WO2012030512A1 - Flow-through protein purification process - Google Patents

Abstract

A process for isolating a protein of interest from a sample includes the steps of providing a sample comprising the protein of interest; and subjecting the sample to chromatography in flow-through mode, wherein the sample is applied to a chromatography material under conditions that highly bind the protein of interest but where the amount of protein of interest exceeds the binding capacity of the chromatography material such that a substantial amount of the protein of interest does not adsorb to the material; and collecting at least a portion of the sample that does not adsorb to the material, to thereby isolate the protein of interest.

Description

Flow-Through Protein Purification Process

Field of the Invention

The present invention relates to the field of protein purification. In the large-scale production of pharmaceutically important proteins, which production typically uses prokaryotic or eukaryotic cell lines engineered to express the protein of interest, biologies manufacturers face formidable challenges in the separation of the desired protein from cellular byproducts and other impurities created in the production process. These challenges include compliance with strict regulatory standards, which typically require that protein-based pharmaceutical products be substantially free from impurities, such as product related contaminants, e.g., aggregates, fragments and variants of the recombinant protein, and process related contaminants, e.g., host cell proteins (HCPs), media components, viruses, DNA, and endotoxins. Reported Developments

While various protein purification schemes are available to the biopharmaceutical industry, these schemes typically include one or more "bind-and-elute" steps and/or affinity purification steps in order to reach a pharmaceutically acceptable degree of purity. The bind-and-elute processes adsorb the protein of interest to a chromatography medium, which is then washed to remove impurities, followed by elution of the protein of interest from the chromatography medium by altering solvent conditions. Because these methods employ multiple steps and washes, they result in high costs.

An example of these processes is disclosed in US Pat. No. 7,323,553, which describes a method for purifying antibodies from a mixture containing host cell proteins using non-affinity chromatography purification steps, including cation exchange chromatography in a bind-and-elute mode, followed by a high-performance tangential- flow filtration (HPTFF) step. Another example is disclosed in WO 2007/108955, which describes the purification of antibodies or antibody-like proteins, from a cell culture supernatant, using cation exchange in a bind-and-elute mode followed by anion exchange chromatography.

The present invention relates to the flow-through protein purification system, eliminating the expensive and time-consuming bind-and-elute protein purification process.

Summary of the Invention

The present invention is based, at least in part, on the surprising discovery that significant purification of proteins from mixtures containing multiple contaminants can be obtained by using a chromatographic medium under conditions where the protein of interest binds to the medium, but where the amount of protein of interest contacted with the medium exceeds the capacity of the medium. This discovery permits the design of a downstream flow-through process, eliminating the typical bind-and-elute process, for the purification of proteins. The processes described herein can, as compared to prior systems, result in a shortening of processing time and lower costs due to, e.g., reduced buffer consumption, faster purification time, reduced cleaning validation cost, and/or lower capital expenditure (e.g., due to smaller footprint facilities with simple equipment).

More particularly, the present invention relates to a process for purifying a protein of interest, comprising:

(a) providing a sample comprising an amount of protein of interest and impurities;

(b) contacting said sample with a cation exchange, anion exchange or hydrophobic interaction material under chromatography conditions, which absorb said protein of interest, wherein said amount of protein of interest in said sample exceeds the capacity of said material to adsorb more than an insubstantial portion of said amount of protein of interest, and wherein a substantial amount of said impurities adsorb to said material; and

(c) separating said material to which impurities have been adsorbed from the sample containing the substantial portion of said protein of interest that has not adsorbed to said material, to result in a purified sample.

In a preferred aspect of the present invention, the process is conducted in a flow- through mode.

In a particularly preferred aspect of the present invention, the material is a cation exchange material and wherein said chromatography conditions of step (b) provide for said protein of interest to exhibit a net positive charge. In another aspect of the present invention, the pH and conductivity of said sample are selected to (i) maximize the purification yield of said protein of interest, and (ii) reduce the impurities retained in said purified sample. In some embodiments, the protein of interest has a pi between 6.5 and 9.5, and the pH and conductivity values of the chromatography conditions are set based on such pi.

In another preferred aspect of the present invention, the material is hydrophobic interaction chromatography material, and wherein said chromatography conditions of step (b) provide for said sample to contain concentration of lyotropic salt selected to maximize binding of said protein of interest. A preferred sample contacted with said hydrophobic interaction chromatography material contains the protein of interest together with aggregate impurities from about 1% to about 20%, and of host cell protein impurities from about 10 ppm to about 1000 ppm. In another preferred aspect of the present invention, the material is anion exchange material, and wherein said chromatography conditions of step (b) provide for said protein of interest to exhibit a net positive charge.

In a further preferred aspect of the present invention, the purified sample obtained from the cation exchange chromatography (CEX) is subjected to anion exchange chromatography (AEX) in a flow-through mode to form a second purified sample.

In a most preferred aspect of the present invention, the second purified sample is subjected to hydrophobic interaction chromatography (HIC) in a flow-through mode to form a further purified sample.

In some embodiments of the present invention, the insubstantial portion of said amount of protein of interest that binds to said material is less than about 20%, preferably less than about 15%, more preferably less than about 10%, and most preferably less than about 5% of the total amount of the protein of interest in the sample.

In other embodiments, the present invention process includes at least two additional processing steps prior to step (b) above, such as (i) filtering the sample, and/or (ii) removing cells from the sample by sedimentation, flocculation, enhanced cell settling, and/or centrifugation, and/or (iii) precipitating the protein of interest with polyethylene glycol (PEG). In further embodiments, the process includes inactivating a virus present in the sample or a portion thereof, as well as filtering the sample or a portion thereof to remove viruses.

In some embodiments of the present invention, at least 50%, preferably at least 60%, more preferably at least about 75%, and most preferably at least about 90% of the protein of interest originally present in the sample is collected at step (c).

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below.

Description Of Drawings

FIG. 1 is a schematic diagram of an exemplary purification process including cation exchange, anion exchange, and hydrophobic interaction steps in flow-through mode. FIG. 2 A is a contour plot showing the effects of solution pH and conductivity on antibody yield for the Natrix Adsept™ S CEX membrane operated in flow-through mode.

FIG. 2B is a contour plot showing the effects of solution pH and conductivity on HCP reduction for the Natrix Adsept™ S CEX membrane operated in flow-through mode.

FIG. 3 A is a contour plot showing the effects of solution pH and conductivity on antibody yield for the Natrix Adsept™ C CEX membrane operated in flow-through mode.

FIG. 3B is a contour plot showing the effects of solution pH and conductivity on HCP reduction for the Natrix Adsept™ C CEX membrane operated in flow-through mode.

FIG. 3C is a contour plot showing the effects of solution pH and conductivity on aggregate reduction for the Natrix Adsept™ C CEX membrane operated in flow-through mode.

FIG. 4 A is a contour plot showing the effects of solution pH and conductivity on antibody yield for the Sartobind S CEX membrane operated in flow-through mode.

FIG. 4B is a contour plot showing the effects of solution pH and conductivity on HCP reduction for the Sartobind S CEX membrane operated in flow-through mode.

FIG. 4C is a contour plot showing the effects of solution pH and conductivity on aggregate reduction for the Sartobind S CEX membrane operated in flow-through mode.

FIG. 5 shows the effluent levels of aggregates as a function of antibody loading when the Sartobind Phenyl membrane adsorber is loaded with antibody at 0.75 M ammonium sulfate.

FIG. 6 shows the effluent levels of aggregates as a function of antibody loading when the Sartobind Phenyl membrane adsorber is loaded with antibody at 0.85 M ammonium sulfate. Detailed Description

Definitions

The following definitions are used throughout the specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

"Anion exchange material" means a solid phase that is positively charged, for example, from a solid phase having one or more positively charged ligands attached thereto, and which has free anions for exchange with anions in an aqueous solution passed over or through the solid phase. Any positively charged ligand can be used to attach to a solid phase, such as quaternary amino groups, including for example a quaternary amine, such as quaternary alkylamine and quaternary alkylalkanol amine, or amine, diethylamine, diethylaminoethyl (DEAE) diethylaminopropyl, amino, timethylammoniumethyl, trimethylbenzyl ammonium, dimethylethanolbenzyl ammonium, and polyamine. The anion exchange material may be in the form of a resin or alternatively, a membrane.

"Antibody" means monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they retain, or are modified to comprise, a ligand- or antigen-specific binding domain. Antibodies and fragments thereof can categorized by class, e.g., IgG (e.g., Igd, IgG₂, IgG₃, IgG₄), IgA (e.g., IgA_h IgA₂), IgM, IgD, and IgE.

