and FAST Q SEPHAROSE® (Pharmacia). [0033] A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. Various buffers which can be employed depending, for example, on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975) . In one embodiment, the buffer has a pH in the range from about 2 to about 9, alternatively from about 3 to about 8, alternatively from about 4 to about 7 alternatively from about 5 to about 7. Non-limiting examples of buffers that will control the pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, as well as combinations of these.

[0034] The term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Therefore, with an increasing amount of ions present in the aqueous solution, the solution will have a higher conductivity. The unit of measurement for conductivity is milliSiemens per centimeter (mS/cm), and can be measured using a conductivity meter sold, e.g., by Orion. The conductivity of a solution may be altered by changing the concentration of ions therein. For example, the concentration of a buffering agent and/or concentration of a salt (e. g. NaCl or KCl) in the solution may be altered in order to achieve the desired conductivity. [0035] The "pi" or "isoelectric point" of a polypeptide refer to the pH at which the polypeptide's positive charge balances its negative charge, pi can be calculated from the net charge of the amino acid residues or sialic acid residues of attached carbohydrates of the polypeptide or can be determined by isoelectric focusing. [0036] By "binding" a molecule to an ion exchange material is meant exposing the molecule to the ion exchange material under appropriate conditions (pH/conductivity) such that the molecule is reversibly immobilized in or on the ion exchange material by virtue of ionic interactions between the molecule and a charged group or charged groups of the ion exchange material.

[0037] By "washing" the ion exchange material is meant passing an appropriate buffer through or over the ion exchange material.

[0038] To "elute" a molecule (e.g. polypeptide or impurity) from an ion exchange material is meant to remove the molecule therefrom by altering the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for the charged sites on the ion exchange material.

[0039] "Flow through" in the context of column chromatography, mean the sample collected that does not bind to the column resin. In particular, the flow through from a hydrophobic interaction column will contain an antibody or antibody fragment thereof of interest, while the material bound to the column will be hydrophobic variants of the antibody. [0040] "Hydrophobic variant or variants" as used herein encompasses proteins with polar charges different from native proteins, thereby resulting in aggregation and/or reduced solubility in aqueous solutions. Hydrophobic variants are those that differ from the mature, correctly folded protein in hydrophobicity, including partially processed precursor sequences, glycosylated mature and precursor-containing forms and misfolded and partially folded variants.

II. General.

[0041] The present invention provides methods of removing impurities from antibody preparation. In particular, Reverse Phase HPLC (RP-HPLC) is used to detect hydrophobic variants in a sample containing antibodies. If hydrophobic variants are detected, the sample is then subjected to a series of column chromatography steps.

[0042] A particular aspect of the present invention is directed to a method for analyzing an antibody or a fragment thereof, the method comprising preparing a sample comprising the antibody or fragment thereof for loading onto a high performance liquid chromatography (HPLC) column; separating the antibody or fragment thereof from the sample by reversed-phase HPLC on the column, wherein the eluate from the reversed-phase HPLC is introduced into the ion source of a mass spectrometer, wherein the mass spectrometer is in-line with the HPLC column; and obtaining mass fragmentation data of the antibody or fragment thereof by mass spectrometry; wherein the HPLC column is heated to a temperature of from about 50° C. to about 90° C; and wherein the mobile phase of the reversed-phase HPLC comprises a water miscible organic solvent having a C 18 eluotropic strength coefficient of at least 6.0. Preferably, the UV cutoff of the solvent is one which allows the solvent to be used in UV detection of proteins especially at 215 nm, 245 nm or 280 nm.

[0043] An antibody comprises a constant domain and two variable regions. More particularly, in exemplary embodiments, an antibody analyzed in the methods of the invention is of an IgG class selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. The method of the invention also contemplates analysis of an antibody that is a single chain antibody, e.g., scFv. In alternative embodiments the methods of the invention are used to analyze a humanized antibody. In particular embodiments, the antibody is a humanized IgG2 antibody. In yet further embodiments, the methods of the invention are used to analyze fusion proteins or human or humanized antibodies.