"Antibody fragment" means 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.

"Cation exchange material" means a solid phase that is negatively charged, and , for example, from a solid material having one or more negatively charged ligands attached thereto, and which has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. Any negatively charged ligand attached to the solid phase suitable to form the cation exchange sorbent can be used, e.g., a carboxylate, sulfonate and others as described below. The cation exchange material may be in the form of a resin or alternatively, a membrane.

"Chromatography" means the process by which a solute of interest in a mixture is separated from other solutes in said 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. Chromatography separates a solute of interest, e.g., a protein of interest, in a mixture from other solutes in the mixture by percolation of the mixture through an resin or membrane, which adsorbs or retains, under particular buffering conditions, a solute more or less strongly due to properties of the solute, such as the pi, hydrophobicity, size and structure, of the solute of interest. Chromatography includes column- and membrane-type chromatography.

"Clarification" means the method of separating a portion of the solid material, such as cells and cell debris, from an aqueous mixture containing said solid material.

"Conductivity" means the ability of an aqueous solution to conduct an electric current between two electrodes, which is measured in milliSiemens per centimeter (mS/cm), using a conductivity meter, for example sold by Orion. Conductivity is a measure of ion transport; therefore, an increasing concentration of ions in an aqueous solution, will increase the solution's conductivity. 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 KC1) in the solution may be changed to achieve a desired conductivity.

"Hydrophobic interaction" and "hydrophobic interaction chromatography" means a process in which a solute with hydrophobic surfaces (e.g., a protein of interest in a mixture or a protein aggregate in a mixture) interacts with a hydrophobic ligand (typically aliphatic or aromatic groups) in the presence of an appropriate amount of a lyotropic salt such that the solute of interest interacts non-specifically with the hydrophobic ligand more or less than other solutes in the mixture. The contaminating solutes in the mixture may be washed from a column of the hydrophobic interaction material, or be bound to or excluded from the hydrophobic interaction material, with different affinities than the solute of interest. Lyotropic salts are characterized by their ability to "salt out," or precipitate, proteins from aqueous solutions by promoting hydrophobic interactions. They are ranked in their ability to induce precipitation according to the Hofmeister series (F.Hofmeister Arch. Exp. Pathol. Pharmacol. 24, (1888) 247-260.). Typical lyotropic salts used in HIC are ammonium sulfate, sodium sulfate, sodium citrate, and potassium or sodium phosphate. Commonly used hydrophobic interaction materials include "HighSub" and "LowSub" Phenyl Sepharose and Butyl Sepharose from GE Healthcare, Toyopearl^® PPG-600M and Toyopearl^® Butyl- 650 among other HIC resins from Tosoh Bioscience.

A "homogeneous" composition, means a composition comprising the protein of interest and less than 1000 ppm impurities (e.g., HCP), alternatively less than 900 ppm, less than 800 ppm, less than 700 ppm, less than 600 ppm, less than 500 ppm, less than 400 ppm, less than 350 ppm, less than 300 ppm, less than 250 ppm, less than 200 ppm, less than 150 ppm, less than 100 ppm, less than 90 ppm, less than 80 ppm, less than 70 ppm, less than 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm, or less than 3 ppm.

"Host cell protein" or "HCP" means any of the proteins derived from the metabolism (intra and extra-cellular) of a host cell that expresses the protein of interest, including any proteins expressed from the genome of the host cell.

"Impurity" and "contaminant," and grammatical variations thereof, are used interchangeably, and mean any material, other than the protein of interest. Exemplary contaminants include biological macromolecules such as HCPs, polypeptides other than the protein of interest, nucleic acids (e.g., DNA and RNA), lipids, saccharides, endotoxins, microorganisms such as bacteria, yeast, media components, and any molecule leached from an adsorbent used in chromatography.

"Ion-exchange" and "ion-exchange chromatography" means a chromatographic process, such as cation exchange, anion exchange, and mixed mode chromatographies, in which an ionizable solute of interest (e.g., a protein of interest in a mixture) interacts with an oppositely charged ligand in an ion exchange material under appropriate conditions of pH and conductivity, such that the solute of interest interacts non-specifically with the charged ligand more or less than the impurities in the mixture. The contaminating solutes in the mixture may be washed from a column of the ion exchange material, or be bound to or excluded from the ion exchange material, with different affinities than the solute of interest.

"Ion exchange material" means a solid phase that is negatively charged (i.e., cation exchange material) or positively charged (i.e., anion exchange material). The negative or positive charge may be provided by covalently linking one or more charged ligands (or adsorbents) to the solid phase, or may be an inherent property of the solid phase (e.g., silica, which has an overall negative charge).

"Isolated," "purified", or "substantially pure" are used herein interchangeably, and means that the protein of interest has been enriched or separated from impurities in the mixture in which the protein was present before being subjected to purification, and in some contexts, may also mean that the protein of interest is the predominant macromolecule present (i.e., on a mass basis it is more abundant than any other individual molecules in a composition), and in some instances comprises at least about 50 percent (on a mass basis) of all macromolecules present. Thus, after being subjected to purification, a protein of interest is "isolated" notwithstanding that it may be present together with other molecules or other cellular components.

"Mixture" means a protein of interest (for which purification is desired) in combination with one or more contaminant. Preferred mixtures are obtained directly from a host cell or organism producing the protein of interest. Without intending to be limiting, examples of mixtures include cell culture fluid, cell culture supernatant and conditioned cell culture supernatant.

"Monoclonal antibody" means an antibody that is highly specific, being directed against a single antigenic site. Monoclonal antibodies may be 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. 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 ah, 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 ah, Nature 352:624-628 (1991) and Marks et ah, J. Mol. Biol. 222:581-597 (1991), for example.

"Parts per million" and "ppm" mean a measure of purity of the protein of interest. The units ppm refer to the amount of impurity, e.g., HCP, in nanograms/ milliliter per protein of interest in milligrams/milliliter (i.e., HCP ppm = (HCP ng/ml) / (protein of interest mg/ml), where the proteins are in solution). Where proteins are dried (such as by lyophilization), ppm can refer to (HCP ng)/(protein of interest mg)).

"pi" or "isoelectric point" of a protein means the pH at which the positive charge of the protein 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 protein or can be determined experimentally, e.g., by isoelectric focusing.

"Protein" means a polypeptide having at least 5 amino acids which are linked together by peptide bonds, and is preferably a complex polypeptide. Exemplary proteins are antibodies and fragments thereof.

"Protein of interest" and "target protein" are used interchangeably and mean a protein such as an antibody (as defined herein) that is to be purified by a method of the invention from a mixture.

A purification "step" means a discrete part of an overall purification process.

"Purifying" means increasing the degree of purity and reducing the amounts of at least one impurity.

Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples are illustrative only and not intended to be limiting. Process Overview

The processes described herein employ various purification steps, including one or more of cation exchange chromatography, anion exchange chromatography, mixed mode chromatography, (ceramic) hydroxyapatite chromatography, and hydrophobic interaction chromatography.

Skilled practitioners will appreciate that the separation of a mixture for protein purification from cellular debris initially depends on the manner of expression of the protein, and that processes described herein do not rely on any particular methodology for removing cellular debris. For intracellular proteins, the cell can be disrupted using any method known in the art, such as mechanical shear and/or osmotic shock followed by centrifugation and/or filtration. For proteins secreted into the medium, the recombinant host cells may be separated from the cell culture medium by, e.g., tangential flow filtration (TFF), centrifugation, sedimentation, enhanced cell settling, and/or depth filtration.

Once a mixture containing the protein of interest has been obtained, its separation from contaminants in the mixture is performed using a variety of processing steps (e.g., flow-through steps). In some embodiments, one or more of the processing steps utilize ion exchange chromatography. In ion exchange chromatography the interaction between the solute of interest and the solid phase (ion exchange material) is based on electrostatics, i.e., the net surface charge on the solute and the charge of the ligand chemistry. The surface charge of the protein is determined by the electrostatic contribution of positive and egative ioiiogenic groups on the surface of the solute. The overall charge of a biological substance is based on the p a of the acidic and basic residues and the pH of the solution. For example, for the solute to have a net positive charge in the sample, the pi (the pH where the net. charge Is zero) of the substance must be higher than the pH of the medium.

The buffering systems chosen for a given ion exchange membrane can be adjusted in terms of pH and conductivity to alter the net surface charge of the protein of interest, allowi g the above described process to be adapted to different proteins.