[0044] The RP-HPLC analysis of the present invention of a protein further comprises subjecting the protein to cleavage by limited proteolysis or chemical cleavage. Preferably, the limited proteolysis is conducted prior to loading the sample on the HPLC column. In more particular embodiments, the limited proteolysis comprises digestion with an enzyme during a relatively short period of time, typically less than 1 hour. In another embodiment, the chemical cleavage was performed by reducing the disulfide bonds in the protein or fragments thereof. For example, the reduction of the disulfide bonds comprises contacting the sample with a reducing agent. Exemplary reducing agents include but are not limited to dithiothreitol, mercaptoethanol, tributylphosphine, and tri(2-carboxyethyl)phosphine hydrochloride. Alternatively, the protein may be subjected to chemical cleavage. As an alternative to chemical cleavage, the methods of the invention also contemplate enzymatic proteolysis using enzymes such as papain, pepsin, or Lys-C protease. [0045] The methods of analyzing the proteins described herein will be particularly useful in determining the structural integrity of a protein. Thus, the RP-HPLC/MS methods of the invention specifically contemplate determining the presence of a protein degradation product in an antibody sample, the method comprising performing RP-HPLC on the protein under conditions wherein the HPLC column is heated to a temperature of from about 50° C. to about 90° C; and wherein the mobile phase of the reversed-phase HPLC comprises a water miscible organic solvent having a Cl 8 eluotropic strength coefficient of at least 6.0, and determining the molecular weight data of the protein using ESI-MS. Comparing the molecular weight data from the protein to data generated from known standards may be an effective method of determining the presence of any degradation products. For example, performing the method on an antibody sample known not to have undergone degradation will provide an effective standard against which to measure the data produced from an antibody sample that is being tested for degradation products as the presence of degradation products will be detectable as differences compared to the measurements produced by the standard. Likewise, molecular weight profiles may be generated for common moieties normally present and change in molecular weight values thereof due to e.g., alteration or loss may be indicative of degradation. Similarly, profiles of dimer formation, cleavage product, oxidation, deamidation, N-terminal pyroglutamation and disulfide bond scrambling may be generated or known to those of skill in the art and the presence of such a profile may be indicative of the degradation.

[0046] In certain preferred embodiments, the methods described herein may be used as methods of determining disulphide bond rearrangement of an IgG2 sample, where the method comprises performing RP-HPLC on the IgG2 sample under conditions wherein the HPLC column is heated to a temperature of from about 50° C. to about 90° C; and wherein the mobile phase of the reversed-phase HPLC comprises a water miscible organic solvent having a Cl 8 eluotropic strength coefficient of at least 6.0; detecting the presence of heterogeneous peaks from the RP-HPLC of the IgG2 sample; and determining the molecular weight data of the components of the heterogeneous peaks of the RP-HPLC of the IgG2 sample using ESI-MS, wherein identical or similar molecular weight data is indicative of disulphide bond rearrangement in the IgG2 sample. In more specific embodiments, the disulphide bond rearrangement may be monitored as a mass difference of two mass units. [0047] As noted above, if hydrophobic variants are detected, the antibody sample is then subjected to a series of column chromatography purification steps. Protein A is a group specific ligand which binds to the Fc region of most IgG. It is synthesised by some strains of staphylococcus aureus and can be isolated from culture supernatants then insolubilised by coupling to agarose beads or silica. An alternative method is to use whole bacteria of a strain which carries large amounts of protein A on the bacterial cell surface. Both types of gel preparation are available commercially. (Protein A— Pharmacia. Whole bacteria Calbiochem, IgG sorb). (Alan Johnstone and Robin Thorpe Immunochemistry in practice, Blackwell Scientific Publn. Chpt.lO). An alternative to Protein A is Protein G (Analytical Chem. Vol. 61 (13) 1989 1317).

[0048] The column which is most preferably used is a Protein A Sepharose column, particularly Protein A Sepharose Fast Flow™ , Protein A MabSelect™, or MabSure™. Ideally the column is washed with tris(hydroxyaminomethane), citrate or phosphate buffered saline in a pH range from pH 5.0 to pH 7.5 and the antibody is eluted at acid pH 3.0-3.5 advantageously pH 3.0 using an acid such as citric or acetic acid for example in a concentration of about 1.0M.