In the exemplary method shown in FIG, 1, cell culture fluid from a fed-batch or XD^® (registered trademark of DSM N.V.) reactor is clarified by sedimentation or enhanced cell settling followed by depth filtration. The clarified media is titrated close to the pi of the protein of interest (e.g., an antibody) and PEG is added to cause the protein to precipitate. The precipitate is concentrated and washed by microfiltration to remove soluble impurities and then redissolved in a suitable buffer that facilitates the subsequent purification step. This material is then passed through one or more chromatography steps (e.g., cation exchange followed by anion exchange and hydrophobic interaction chromatography) in which a substantial fraction (e.g., > 90%) of the protein does not bind to the chromatography medium. Viral inactivation and filtration may also be performed in this sequence of operations. Finally the product is concentrated by ultrafiltration and formulated into an appropriate buffer by diafiltration.

Once a clarified sample has been obtained, the subsequent processing steps can typically be performed in any order. For example, a cation exchange step can be followed by an anion exchange step and a hydrophobic interaction step. In another example, an anion exchange step is followed by a cation exchange step and a hydrophobic interaction step. Typically, the effluent or resulting product from one purification step is used as the input for the next processing step. In some cases, one or more properties of the effluent or resulting product (e.g., pH, conductivity, ionic strength, sample concentration) can be modified prior to the subsequent processing step. Samples for Purification

In some embodiments, the protein of interest can be produced or expressed by living host cells that have been genetically engineered to produce the protein. Methods of genetically engineering cells to produce proteins are well known in the art. See e.g. Ausubel et ah, eds. (1990), Current Protocols in Molecular Biology (Wiley, New York) and U.S. Pat. Nos. 5,534,615 and 4,816,567, each of which is specifically incorporated herein by reference. Such methods include introducing nucleic acids that encode and allow expression of the protein into living host cells. The nucleic acids can be expressed stably or transiently, as disclosed, e.g., in U.S. Pat. No. 7,604,960. These host cells can be bacterial cells, fungal cells, or, preferably, animal cells grown in culture. In some embodiments, the cells are cultured using a high density culture method, e.g., as described in WO 2008/006494 and U.S. Patent No. 7,291,484. Bacterial host cells include, but are not limited to E. coli cells. Examples of suitable E. coli strains include: HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that fails to cleave foreign DNA. Fungal host cells that can be used include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus cells. A few examples of animal cell lines that can be used are CHO, VERO, DXB1 1, BHK, HeLa, Cos, MDCK, HEK293, 3T3, NSO ,PER.C6^®, and WI 138. In particular embodiments, the cells are PER.C6^® cells or El -immortalized HER (human embryonic retina) cells (US Patent No. 5,994,128). New animal cell lines can be established using methods well know by those skilled in the art (e.g., by transformation, viral infection, and/or selection). In particular embodiments, the protein of interest is produced in a CHO cell (see, e.g., WO 94/11026). Various types of CHO cells are known in the art, e.g., CHO- Kl, CHO-DG44, CHO-DXB1 1, CHO/dhfr and CHO-S. A host cell that has been engineered with nucleic acid encoding the protein of interest can be cultured under conditions well known in the art that allow expression of the protein.

Methods of using immortalized cells to produce recombinant proteins are described, e.g., in US Patents Nos. 6,855,544; 7,132,280; 7,470,523; and 7,491,532.

In some embodiments, the sample containing the protein of interest can be a plasma fraction, milk (e.g., containing a transgenically or transiently expressed protein (see Baldassarre et al., 2004, Reprod. Fertil. Dev., 16:465-470), plant extract, or cell (e.g., microbial lysate).

Clarification

A sample comprising a protein of interest (e.g., a cell culture or broth) can be clarified prior to subjecting the sample to one or more purification steps. Clarification, as used herein, refers to methods of separating at least a portion of the solid material, such as cells and cell debris, present in a fluid sample from the remaining fluid. When the sample is a cell culture, the cell culture can have a density of greater than 1 million cells/ml, e.g., greater than 5 million, 10 million, 15 million, 20 million, 25 million, 50 million, 75 million, 100 million, or 150 million cells/ml. Exemplary clarification methods include sedimentation, centrifugation, filtration (e.g., depth filtration, microfiltration, tangential flow filtration, and filtration through absolute pore size membranes), expanded bed chromatography, and hydrocyclonic methods (see Elsayed et ah, 2006, Eng. Life Sci., 6: 347-354). In some embodiments, a combination of clarification methods can be used in series to clarify the sample. Methods of clarification are reviewed in Roush et ah, 2008, Biotechnoh Prog., 24:488-495.

In some embodiments, the sample is clarified by one or more gravitational sedimentation or centrifugation steps. In these methods, the sample is incubated for a period of time sufficient for at least a portion of the solid material to settle and separate from the fluid sample. Centrifugation methods can also be used, wherein the effective gravitation force on the sample is increased by rapidly rotating the sample. Centrifugation can decrease the time required for solid material to separate from a sample as compared to standard sedimentation.

In some embodiments, the sample is clarified by one or more enhanced cell settling steps. Enhanced cell settling utilizes ion exchange matrices to induce and enhance the settling of cells in situ. Exemplary ionic exchange matrices that can be used in enhanced cell settling methods include Bakerbond wide-pore polyethylenimine, BAKERBOND SiPEI (15 Dm) (JT Baker); DEAE HyperD, CM HyperD, and HyperZ (Pall); Streamline DEAE (GE Healthcare), TP SuperQ-650M, TP DEAE-650M, TP SuperQ-650S, and TP DEAE-650S (Tosoh). Methods of performing enhanced cell settling are described in Schirmer et ah, 2010, BioProcess Int., 8:32-39; WO 2010/043700; WO 2010/043701; and WO 2010/043703, all of which are incorporated by reference herein.

In some embodiments, the sample is clarified by one or more filtration steps, e.g. depth filtration, microfiltration, tangential flow filtration, and filtration through absolute pore size membranes, all of which are known to one of ordinary skill in the art.

Depth filters contain filtration media having a graded density. Such graded density allows larger particles to be trapped near the surface of the filter while smaller particles penetrate the larger open areas at the surface of the filter, only to be trapped in the smaller openings nearer to the center of the filter. Non-limiting examples of depth filters that can be used in the context of the methods described herein include the Cuno depth filters (e.g., models 30/60ZA, 10M02, and 60ZA05A) (3M Corp.), SartoclearP (Sartorius), SUPRACap (Pall), and Millistak+ HC (Millipore) filters. Microfiltration separates solid material by use of a microporous membrane. Typically, microfiltration membranes have pore sizes ranging 0.1 to 10 μιη, although membranes with pores of other sizes can be used.

Tangential flow filtration differs from other filtration methods in that the majority of the feed flow travels tangentially across the surface of the filter, rather than into the filter.

In some embodiments, an absolute pore size membrane can be used for filtration. Such membranes are typically made of a solid film, e.g., polycarbonate, with cylindrical pores etched through the membrane. Exemplary membranes include GE PCTE (polycarbonate) membranes (GE Healthcare).

In some embodiments, flocculation can be used in combination with other clarification procedures. Common flocculation agents include electrolytes, polyionic polymers (such as DEAE dextran, acryl-based polymers, polyethylenimine, and polyethylene amine), other polymers (see US 2009/0232737, US 2009/0036651, US 2008/0255027), chitosan, and inorganic materials such as calcium phosphate, diatomaceous earth, and perlites. Additionally, cells can be flocculated by treatment with low pH (e.g., < 4.0).

Precipitation

In some embodiments, a sample can be subjected to one or more precipitation steps to remove impurities. To perform the precipitation a solute, e.g., polyethylene glycol (see Kuczewski et al., BioPharm International Supplements, March 2010), polyvinyl sulfonate (see McDonald et al., 2009, Biotechnol. Bioeng. ,\02: \ 141-51), ammonium sulfate, short-chain fatty acids (e.g., caprylate) (see Habeeb et al., 1984, Prep. Biochem., 14:1-17), or other polymer (see US 2009/0232737, US 2009/0036651, US 2008/0255027), is added to a solution containing the protein of interest to form a precipitate. In some embodiments, the precipitate contains the majority of the protein of interest and is collected, e.g., by filtration or other means, for re-solubilization and further processing. In some embodiments, the precipitate contains a portion of the impurities present in the sample, whereas a majority of the protein of interest remains in solution. The precipitate can then be removed form the sample, e.g., by filtration or other means. Cation Exchange Chromatography

A sample comprising a protein of interest can be subjected to one or more cation exchange purification steps. At least one of the cation exchange purification steps can be run in flow-through mode under conditions in which the protein of interest is capable of binding to the medium (e.g., the protein of interest has a net positive charge) but the total binding capacity of the medium for the protein of interest is exceeded. In some embodiments, the cation exchange step is run using conditions under which the protein of interest is capable of binding to the medium but the total binding capacity of the medium is exceeded, such that a substantial fraction of the protein of interest originally present in the sample (e.g., at least 75%, 80%, 85%, 90%, 95%) flows through the medium. Typically, in these conditions the pH of the buffer is less than the pi of the protein (e.g., by at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, or 3.5 pH units). In some embodiments, the conductivity of the buffer is from 2-10 mS/cm (e.g., from 3-9 mS/cm, 4-8 mS/cm or 5-7 mS/cm). In some embodiments, the protein of interest comprises at least 95% (e.g., at least 96%>, 97%>, 98%>, 99%>, or 99.5%>) of the total protein present in the sample applied to the cation exchange medium. In some embodiments, the sample applied to the cation exchange medium comprises at most 200,000 ppm (e.g., at most 150,000 ppm, 100,000 ppm, 80,000 ppm, 60,000 ppm, 50,000 ppm, 40,000 ppm, 30,000 ppm, 20,000 ppm, 15,000 ppm, 10,000 ppm, 9,000 ppm, 8,000 ppm, 7,000 ppm, 6,000 ppm, 5,500 ppm, or 5,000 ppm) HCPs relative to the protein of interest.

Commercially available cation exchange materials include, but are not limited to, for example, those having a sulfonate based group (e.g., MonoS, MiniS, Source 15S and 30S, SP Sepharose Fast Flow™, SP Sepharose High Performance from GE Healthcare, Toyopearl^® SP-650S and SP-650M from Tosoh, Macro-Prep High S from BioRad, Ceramic HyperD S, Trisacryl M and LS SP and Spherodex LS SP from Pall Technologies); a sulfoethyl based group (e.g., Fractogel EMD SE from EMD, Poros S-10 and S-20 from Applied Biosystems); a sulphopropyl based group (e.g., TSK Gel SP 5PW and SP-5P W-HR from Tosoh, Poros HS-20 and HS 50 from Applied Biosystems); a sulfoisobutyl based group (e.g., Fractogel^® EMD S0₃ from EMD); a sulfoxyethyl based group (e.g., SE52, SE53 and Express-Ion S from Whatman), a carboxymethyl based group (e.g., CM Sepharose Fast Flow from GE Healthcare, Hydrocell CM from Biochrom Labs Inc., Macro-Prep CM from BioRad, Ceramic HyperD CM, Trisacryl M CM, Trisacryl LS CM, from Pall Technologies, Matrex Cellufme C500 and C200 from Millipore, CM52, CM32, CM23 and Express - Ion C from Whatman, Toyopearl^® CM- 650S, CM-650M and CM-650C from Tosoh); sulfonic and carboxylic acid based groups (e.g. BAKERBOND Carboxy-Sulfon from J.T. Baker); a carboxylic acid based group (e.g., WP CBX from J.T Baker, DOWEX MAC-3 from Dow Liquid Separations, Amberlite Weak Cation Exchangers, DOWEX Weak Cation Exchanger, and Diaion Weak Cation Exchangers from Sigma- Aldrich and Fractogel EMD COO- from EMD); a sulfonic acid based group (e. g., Hydrocell SP from Biochrom Labs Inc., DOWEX Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations, UNOsphere S from BioRad, WP Sulfonic from J. T. Baker, Amberlite Strong Cation Exchangers, DOWEX Strong Cation and Diaion Strong Cation Exchanger from Sigma- Aldrich); and a orthophosphate based group (e.g., PI 1 from Whatman). Commercially available high- capacity resins include GigaCap S-650M (Tosoh), Eshmuno™ S (EMD), Nuvia™ S (BioRad), Poros^® XS (Applied Biosystems), and Capto S (GE Healthcare). In some embodiments, a cation exchange membrane can be used, e.g., Sartobind S (Sartorius; Edgewood, NY), Natrix Adsept^™ S (also referred to as "Natrix S") and Natri Adsept™ C (also referred to as "Natrix C", and Mustang S (Pall).

Anion Exchange Chromatography

A sample comprising a protein of interest can be subjected to one or more anion exchange purification steps. At least one of the anion exchange purification steps can be run in flow-through mode under conditions in which the protein of interest does not bind to the medium (e.g., the protein of interest has a net positive charge).

Commercially available anion exchange materials include, but are not limited to, DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose High Performance, QAE SEPHADEX™ and FAST Q SEPHAROSE™ from GE Healthcare, WP PEI, WP DEAM, WP QUAT from J.T. Baker, Hydrocell DEAE and Hydrocell QA from Biochrom Labs Inc., UNOsphere Q, Macro- Prep DEAE and Macro-Prep High Q from BioRad, Ceramic HyperD Q, Ceramic HyperD DEAE, Q HyperZ, Trisacryl M and LS DEAE, Spherodex LS DEAE, QMA Spherosil LS, QMA Spherosil M from Pall Technologies, DOWEX Fine Mesh Strong Base Type I and Type π Anion Resins and DOWEX MONOSPHERE 77, weak base anion from Dow Liquid Separations, Matrex Cellufme A200, A500, Q500, and Q800, from Millipore, Fractogel^® EMD TMAE, Fractogel^® EMD DEAE, and Fractogel^® EMD DMAE from EMD, Amberlite weak and strong anion exchangers type I and II, DOWEX weak and strong anion exchangers type I and II, Diaion weak and strong anion exchangers type I and II, Duolite from Sigma- Aldrich, TSK gel Q and DEAE 5PW and 5PW-HR, Toyopearl^® SuperQ-650S, 650M and 650C3 QAE-550C and 650S, DEAE- 650M and 650C from Tosoh, and QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D and Express-Ion Q from Whatman. Commercially available high-capacity resins include GigaCap Q-650M (Tosoh), Capto Q (GE Healthcare), Eshmuno™ Q from EMD, and Nuvia™ Q from Bio-rad.

In some embodiments, an anion exchange membrane can be used. Commercially available anion exchange membranes include, but are not limited to, Sartobind Q and Sartobind STIC from Sartorius, Mustang Q from Pall Technologies, ChromaSorb membrane from Millipore, and Adsept™ Q f rom Natrix. Hydrophobic Interaction Chromatography

A sample comprising a protein of interest can be subjected to one or more hydrophobic interaction separation steps (see Kuczewski et ah, 2010, Biotechnol Bioeng., 105:296-305; Fraud et ah, BioPharm International Supplements, October 2, 2009) Hydrophobic interaction chromatography steps, such as those disclosed herein, can be performed to remove protein aggregates, such as antibody aggregates, and process-related impurities. Samples containing the protein of interest and that are appropriate for use in this process may contain amounts of aggregate impurities from about 1% to about 10%, and as high as about 20%, and of host cell protein impurities from about 10 ppm, more preferably about 100 ppm, to about 1000 ppm. It is understood that the actual amounts of impurities in the samples will depend on the proteins of interest, and the purification processes to which the samples are subjected prior to application of the flow through hydrophobic interaction chromatography process.

In performing the separation, the sample mixture is contacted with the HIC material, e.g., using a batch purification technique or using a column. Whereas ion exchange chromatography relies on the charges of the proteins to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of the proteins. Hydrophobic groups on the protein interact with hydrophobic groups on the column. The more hydrophobic a protein is the stronger it will interact with the column. Thus the HIC step removes host cell derived impurities (e.g., DNA, HCP, and other high and low molecular weight product-related species).

Adsorption of the protein to a HIC material is favored by high lyotropic salt concentrations, since hydrophobic interactions are strongest in the presence of high levels of lyotropic salts, but the actual concentrations can vary over a wide range depending on the nature of the protein and the particular HIC ligand chosen. Exemplary lyotropic salt concentrations may be from 0.7M, preferably 0.75M, more preferably about 0.8M, and most preferably about 0.85M. Therefore, high concentration of lyotropic salt in the sample is required to maximize protein binding. As such, this form of separation may be conveniently performed following salt precipitations and/or ion exchange procedures.

As in the case of cation-exchange flow-through chromatography with strong product-binding conditions, hydrophobic interaction chromatography of a sample may be performed where the lyotropic salt concentration of the sample is selected to maximize product binding to the hydrophobic interaction material, provided that the mass loading of the protein of interest on the material is selected to be high enough to saturate, or exceed, the capacity of the material for the protein of interest. Under these conditions, the HIC material has been found to still bind impurities such as aggregates, and a highly purified sample is obtained in the flow-through and wash fractions while impurities remain bound to the HIC material.