[0049] Ion-exchange chromatography exploits interactions between charged groups in a stationary phase and the sample which is in a mobile phase. The stationary phase of an ion- exchange column may be a positively charged cation exchanger or a negatively charged anion exchanger. The charged groups are neutralised by oppositely charged counter ions in the mobile phase, the counter ions being replaced during chromatography by more highly charged sample molecules. It is preferable to use cross-linked chromatography resins based for example on agarose for example S-Sepharose Fast Flow (Trademark) cation exchange column particularly S. Sepharose Fast Flow™ cation exchange. Alternatively a membrane- based column could be employed. The column is usually washed after application of the eluate from the Protein A column, with 20 mM HEPES buffer pH 7.5 and the antibody is eluted with the same buffer containing sodium chloride in the range 0.2M to 0.075M. [0050] Size exclusion chromatography as its name suggests separates on the basis of the size of proteins. In general separation occurs when large molecules are excluded from entering the porous stationary phase and are carried straight through the column while progressively smaller molecules are increasingly able to enter the stationary phase and consequently have particularly longer elution times. It is the porosity of the stationary phase which therefore determines the separation achieved. Suitable materials are chemically bonded and provide resistance to compression for example an agarose and/or dextran composition such as Superdex (Trademark) . A preferred column is a Superdex 200 size exclusion medium. The eluate from the ion exchange column is preferably applied to the Superdex column and developed in buffer in the range pH5-8 preferably PBS pH 7.2. [0051] Each column is preferably protected by a filter which may be a 0. 2μ Gelman

Aero sterilising filter or in the case of the Protein A column a PALL posidyne SLK 7002 NFZP or a PALL DSLK2 filter (available from Pall Process Filtration Ltd. European House, Havant Street, Portsmouth 301 3PD) and for the other two columns a Millipak filter preferably Millipak 100 for the ion exchange column and Millipak 20 or 60 for the size exclusion column (available from Millipore, The Boulevard, Blackmore Lane, Warford, Herts. The columns are preferably sanitised before use with an appropriate sanitant for example 0.5M NaOH for for 0.5-3.0 hours for any of the columns, or 2% hibitane gluconate in 20% ethanol for the Protein A column or IN NaOH for the other two columns. Sanitants were washed out with the appropriate sterile buffers before applying the protein solution. All solutions used in the process were preferably sterile and endotoxin free. [0052] Where ion exchange chromatography relies on the charges of proteins to isolate them, hydrophobic interaction chromatography (HIC) uses the hydrophobic properties of some proteins. Hydrophobic groups on the protein bind to hydrophillic groups on the column. The more hydrophobic a protein is, the stronger it will bind to the column. [0053] Proteins are loaded on the HIC column in the presence of a high concentration of ammonium sulfate. Ammonium sulfate is a chaotropic agent that increases hydrophobic interactions. Ammonium sulfate also stabilizes proteins. So as a result of using an HIC column proteins can be in their most stable form.

[0054] The hydrophobic column is packed with a butyl or a methacyrlate based resin matrix (Butyl 650M or 600M available from, e.g., Tosoh). In the presence of high salt concentrations the phenyl groups on this matrix binds hydrophobic portions of proteins. Conventionally, proteins of interest are bound to the HIC resin, impurities are washed out, and the protein of interest is eluted using standard elution buffers with decreasing salt concentrations. The present invention binds the hydrophobic variants to the HIC resin and the proteins of interest, e.g., antibodies, are obtained by collecting the flow through. [0055] Additional steps may be added to the purification procedure set out above.

Ultrafiltration may be used to further reduce viral and host cell nucleic acid contamination. This may be carried out using commercially available ultrafiltration units such as Viresolve/70' or Viresolve/180' membranes additionally, PLMK regenerated cellulose 300 k cut off membrane all available from Millipore, The Boulevard, Blackmore Lane, Watford, Herts or Piano va 2ON (Asahi). An alternative method to reduce virus contamination is microfϊltration using a Nylon membrane in cartridge form for example Nylon 66,0.04M membrane from PALL.

II. Antibodies

[0056] The preferred protein to be purified according to the present invention is an antibody. The antibody herein is directed against an antigen of interest. Preferably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal. However, antibodies directed against nonpolypeptide antigens (such as tumor- associated glycolipid antigens; see U.S. Pat. No. 5,091,178) are also contemplated. Where the antigen is a polypeptide, it may be a transmembrane molecule (e.g. receptor) or ligand such as a growth factor.

[0057] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N- hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N= C=NR, where R and R¹ are different alkyl groups.

[0058] Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with Vs to 1/10 the original amount of antigen or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. [0059] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0060] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59- 103 (Academic Press, 1986)).

[0061] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. [0062] Preferred myeloma cells are those that fuse efficiently, support stable high- level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63- Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0063] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). [0064] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI- 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

[0065] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, Protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Preferably the Protein A chromatography procedure described herein is used.

[0066] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.

[0067] The DNA also may be modified, for example, by substituting the coding sequence for human heavy-and light-chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al, Proc. Natl. Acad. Sci. USA, 81 :6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0068] Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

[0069] In a further embodiment, monoclonal antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. MoI. Biol, 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21 :2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional hybridoma techniques for isolation of monoclonal antibodies.

[0070] A humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0071] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best- fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human FR for the humanized antibody (Sims et al. , J. Immunol, 151 :2296 ( 1993)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol, 151 :2623 (1993)).

[0072] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three- dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. [0073] Alternatively, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J _H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ- line immunoglobulin gene array in such germ- line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. ScL USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and Duchosal et al. Nature 355:258 (1992). Human antibodies can also be derived from phage-display libraries (Hoogenboom et al., J. MoL Biol., 227:381 (1991); Marks et al., J. MoL Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech 14:309 (1996)).

[0074] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185.

[0075] Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecifϊc antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein.

[0076] Methods for making bispecifϊc antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecifϊc structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

[0077] According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the C_H3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0078] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0079] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0080] Recent progress has facilitated the direct recovery of Fab'-SH fragments from

E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')₂ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0081] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol, 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (V_L) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V_H and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol, 152:5368 (1994). Alternatively, the antibodies can be "linear antibodies" as described in Zapata et al. Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V_H-C_H 1 -V_H-C_HI) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. [0082] Antibodies of interest include, but are not limited to, humanized and parental versions of anti -cytokine antibodies, e.g., anti-human IL-10, anti-human IL- 17, anti-human IL-23, etc., anti-cytokine receptors, e.g., IL-23R, IL-10R, IL-17R, etc., antibodies against cell surface expressed proteins, e.g., IGFR.

[0083] The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific embodiments.

Examples

I. General Methods

[0084] Standard methods of biochemistry and molecular biology are described or referenced, e.g., in Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA. Standard methods also appear in Ausbel et al. (2001) Current Protocols in Molecular Biology, VoIs.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. T), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0100] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described. Coligan et al. (2000) Current Protocols in Protein Science, Vol. I, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described. See, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391. Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra. Standard techniques for characterizing ligand/receptor interactions are available. See, e.g., Coligan et al. (2001) Current Protcols in Immunology, Vol. 4, John Wiley, Inc., New York. [0101] Methods for flow cytometry, including fluorescence activated cell sorting detection systems (F ACS®), are available. See, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2^nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO. [0102] Standard methods of histology of the immune system are described. See, e.g.,

Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY.

[0103] Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available. See, e.g., GenBank, Vector NTI^® Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher^® (TimeLogic Corp., Crystal Bay, Nevada); Menne et al. (2000) Bioinformatics 16: 741-742; Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690.

II. Reverse Phase HPLC Assay

[0104] A reverse phase HPLC analysis of monoclonal antibodies was designed that allows quantification and analysis of hydrophobic variant contamination of an antibody containing sample in a brief assay. Prior to subjecting an aliquot of the antibody containing sample free cell samples were 0.2μ filtered. The antibody containing sample could be directly from a cell culture or after elution from a Protein A column as described below. Five microliters of antibody containing sample or standard were injected onto a POROS® R2 10

Micron 2.1mmD/30mmL PEEK™ HPLC column (for samples with concentrations of less than 35 μg/ml a 100 μl injection was performed).

[0105] An Agilent 1100 or 1200 HPLC system equipped with the following was used:

Binary pump, auto-injector with thermostat, column compartment with switching valve, and a multi-wavelength detector. The column temperature was maintained at 70⁰C.

[0106] Buffer gradient was 32% Buffer B (0.2 trfluoroacetic acid in 90% acetonitrile,

10% water) to 60% Buffer B in five minutes followed by a gradient to 100% Buffer B in 1 minute. A gradient back o 32% Buffer B in 1 minute followed. Gradients started as low as

25% Buffer B for hydrophilic proteins to 38% Buffer B for more hydrophobic proteins. The starting percentage of Buffer B was determined so that the protein peak of interest elueted between 2 and 3 minutes. The column was equilibrated for 2 minutes at the end of each run in preparation for the next sample.

[0107] The flow rate was maintained at 2 ml/minute. Pressure were typically run from 105 barometers (bar) at 32% Buffer B to 65 bar at 60% Buffer B. The detector was set at 280 nm. A wavelength of 360 nm with a bandwidth of 100 nm was used as a reference wavelength.