HIC materials typically comprise a base matrix (e.g., cross-linked agarose or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. Many HIC materials are available commercially. Examples include, but are not limited to, Phenyl Sepharose 6 Fast Flow column with low or high substitution (GE Healthcare); Phenyl Sepharose High Performance column (GE Healthcare); Octyl Sepharose High Performance column (GE Healthcare); Fractogel^® EMD Propyl or Fractogel^® EMD Phenyl columns (EMD, Germany); Macro-Prep Methyl or Macro-Prep t-Butyl Supports (BioRad, California); WP Hl-Propyl (C₃) column (J. T. Baker, New Jersey); and Toyopearl^® ether, PPG, phenyl or butyl columns (Tosoh, PA). In some embodiments, a hydrophobic interaction membrane can be used as a HIC material. Commercially available hydrophobic interaction membranes include, but are not limited to, Sartobind Phenyl from Sartorius.

Virus Neutralization

If a therapeutic grade protein formulation is desirable, then a viral clearance step is employed, e.g., a virus filtration step, which is, however, not required to achieve the levels of purity attainable by a method of the invention. Filtration devices useful in viral clearance are well-known in the art (e.g., Ultipor® VF Grade DV20 or DV50 and Filtron® TFF (Pall Corporation, East Hills, NY); Viresolve 180, Viresolve NFP, Viresolve NFR, and Viresolve Pro/Pro+ (Millipore, Billerica, MA); and Planova® (Asahi Kasei Pharma, Planova Division, Buffalo Grove, IL). To remove viruses and other biological materials, pore sizes smaller than 20 nm, which can remove polioviruses, are typically used. Viral filtration can be included at any point in the process, but is it typically performed once the product has been purified and the processing volume has been minimized.

The methods can also include one or more virus inactivation treatments, e.g., pH inactivation, solvent/detergent inactivation, or UV inactivation. Methods of pH viral inactivation include, but are not limited to, incubating the mixture for a period of time at low pH, and subsequently neutralizing the pH and removing particulates by filtration. In certain embodiments the mixture will be incubated at a pH of 2 to 5, preferably at a pH of 3 to 4, and more preferably at a pH of 3.5. The pH of the sample mixture may be lowered by any suitable acid including, but not limited to, citric acid, acetic acid, caprylic acid, or other suitable acids. The choice of pH level largely depends on the stability profile of the antibody product and buffer components. It is known that the quality of the target antibody during low pH virus inactivation can be affected by pH and the duration of the low pH incubation. In certain embodiments the duration of the low pH incubation will be from 0.5 hours to 2 hours, preferably 0.5 hours to 1.5 hours, and more preferably the duration will be 0.5 hour. Virus inactivation is dependent on these same parameters in addition to protein concentration, which may reduce inactivation at high concentrations. Thus, the proper parameters of protein concentration, pH, and duration of inactivation can be selected to achieve the desired level of viral inactivation.

In solvent/detergent inactivation methods, organic solvent/ detergent mixtures can disrupt the lipid membrane of enveloped viruses. Exemplary conditions that can be used are 0.3% tri(n-butyl) phosphate (TNBP) and 1% nonionic detergent, e.g., Tween 80 or Triton X-100, at 24 °C. for a minimum of 4 hours with Triton X-100 or 6 hours with Tween 80.

Viruses can also be inactivated by UV treatment using commercially available systems, e.g., UVivatec® Lab System (Sartorius Stedim Biotech). Ultrafiltration/Diafiltration

A sample purified or isolated by the methods described herein can be concentrated or further purified by ultrafiltration and/or diafiltration. Protein ultrafiltration is a pressure-driven membrane process used for the concentration or purification of protein solutions (Robert van Reis and Andrew L. Zydney, "Protein Ultrafiltration" in Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, M. C. Flickinger and S. W. Drew, eds., John Wiley & Sons, Inc. (1999), p. 2197). UF membranes typically have a mean pore size between 10 and 500 Angstroms, which is between the mean pore size of reverse osmosis and microfiltration membranes. Ultrafiltration separates solutes based on differences in the rate of filtration of different components across the membrane in response to a given pressure driving force (R. van Reis and A. L. Zydney, supra, p. 2197). Solute filtration rates, and thus membrane selectivity, are determined by both thermodynamic and hydrodynamic interactions (R. van Reis and A. L. Zydney, supra, p. 2197). Ultrafiltration is frequently used in downstream processing for protein concentration, buffer exchange and desalting, protein purification, virus clearance, and clarification (R. van Reis and A. L. Zydney, supra, p. 2197). Diafiltration is a method of using ultrafilters to remove and exchange salts, sugars, and non-aqueous solvents, to separate free from bound species, to remove low molecular- weight material, and/or to cause the rapid change of ionic and/or pH environments. Microsolutes are removed most efficiently by adding solvent to the solution being ultrafiltered at a rate approximately equal to the ultrafiltration rate. This washes microspecies from the solution at a constant volume, effectively purifying the retained antibody. In certain embodiments of the present invention, a diafiltration step is employed to exchange the various buffers used in connection with the instant invention, optionally prior to further chromatography or other purification steps, as well as to remove impurities from the antibody preparations.

Analysis of Purified Sample

The present invention also provides methods for determining the residual levels of impurities (e.g., HCPs) in an isolated/purified protein composition. As described above, impurities are desirably excluded from the final product. Exemplary impurities include proteins originating from the source of the protein production. Failure to identify and sufficiently remove HCPs from the target antibody may lead to reduced efficacy and/or adverse subject reactions.

The presence and quantity of impurities and/or aggregates can be assayed by means known in the art, e.g., size exclusion chromatography, ELISA, reducing and non- reducing SDS-PAGE, isoelectric focusing (IEF), reverse phase chromatography (e.g., RP-HPLC), 2D gel electrophoresis, rtPCR, nucleic acid-binding dyes (e.g., pico green), dynamic light scattering, threshold^® (Molecular Dynamics), etc. ELISA kits specific for HCP of various host cells are commercially available, such as the PER.C6^® HCP ELISA kit (Cygnus Technologies). Additionally, the activity of an isolated/purified protein composition can be measured. Exemplary activities include binding activities of antibodies and other binding proteins and catalytic activity of purified enzymes.

The concentration of a protein in an isolated/purified protein composition can be measured or estimated using standard methods. Colorimetric assays (e.g., Lowry method, Bradford assay, bicinchoninic acid (BCA) assay) can be used to determine protein concentration by comparison to a control sample of known concentration. Additionally, protein concentration can be estimated by measuring absorbance at 280 nm (A₂8o) using an experimentally derived extinction coefficient for the protein of interest. For increased precision of protein concentration, absorbance at 320 nm (A320, corresponding to light scattering due to turbidity in the sample) can be subtracted from the A₂8o value.

Proteins of Interest

Any protein of interest may be purified using the methods described herein. In some embodiments, the protein of interest has a pi ranging from 4 to 10, e.g., from 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, or 6 to 8. In some embodiments, the protein of interest is between 20 and 1,000 kDa in size, e.g., between 30 and 800 kDa, between 40 and 600 kDa, between 50 and 500 kDa, between 60 and 400 kDa, between 80 and 300 kDa, or between 100 and 200 kDa. Antibodies

Exemplary proteins of interest are antibodies. Antibodies within the scope of the present invention include, but are not limited to: anti-HER2 antibodies including Trastuzumab (HERCEPTIN^®) (Carter et al, Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992), U.S. Pat. No. 5,725,856); anti-CD20 antibodies such as chimeric anti-CD20 "C2B8" as in U.S. Pat. No. 5,736,137 (RITUXAN^®), a chimeric or humanized variant of the 2H7 antibody as in U.S. Pat. No. 5,721,108 or Tositumomab (BEXXAR^®); anti-IL-8 (St John et al, Chest, 103:932 (1993), and International Publication No. WO 95/23865); anti-VEGF antibodies including humanized and/or affinity matured anti-VEGF antibodies such as the humanized anti-VEGF antibody huA4.6.1 AVASTIN® (Kim et al, Growth Factors, 7:53-64 (1992), International Publication No. WO 96/30046, and WO 98/45331,); anti-PSCA antibodies (WO 01/40309); anti-CD40 antibodies, including S2C6 and humanized variants thereof (WO 00/75348); anti-CDl la (U.S. Pat. No. 5,622,700, WO 98/23761, Steppe et al, Transplant Intl. 4:3-7 (1991), and Hourmant et al, Transplantation 58:377-380 (1994)); anti-IgE (Presta et al, J. Immunol. 151 :2623- 2632 (1993), and International Publication No. WO 95/19181); anti-CD18 (U.S. Pat. No. 5,622,700, issued Apr. 22, 1997, or as in WO 97/26912); anti-IgE (including E25, E26 and E27; U.S. Pat. No. 5,714,338or U.S. Pat. No. 5,091,313, WO 93/04173, or International Publication No. WO 99/01556, U.S. Pat. No. 5,714,338); anti-Apo-2 receptor antibody (WO 98/51793 published Nov. 19, 1998); anti-TNF-Π antibodies including cA2 (REMICADE^®), CDP571, adalimumab, and MAK-195 (See, U.S. Pat. No. 5,672,347, Lorenz et al. J. Immunol. 156: 1646-53 (1996), and Dhainaut et al. Crit. Care Med. 23: 1461-69 (1995)); anti-Tissue Factor (TF) (European Patent No. 0 420 937 Bl); anti-human

integrin (WO 98/06248); anti-EGFR (chimerized or humanized 225 antibody as in WO 96/40210); anti-CD3 antibodies such as OKT3 (U.S. Pat. No. 4,515,893); anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT^®) and (ZENAPAX^®) (See U.S. Pat. No. 5,693,762); anti-CD4 antibodies such as the cM-7412 antibody (Choy et al. Arthritis Rheum 39:52-56 (1996)); anti-CD52 antibodies such as CAMPATH-1H (Riechmann et al. Nature 332:323-337 (1988)); anti-Fc receptor antibodies such as the M22 antibody directed against FcgammaRI as in Graziano et al. J. Immunol. 155:4996-5002 (1995); anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkey et al. Cancer Res. 55(23Suppl): 5935s-5945s (1995); antibodies directed against breast epithelial cells including huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al. Cancer Res. 55(23): 5852s-5856s (1995); and Richman et al. Cancer Res. 55(23 Supp): 5916s-5920s (1995)); antibodies that bind to colon carcinoma cells such as C242 (Litton et al. Eur J. Immunol. 26: 1-9 (1996)); anti-CD38 antibodies, e.g. AT 13/5 (Ellis et al. J. Immunol. 155:925-937 (1995)); anti-CD33 antibodies such as Hu M195 (Jurcic et al. Cancer Res 55(23 Suppl):5908s-5910s (1995) and CMA-676 or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al. Cancer Res 55(23 Suppl):5899s- 5907s (1995)); anti-EpCAM antibodies such as 17-1A (PANOREX^®); anti-GpIIb/IIIa antibodies such as abciximab or c7E3 Fab (REOPRO.RTM.); anti-RSV antibodies such as MEDI-493 (SYNAGIS^®); anti-CMV antibodies such as PROTOVIR^®; anti-HIV antibodies such as PR0542; anti-hepatitis antibodies such as the anti-Hep B antibody OSTAVIR^®; anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitope antibody BEC2; anti-f|v...3 antibody VITAXIN^®; anti-human renal cell carcinoma antibody such as ch- G250; ING-1; anti-human 17-1 A antibody (3622 W94); anti-human colorectal tumor antibody (A33); anti-human melanoma antibody R24 directed against GD3 ganglioside; anti-human squamous-cell carcinoma (SF-25); and anti-human leukocyte antigen (HLA) antibodies such as Smart ID 10 and the anti-HLA DR antibody Oncolym (Lym-1). Exemplary target antigens for the antibody herein are: HER2 receptor, VEGF, IgE, CD20, CD 11a, and CD40.

Aside from the antibodies specifically identified above, the skilled practitioner could generate antibodies directed against an antigen of interest. Where the antigen is a protein, it may be a transmembrane molecule (e.g. receptor) or ligand such as a growth factor. Exemplary antigens include those proteins described in section (3) below. Exemplary molecular targets for antibodies encompassed by the present invention include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22, CD34, CD40; members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Macl, pl50,95, VLA-4, ICAM-1, VCAM and Hv/U3 integrin including either alpha or beta subunits thereof (e.g. anti-CD 11a, anti- CD 18 or anti-CD l ib antibodies); growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, or any of the other antigens mentioned herein. Antibodies directed against non-protein antigens (such as tumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) are also contemplated.

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).

A humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. Accordingly, such "humanized" antibodies can be 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.

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., J. Biochem. Biophys. Meth., 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 ah, 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 antibody fragment (scFv). See, e.g., WO 93/16185.

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

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.

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et ah J. Immunol. 147: 60 (1991).

Immunoadhesins

The simplest and most straightforward immunoadhesin design combines the binding domain(s) of the adhesin (e.g., the extracellular domain (ECD) of a receptor) with the hinge and Fc regions of an immunoglobulin heavy chain. Ordinarily, when preparing the immunoadhesins of the present invention, nucleic acid encoding the binding domain of the adhesin will be fused C-terminally to nucleic acid encoding the N- terminus of an immunoglobulin constant domain sequence, however N-terminal fusions are also possible. Typically, in such fusions the encoded chimeric polypeptide will retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CHI of the heavy chain or the corresponding region of the light chain. The precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion, or binding characteristics of the immunoadhesin.

In a preferred embodiment, the adhesin sequence is fused to the N-terminus of the Fc domain of immunoglobulin Gi (IgGi). It is possible to fuse the entire heavy chain constant region to the adhesin sequence. However, more preferably, a sequence beginning in the hinge region just upstream of the papain cleavage site, which defines IgG Fc chemically (i.e., residue 216, taking the first residue of heavy chain constant region to be 114), or analogous sites of other immunoglobulins is used in the fusion. In a particularly preferred embodiment, the adhesin amino acid sequence is fused to (a) the hinge region and CH2 and CH3 or (b) the CHI, hinge, CH2 and CH3 domains, of an IgG heavy chain.

Other CH2/CH3 region-containing proteins

In other embodiments, the protein to be purified is one which is fused to, or conjugated with, a CH2/CH3 region. Such fusion proteins may be produced so as to increase the serum half-life of the protein. Examples 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 Willebrand 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 A or 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-.beta.; 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-betal, TGF-beta2, TGF- beta3, TGF-beta4, or TGF-beta5; insulin-like growth factor-I and -II (IGF-I and IGF -II); des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., 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 AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins such as CD 11 a, CD l ib, CD 11c, CD 18, 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.

Examples

Example 1. Flow-through Purification - Sartobind membrane CEX Step

A monoclonal IgGi antibody (-150 kDa, pi = 8.5) is expressed using PER.C6^® cells grown in a fed-batch reactor using chemically defined media. The material is harvested by allowing the cells to settle out of suspension at room temperature for 2 hours. The supernatant is recovered and further processed at 50 liters per square meter per hour (LMH) through a filter train consisting of 0.55 m² of Millistak+HC D0HC media (Millipore, Billerica, MA) followed by 0.55 m² of Millistak+HC X0HC media (Millipore, Billerica, MA). The clarified media is then sterile filtered and stored at 4 °C for further processing.

The clarified media is titrated to pH 8.5 using 2 M Tris, and PEG-3350 is added to a final concentration of 14.4% (w/w), causing the antibody to precipitate. The precipitate is concentrated ten-fold using a hollow fiber microfiltration membrane, and then washed with three diavolumes of 20 mM Tris pH 8.5 + 15% (w/w) PEG-3350. The precipitate is then redissolved in 20 mM Tris pH 7.5 + 50 mM NaCl, sterile filtered, and stored at 4 °C for further processing.

The redissolved precipitate is titrated to pH 5.5 with 10% acetic acid and the conductivity is adjusted to 7.5 mS/cm with 25 mM 2-(N-morpholino)ethanesulfonic acid

(MES) pH 5.5 + 1 M NaCl. This material is passed through a Sartobind S CEX membrane to a loading of 600 mg-MAb/mL-membrane at a flow rate of 10 MV/min.

After loading, the membrane is washed with 25 mM MES + 66 mM NaCl buffer at pH

5.5, 7.5 mS/cm until the A₂so reached baseline levels. The combined flow-through and wash fractions ("CEX pool") are pooled together, sterile filtered, and stored at 4 °C for further processing.

The CEX pool is titrated to pH 7.5 with 2 M Tris and the conductivity is adjusted to 6.5 mS/cm. This material is passed through a ChromaSorb AEX membrane at 10 MV/min to a loading of -1.5 g-MAb/mL-membrane. The AEX pool is then titrated to pH 3.4 with 1 N HC1 and held at room temperature for 30 minutes to inactivate viruses before being neutralized to pH 7.0 with 2 M Tris.

The resulting material is blended in-line with a sodium phosphate/ammonium sulfate buffer to a final ammonium sulfate concentration of 0.4 M for loading onto a Sartobind Phenyl HIC membrane. Loading is performed at 3.3 MV/min to a total of -150 mg-MAb/mL-membrane. The adsorber is then washed with 50 mM phosphate + 0.4 M ammonium sulfate until the A₂so reached baseline, and the flow-through and wash fractions ("HIC pool") are pooled and sterile filtered.