[0108] In addition to its capability of monitoring impurities from cell culture fermentation (e.g. antibody fragments), this assay was used to assess if an antibody preparation contained hydrophobic variants that are associated with aggregate and particulate formation. In particular, the humanized anti-IL-10 monoclonal antibody (as described in US

2005/0101770) was analyzed for aggregate formation. Following a three column purification

(Protein A -> Cation Exchange (e.g., Fractogel) -> Anion Exachange (e.g., Sepharose)), a sample of the humanized anti-IL-10 preparation was subjected to the RP-HPLC assay described above. The result showed a hydrophobic variant product that eluted after the main antibody peak. The 3 column purified sample was subjected to Hydrophic Interaction Chromatography (HIC) in flow through mode. The flow through from the HIC column was again analyzed using the RP-HPLC assay. Results of the RP-HPLC demonstrated that the content of hydrophobic variants was reduced. The hydrophobic variant after the three column process was 1-10% of the antibody preparation, typically 4-6%. Typical purity of the main antibody was about 95% +/- 3%. It was also determined that a percentage range of hydrophobic variants in the total preparation could be established to warrant use of the flow through HIC either before or after elution from the Protein A column.

[0109] Table 1 represents the ranges as determined by RP-HPLC, used to determine if hydrophobic variants were present in quantities to warrant the additional flow-through HIC purification. Ranges of hydrophobic variants are percentages of total antibody preparation.

Table 1 : Percentage of hydrophobic variant present in sample following RP-HPLC analysis

III. Protein A Chromotography

[0110] Mabselect chromatography media (GE Healthcare Biosciences, Little

Chalfont, UK) was packed in a bed of 25 cm diameter x 20 cm height (1 bed volume = 9.8 L). The bed was sanitized with 3 bed volumes (BV) of solution J4 (0.1N Sodium Hydroxide, IM Sodium Chloride), equilibrated with 3 BV of solution J3 (100 mM Acetic Acid) and 5 BV of solution Jl (pH 7.2, 10 mM Sodium Phosphate, 125 mM Sodium Chloride). [0111] Approximately 420L of harvested clarified cell culture fluid (HCCF) with a mAb titer of approximately 400 mg/L was loaded onto the column, followed by a 10 BV wash with solution Jl and 5 BV wash with solution J2 (pH 7.2, 10 mM Sodium Phosphate). After washing, elution was carried out by a step gradient using 10 BV of solution J3. When the online absorbance (280 nm) of the eluted peak reached 0.5 AU/cm the collection of the pool was initiated and continues until the online absorbance (280 nm) dropped below 0.5 AU/cm.

[0112] The chromatography media used in this step was highly specific for human

IgGl (such as humanized anti-IL-10). The binding occurred between the constant region (Fc) of the antibody and the proteinaceous ligand (Protein- A). Accordingly, the mAb bound to the column and other contaminants present in the HCCF that exhibited much less affinity for the Protein-A ligand (e.g. host cell proteins and DNA) flow through. All chromatography stages are carried out at a linear velocity of 3 cm/min.

IV. Viral Inactivation

[0113] The pool collected during the Protein-A chromatography step was adjusted immediately to pH 3.5 with solution J3 and held for 1 hour. Afterwards, the pH of the inactivated pool was adjusted to 5.5 with solution TRIS (IM Tris(hydroxymethyl) aminomethane). After pH adjustment, the inactivated pool was 0.2 μm filtered.

V. Ion Exchange Chromatography

[0114] Fractogel SE HiCap chromatography media (Merck KGaA, Darmstadt,

Germany) was packed in a bed of 25 cm diameter x 20 cm height (1 bed volume = 9.8 L). The bed was equilibrated with 12 BV of solution Ll (pH 5.5, 20 mM Sodium Acetate). [0115] Following viral inactivation, antibody samples are diluted with an equal volume of solution Ll in order to decrease its conductivity to less than 3 mS. The adjusted feed was loaded onto the column, followed by a 10 BV wash with solution Ll . After washing, elution was carried out by a linear gradient from 100% of solution Ll to 100% of solution L2 (pH 5.5, 20 mM Sodium Acetate, 250 mM Sodium Chloride) over 20 BV. When the online absorbance (280 nm) of the eluted peak reaches 0.2 AU/cm the collection of the pool is initiated and continues until the online absorbance (280 nm) drops below 0.2 AU/cm. [0116] During this step, the mAb binds to the column. The concentration of contaminants present in the feed such as Protein-A (coeluted with mAb during the Protein-A chromatography step), host cell proteins and DNA is reduced across this step, thus effecting purification. All chromatography stages are carried out at a linear velocity of 2 cm/min. VI. Hydrophobic Interaction Chromatography

[0117] The conductivity of the pool collected during the cation exchange chromatography step was adjusted to 60 mS with solution Ml (2.12M Ammonium Sulfate). Afterwards, the pH of the pool was adjusted to 7.0 with solution TRIS. The pool is subsequently 0.2 μm filtered.