Each process intermediate is assayed for product concentration by analytical protein A HPLC or A₂so/A₃₂o. Purity is measured by size exclusion chromatography HPLC and SDS PAGE. HCPs are quantified using a commercially available ELISA kit specific to PER.C6 HCPs (Cygnus Technologies). Product yield, percent aggregate, and HCP levels at each intermediate step are indicated in the table below.

Recovery HCP HCP Reduction [Aggregates, HMW] [Aggregates reduction]

Step

[%] [ppm] [%] [%] [%]

Clarified harvest 52,000

PEG/TFF 88% 5,040 90% 3.2%

CEX FT 95% 1, 150 77% 1.4% 56%

AEX FT 100% 273 76% 1.2% 13%

VI 96% 254 7% 1.2% 0.9%

HIC FT 82% 343 -35% 0.6% 54%

Final 66% 343 99.3% 0.6% 82%

As can be seen from the table, the cation exchange step in flow-through mode under binding conditions surprisingly resulted in significant purification, with 95% recovery, a 77% reduction in HCP, and a 56% reduction in aggregates. The total process provided substantial purification of the antibody, with 66% total recovery, a 99.3% reduction in HCP, and an 82% reduction in aggregates. The ability to achieve such high purity of the protein of interest at high efficiency, using a process wherein all chromatography steps are in flow-through mode, represents a paradigm shift in the field of industrial protein purification.

Example 2. Flow-through Purification - Natrix Membrane CEX Step w/o HIC Step

The same process as Example 1 is carried out, except that the CEX membrane employed is a Natrix Adsept™ Weak C membrane (Burlington, ON) (referred to herein also as "Natrix C") at a loading of 650 mg-MAb/mL-membrane, and the HIC step is omitted. Recovery HCP HCP Reduction

Step

[%] [ppm] [%]

Clarified harvest 52,000

PEG/TFF 88% 5,040 90%

CEX FT 78% 1 ,142 80%

AEX FT 108% 88 92%

VI 98% 82 8%

Final 73% 82 99.8%

As can be seen from the table, the cation exchange step in flow-through mode under binding conditions surprisingly resulted in significant purification, with 78% recovery and 80% reduction in HCP. The total process provided substantial purification of the antibody, with 73% total recovery and a 99.8% (2.8 log) reduction in HCP.

Example 3. Flow-through Purification - Higher Load CEX Step w/ HIC Step

A monoclonal IgGi antibody (-150 kDa, pi = 8.5) is expressed using PER.C6^® cells grown in a fed-batch reactor using chemically defined media. The material is harvested by allowing the cells to settle out of suspension at room temperature for 2 hours. The supernatant is recovered, and the cell pellet is resuspended in Tris buffered saline and allowed to settled again for 2 hours at room temperature. This second supernatant is also recovered, and both supematants are further processed at 100 liters per square meter per hour (LMH) through a filter train consisting of 0.054 m² of Millistak+HC D0HC media (Millipore, Billerica, MA) followed by 0.054 m² of Millistak+HC X0HC media (Millipore, Billerica, MA). The clarified media is then sterile filtered and stored at 4 °C for further processing.

The clarified media is titrated to pH 8.5 using 2 M Tris, and PEG-3350 is added to a final concentration of 15% (w/w), causing the antibody to precipitate. The precipitate is concentrated 30-fold using a hollow fiber micro filtration membrane, and then washed with 1.5 diavolumes of 20 mM Tris pH 8.5 + 15% (w/w) PEG-3350. The precipitate is then redissolved in 85 mM sodium acetate buffer pH 5.3, sterile filtered, and stored at 4 °C for further processing. The rest of the purification train is carried out as in Example 2, except that the loading on the CEX membrane is approximately 870 g-MAb/mL-membrane, and the HIC step is included.

Recovery HCP HCP Reduction Aggregates, HMW Aggregates reduction

Step

[%] [ppm] [%] [%] [%]

Unclarified media 194,000

Clarified harvest 89% 103,000 47% 0.2%

PEG/TFF 89% 6,770 93% 0.9% 21%

CEX FT 94% 187 97% 0.7%

AEX FT 101% 33 82% 0.8%

VI 100% 29 13% 0.9%

HIC FT 88% <29 0.2% 80%

Final 67% <29 >99.98% 0.2%

As can be seen from the table, the cation exchange step in flow-through mode under binding conditions surprisingly resulted in significant purification, with 94% recovery, a 97% reduction in HCP, and a 21% reduction in aggregates. The total process provided substantial purification of the antibody, with 67% total recovery, a 99.98%) (3.82 log) reduction in HCP, and a final aggregate content of 0.2%.

Example 4. Optimization of Conditions for Flow-through Purification of a Monoclonal Antibody by CEX Membrane Chromatography

A partially purified monoclonal IgGi antibody (-150 kDa, pi = 8.5) is purified by flow-through CEX chromatography using Natrix S, Natrix C, and Sartobind S membranes. Each membrane is tested at three pH values (4.5, 5.0, 5.5) and two conductivities (4 mS/cm, 8 mS/cm) for a total of six conditions for each membrane. In all of the experiments the membrane is equilibrated with 20 membrane volumes of 50 mM sodium acetate buffer + sodium chloride at the desired pH and conductivity. The membranes are then loaded with conditioned antibody containing media. 1 g antibody per 1 mL of membrane volume is loaded, and the flow-through is collected in one fraction. The membrane is then washed with 40 membrane volumes of the equilibration buffer and this effluent is collected in two equal fractions. Finally the membrane is stripped with 40 membrane volumes of 1 M sodium chloride in 50 mM sodium acetate buffer, which is collected in one fraction. The collected fractions from the load, wash, and strip are tested for recovery of antibody, removal of HCP, and aggregate levels.

The recovery, HCP removal, and aggregate levels from each membrane and condition are examined to analyze possible trends.

The Natrix S membranes achieve yields between 92%-97% with no strong trend relative to pH or conductivity. HCP levels are reduced to 700-850 ppm, again with no strong trend relative to the tested variables. Aggregate data are not available for this experiment. See Figures 2A and 2B.

The Natrix C membranes had a yield range of 85%- 100%, generally favoring low pH with minimal changes due to conductivity. HCP levels are reduced to 1000-1300 ppm, with better reduction at low pH across the conductivity range. Aggregates are also reduced to 2.2-3.0% favoring the higher pH range with minimal influence of conductivity. See Figures 3A, 3B and 3C.

The Sartobind S membranes produce a yield range of 88%-100% favoring the middle pH range and higher conductivities. HCP levels of the flow-through pool are 1000->1600 ppm favoring low pH and conductivity. Aggregate levels are 2.6-3.0%) favoring higher pH and conductivity. See Figures 4A, 4B, and 4C.

Overall, the Natrix S membrane operating at pH 4.75 and 6 mS/cm provides the best yield and HCP clearance within the operating ranges supported by these experiments.

Example 5. Optimization of Conditions for Flow-through Purification of a Monoclonal Antibody by Hydrophobic Interaction Membrane Chromatography

A sample of a monoclonal IgGi antibody (-150 kDa, pi = 8.5) containing about

3.6-4.7%) aggregated antibody and about 100-125 ppm HCP is purified by flow-through HIC membrane chromatography using Sartobind Phenyl under two conditions. In the first experiment, the antibody is formulated in a buffer containing 0.75 M ammonium sulfate. In the second case, the antibody is formulated in a buffer containing 0.85 M ammonium sulfate. These concentrations have previously been used for bind-and-elute purification of antibodies on this membrane because they maximize product binding (Kuczewski et ah, 2010, Biotechnol Bioeng., 105:296-305). In both experiments, the membrane is loaded to approximately 500 g of antibody per mL of membrane volume and then washed with 20 membrane volumes of 50 mM phosphate pH 7.0 + 0.75 or 0.85 M ammonium sulfate. The flow-through and wash fractions are tested for recovery and level of aggregate and HCP.

As shown in Figures 5 and 6, the aggregate removal is higher at 0.85 M ammonium sulfate than at 0.75 M. The antibody yields in the flow-through and wash fractions for these two processes are 89% (0.75 M) and 86% (0.85 M) despite the fact that these conditions can be used for bind-and-elute purification. The ammonium sulfate concentration did not strongly influence HCP reduction. The flow-through pools contain 86 ppm (0.75 M) and 82 ppm (0.85 M) of HCP.

Example 6. Flow-through Purification of a Monoclonal Antibody

A monoclonal IgGi antibody (-150 kDa, pi = 8.5) is expressed using PER.C6^® cells grown in a "XD^®" reactor using chemically defined media. The material is harvested by Enhanced Cell Settling as described in Schirmer et ah, 2010, BioProcess Int., 8:32-39; WO 2010/043700; WO 2010/043701; and WO 2010/043703. The clarified media is then sterile filtered and stored at 4 °C for further processing.

The clarified media is titrated to pH 8.5 using 2 M Tris, and PEG-3350 is added to a final concentration of 14.4% (w/w), causing the antibody to precipitate. The precipitate is concentrated ~30-fold using a hollow fiber micro filtration membrane, and then washed with 3 diavolumes of 20 mM Tris pH 8.5 + 14.4% (w/w) PEG-3350. The precipitate is then redissolved in 68 mM sodium acetate buffer pH 5.3, sterile filtered, and stored at 4 °C for further processing.

The redissolved precipitate is titrated to pH 4.75 with 10% acetic acid and the conductivity is adjusted to 6 mS/cm with NaCl. This material is passed through a Natrix S CEX membrane to a loading of 1 g-MAb/mL-membrane at a flow rate of 4 MV/min. After loading, the membrane is washed with 68 mM sodium acetate buffer at pH 4.75, 6 mS/cm until the A₂so reached baseline. The combined flow-through and wash fractions ("CEX pool") are pooled together, sterile filtered, and stored at 4 °C for further processing. The CEX pool is titrated to pH 7.5 with 2 M Tris and the conductivity is adjusted to 6.5 mS/cm. This material is passed through a ChromaSorb AEX membrane at 10 MV/min to a loading of ~2.5 g-MAb/mL-membrane. The AEX pool is then titrated to pH 3.5 with 1 N HCl and held at room temperature for 30 minutes to inactivate viruses before being neutralized to pH 7.0 with 2 M Tris.

The resulting material is blended in-line with a sodium phosphate/ammonium sulfate buffer to a final ammonium sulfate concentration of 0.85 M for loading onto a Sartobind Phenyl HIC membrane. Loading is performed at 3.3 MV/min to a total of -434 mg-MAb/mL-membrane. The adsorber is then washed with 50 mM phosphate + 0.85 M ammonium sulfate until the A₂so reached baseline, and the flowthrough and wash fractions ("HIC pool") are pooled and sterile filtered.

Each process intermediate is assayed for product concentration by analytical protein A HPLC or A₂so/A₃₂o. Purity is measured by size exclusion chromatography HPLC and SDS PAGE. HCPs are quantified using a commercially available ELISA kit specific to PER.C6^® HCPs (Cygnus Technologies). Product yield, percent aggregate, and HCP levels at each intermediate step are indicated in the table below.

Recovery HCP HCP Reduction Aggregates, HMW Aggregates reduction

Step

[%] [ppm] [%] [%] [%]

Clarified harvest 21351

PEG/TFF 87% 1962 90% 0.24%

CEX FT 90% 282 86% 0.21% 13%

AEX FT 90% 125 51% 0.19% 10%

VI 96% 132 -5% 0.19% 0%

HIC FT 97% 89 33% 0.15% 21%

Final 66% 89 99.6% 0.15% 38%

As can be seen from the table, the cation exchange step in flow-through mode under binding conditions surprisingly resulted in significant purification, with 90% recovery, an 86% reduction in HCP, and a 13% reduction in aggregates. The total process provided substantial purification of the antibody, with 66% total recovery, a 99.6% (2.4 log) reduction in HCP, and a final aggregate content of 0.15%. It will be understood that the processes described herein for the practice of the present invention may be varied without departing from the spirit and scope of the invention.

Claims

We claim:

1. The process for purifying a protein of interest, comprising:

(a) providing a sample comprising an amount of protein of interest and impurities;

(b) contacting said sample with a cation exchange, anion exchange or hydrophobic interaction chromatography material under chromatography conditions, which provide for said protein of interest to adsorb to said cation exchange, anion exchange or hydrophobic interaction chromatography material, wherein said amount of protein of interest in said sample exceeds the capacity of said cation exchange, anion exchange or hydrophobic interaction chromatography material to adsorb more than an insubstantial portion of said amount of protein of interest, and wherein a substantial amount of said impurities adsorb to said cation exchange, anion exchange or hydrophobic interaction chromatography material; and

(c) separating said cation exchange, anion exchange or hydrophobic interaction chromatography material to which impurities have been adsorbed from the sample containing the substantial portion of said amount of protein of interest.

2. The process according to claim 1 wherein said process is conducted in a flow-through mode.

3. The process according to claim 2, wherein said material is hydrophobic interaction chromatography material, and said sample contains a concentration of lyotropic salt selected to maximize binding of said protein of interest.

4. The process according to claim 3 wherein said sample contains aggregate impurities from about 1% to about 20%, and of host cell protein impurities from about 10 ppm to about 1000 ppm.

5. The process according to claim 2, wherein said material is a cation exchange material and wherein said chromatography conditions of step (b) provide for said protein of interest to exhibit a net positive charge.

6. The process of claim 1, wherein said protein of interest has a pi between 6.5 and 9.5.

7. The process of claim 1, wherein said protein of interest is an antibody.

8. The process of claim 7, wherein said antibody is an IgG antibody.

9. The process of claim 5, wherein the pH and the conductivity of said sample are selected to (i) maximize the purification yield of said protein of interest, and (ii) reduce the impurities retained in said purified sample.

10. The process of claim 9, wherein less than about 15% of said amount of said protein of interest adsorbs to said cation exchange material.

11. The process of claim 10, wherein less than about 10% of said amount of said protein of interest adsorbs to said cation exchange material.

12. The process of claim 1, wherein said sample is a clarified cell broth, plasma fraction, milk, plant extract, or clarified microbial lysate.

13. The process of claim 12, which further comprises filtering said sample prior to step (b).

14. The process of claim 13, wherein said sample contains cells, and further comprising step (a)(1) removing said cells from said sample by sedimentation, flocculation, enhanced cell settling, and/or centrifugation, prior to step (b).

15. The process of claim 14, further comprising step (a)(2) precipitating said protein of interest with polyethylene glycol (PEG) after step (a)(1) and prior to step (b).

16. The process of claim 10, which further comprises filtering said sample prior to step (b).

17. The process of claim 16, wherein said sample contains cells, and further comprising step (a)(1) removing said cells from said sample by sedimentation, flocculation, enhanced cell settling, and/or centrifugation, prior to step (b).

18. The process of claim 17, further comprising step (a)(2) precipitating said protein of interest with polyethylene glycol (PEG) after step (a)(1) and prior to step (b).

19. The process of claim 18, further comprising step (d) subjecting said purified sample to anion exchange chromatography in a flow-through mode to form a second purified sample.

20. The process of claim 19, further comprising step (e) subjecting said second purified sample to hydrophobic interaction chromatography in a flow-through mode to form a third purified sample.

21. The process of claim 20, further comprising subjecting said purified sample, said second purified sample, or said third purified sample to a virus inactivation step.

22. The process of claim 21, wherein also comprising filtering to remove viruses.

21. The process of claim 20, wherein at least 50% of said protein of interest originally present in said sample is collected after step (e).

22. A process for purifying a protein of interest comprising:

(a) providing a sample comprising an amount of protein of interest and impurities;

(b) subjecting said sample to anion exchange chromatography in a flow-through mode and conditions, which provide for said protein of interest to exhibit a net negative charge, to form a purified sample;

(c) contacting said purified sample with a cation exchange material under chromatography flow-through mode and conditions, which provide for said protein of interest to exhibit a net positive charge, and wherein said amount of protein of interest in said sample exceeds the capacity of said cation exchange material to adsorb more than an insubstantial portion of said amount of protein of interest, and wherein a substantial amount of said impurities adsorb to said cation exchange material; and

(d) separating said cation exchange material to which impurities have been adsorbed from said purified sample containing the substantial portion of said protein of interest that has not adsorbed to said material, to result in a second purified sample.

23. The process of claim 22, further comprising step (e) subjecting said second purified sample to hydrophobic interaction chromatography in a flow-through mode to form a third purified sample.

24. The process of claim 23, wherein said sample is a clarified cell broth.

25. The process of claim 24, wherein said sample has been treated to precipitate said protein of interest prior to step (a).

26. The process of claim 25, wherein said protein of interest has a pi between 6.5 and 9.5.

27. The process of claim 22, wherein the pH and the conductivity of said sample are selected to (i) maximize the purification yield of said protein of interest, and (ii) reduce the impurities retained in said second purified sample.