[0118] Toyopearl Butyl-650M chromatography media (Tosoh Corporation, Tokyo,

Japan) was packed in a bed of 25 cm diameter x 20 cm height (1 bed volume = 9.8 L). The bed was equilibrated with 5 BV of solution M2 (pH 7.0, 20 mM Sodium Phosphate, 376.9 mM Ammonium Sulfate). The feed was loaded onto the column, followed by a 10 BV wash with solution M2. When the online absorbance (280 nm) of the eluted peak reached 0.2 AU/cm the collection of the pool was initiated and continued until the online absorbance (280 nm) dropped below 0.2 AU/cm.

[0119] During this step, product-related impurities which are more hydrophobic than the antibody of interest displayed a stronger interaction with the resin, thus producing the separation and purification in the flow through rather than the eluate. All chromatography stages were carried out at a linear velocity of 2.5 cm/min.

VII. First Ultrafiltration/Diafiltration

[0120] Pellicon-2 cassettes (Millipore Corporation, Billerica, MA) with a total filtration area of 2.5 m² of Biomax 50 membranes (5OkD nominal molecular weight cutoff) were assembled using a Pellicon-2 holder. The assembly was flushed with at least 5OL of solution Ol (pH 8.0, 20 mM Tris(hydroxymethyl) aminomethane hydrochloride). [0121] The pool collected during the hydrophobic interaction chromatography step was concentrated by tangential flow ultrafiltration until the retentate mass reaches 20 kg. After concentration, the retentate was diafiltered against 10 volumes of solution 01. The transmembrane pressure was maintained at 10-20 psi during both concentration and diafiltration. Following diafiltration, the retentate was 0.2 μm filtered and the mAb was recovered by flushing the system with solution Ol .

[0122] During this step, the mAb was retained by the membrane. The main goal of the step was to adjust the composition, pH and conductivity of the feed going into the next chromatography step. The step also reduced the concentration of host cell proteins and DNA. VIII. Anion Exchange Chromatography

[0123] Q Sepharose Fast Flow chromatography media (GE Healthcare Biosciences,

Little Chalfont, UK) was packed in a bed of 25 cm diameter x 20 cm height (1 bed volume = 9.8 L). The bed is equilibrated with 20 BV of solution 01.

[0124] The feed was loaded onto the column, followed by a 5 BV wash with solution

Ol . When the online absorbance (280 nm) of the eluted peak reached 0.2 AU/cm the collection of the pool was initiated and continues until the online absorbance (280 nm) dropped below 0.2 AU/cm. The pH of the pool was adjusted to 5.5 with solution J3. After pH adjustment, the pool was 0.2 μm filtered.

[0125] During this step the mAb did not bind to the column since its pi is higher than the pH at which the chromatography was run. Negatively-charged contaminants present in the feed such as viruses, DNA or host cell proteins bound to the resin, thus enhancing purification. Column equilibration was performed at 3 cm/min. All other chromatography stages were carried out at a linear velocity of 2 cm/min.

IX. Virus Filtration

[0126] A Planova 2ON filter (Asahi Kasei Pharma, Tokyo, Japan) with 1.0 m² of filtration area was used in this step. The filter was flushed with solution Ql (pH pH 5.5, 20 mM Sodium Acetate, 75 mM Sodium Chloride). Solution Ql was tested prior to use to ensure that it had an endotoxin level less than 1 EU/ml.

[0127] The pool from the anion exchange chromatography step was filtered at a pressure drop of less than 14 psi. The filter was operated in the dead-end mode. Once all the pool volume was fed to the filter, the device was flushed with solution Ql . During this step, viruses that might be present in the pool were retained by the filter (19 nm mean pore size) while the mAb flowed through the membrane.

X. Second Ultrafiltration/Diafiltration

[0128] Pellicon-2 casettes (Millipore Corporation, Billerica, MA) with a total area of

1.5 m² of Biomax 50 membranes (5OkD nominal molecular weight cutoff) were assembled using a Pellicon-2 holder. The assembly was then flushed with at least 5OL of solution Ql. Solution Ql was tested prior to use to ensure that it had an endotoxin level less than 1 EU/ml. [0129] The filtrate generated during the virus filtration was concentrated by tangential flow ultrafiltration. After concentration, the retentate was diafiltered against 10 volumes of solution Ql . The transmembrane pressure was maintained at 10-20 psi during both concentration and diafiltration. Following diafiltration, the retentate was concentrated further, 0.2 μm filtered and the mAb was recovered by flushing the system with solution Ql . [0130] During this step, the mAb was retained by the membrane. The main goal of the step was to adjust the mAb concentration (20-30 g/L), solution composition, pH and conductivity of the API composite.

Claims

CLAIMSWhat is claimed is:

1. A method of purifying a protein in a sample comprising: a) loading the sample on a Protein A column; b) eluting the sample from the Protein A column; c) loading the sample on an ion exchange column; d) eluting the sample from the ion exchange column; e) determining if the sample contains hydrophobic variants of the protein; f) if the sample contains hydrophobic variants of the protein, loading the sample on a hydrophobic interaction chromatography (HIC) column, wherein the HIC column is in a flow through mode; and g) collecting flow through from the HIC column.

2. The method of Claim 1 , wherein the protein is an antibody or antibody fragment thereof.

3. The method of Claim 2, wherein the antibody or antibody fragment is humanized.

4. The method of Claim 2, wherein the antibody or antibody fragment is binds to a human cytokine.

5. The method of Claim 4, wherein the human cytokine is IL-IO or IL-17.

6. The method of Claim 1, wherein the ion exchange column is a cation exchange column.

7. The method of Claim 1, wherein the HIC column is a Butyl HIC column.

8. The method of Claim 1, wherein the sample is subjected to viral inactivation between steps b and c.

9. The method of Claim 1, wherein the sample is subjected to ultrafϊltration/diafiltration after step g.

10. The method of Claim 9, wherein the sample is loaded on and eluted from a second ion exchange column after ultrafiltration/diafiltration.

11. The method of Claim 10, wherein the second ion exchange column is an anion exchange column.

12. The method of Claim 10, wherein the sample is further subjected to virus filtration.

13. The method of Claim 12, wherein the sample is subjected to ultrafiltration/diafiltration after the virus filtration.

14. The method of Claim 1, wherein the sample is from a cell culture.

15. The method of Claim 1 , wherein reverse phase high pressure liquid chromatography (RP-HPLC) is used to determine if the sample contains hydrophobic variants.

16. A method of purifying a protein in a sample comprising: a) loading the sample on a Protein A column; b) eluting the sample from the Protein A column; c) determining if the sample contains hydrophobic variants of the protein; d) loading the sample on an ion exchange column; e) eluting the sample from the ion exchange column; f) loading the sample on a hydrophobic interaction chromatography (HIC) column, wherein the HIC column is in a flow through mode; and g) collecting flow through from the HIC column.

17. The method of Claim 16, wherein the protein is an antibody or antibody fragment thereof.

18. The method of Claim 17, wherein the antibody or antibody fragment is humanized.

19. The method of Claim 17, wherein the antibody or antibody fragment is binds to a human cytokine.

20. The method of Claim 19, wherein the human cytokine is IL-10 or IL-17.

21. The method of Claim 16, wherein the ion exchange column is a cation exchange column.

22. The method of Claim 16, wherein the HIC column is a Butyl HIC column.

23. The method of Claim 16, wherein the sample is subjected to viral inactivation between steps c and d.

24. The method of Claim 16, wherein the sample is subjected to ultrafϊltration/diafiltration after step g.

25. The method of Claim 24, wherein the sample is loaded on and eluted from a second ion exchange column after ultrafiltration/diafiltration.

26. The method of Claim 25, wherein the second ion exchange column is an anion exchange column.

27. The method of Claim 25, wherein the sample is further subjected to virus filtration.

28. The method of Claim 27, wherein the sample is subjected to ultrafiltration/diafiltration after the virus filtration.

29. The method of Claim 16, wherein the sample is from a cell culture.

30. The method of Claim 16, wherein reverse phase high pressure liquid chromatography (RP-HPLC) is used to determine if the sample contains hydrophobic variants.