FIELD OF THE INVENTION

The present invention relates to a method of purification of antibodies comprising an anion exchange chromatography.

BACKGROUND OF THE INVENTION

Large-scale purification of proteins remains a significant challenge in the biopharmaceutical industry as efficient and cost-effective methods are required to achieve desired yields and purity levels. Therapeutic proteins are primarily products of recombinant DNA technology, i.e., cloning and expression of a heterologus gene in prokaryotic or eukaryotic systems. However proteins expressed by recombinant DNA methods are typically associated with impurities such as host cell proteins (HCP), host cell DNA (HCD), viruses, etc. Also, there is significant heterogeneity in the expression of the desired protein, in the form of charged variants (typically acidic, lower pi variants and basic, higher pi variants). Further, multimeric proteins, such as antibodies, have a higher tendency to aggregate, contributing to significantly increased impurity levels.

The presence of these impurities, including aggregates and undesirable charged variants, is a potential health risk, and hence their removal from a final product is a regulatory requirement. Thus drug regulatory agencies such as United States Food and Drug administration (FDA) require that biopharmaceuticals be free from impurities, both product related (aggregates or degradation products) and process related (media components, HCP, DNA, chromatographic media used in purification, endotoxins, viruses, etc). See, Office of Biologies Research and Review, Food and Drug Administration, Points to consider in the production and testing of new drugs and biologicals produced by recombinant DNA technology (Draft), 1985. Thus, elimination of impurities from a final product is a requirement and poses a significant challenge in the development of methods for the purification of therapeutic proteins.

Antibodies constitute one of the most important classes of therapeutic proteins, especially in the areas of oncology, arthritis and other chronic diseases.

Purification of antibodies often involves a combination of different chromatographic steps, that generally begins with "a capture step" by protein A affinity chromatography, followed by one or more additional separation steps. A final "polishing step" is often necessary for the removal of trace amounts of HCP, HCD, viruses or endotoxins. The polishing step is typically carried out by anion exchange chromatography performed in a flow-through mode.

The prior art discloses various methods for purification of crude or partially purified antibodies using ion/anion exchange chromatography. WO 2004/024866 describes a method of purifying a polypeptide by ion exchange chromatography in which a gradient wash with differing salt concentrations is used to resolve the polypeptide. WO 1999/057134 describes the use of ion exchange chromatography for purification of polypeptides by varying conductivity and/or pH . US 51 10913 claims purification of murine antibodies using low pH and at least three different pH conditions in the ion-exchange chromatographic step.

US 7847071 describes purification of antibodies using series of chromatographic techniques wherein the anion-exchange chromatography is performed in the flow-through mode using a buffer of pH 8.0 and a displacer salt.

WO 2010072381 reports purification of immunoglobulin in the flow-through from the anion exchange chromatography wherein the buffer has pH value of from 8.0 to 8.5.

However the prior art's use of very low or high pH, in a chromatographic step may result in considerable reduction in antibody yield and stability. Further, as antibody purification generally involves multiple chromatographic steps, use of buffer of either low or high pH in a chromatography necessitates substantial and frequent pH adjustments, or buffer exchanges, between other chromatographic steps, that in turn affect efficient and effective scale up of downstream operations.

Hence, the principle object of the present invention is to provide an improved method for obtaining antibody preparations that avoid use of buffers at very low or high pH during anion exchange chromatography. Interestingly, and contrary to what is taught in the prior-art, the present invention provides an anion exchange chromatography with buffer conditions in the neutral pH range, for the purification of antibodies. The invention results in effective clearance of viruses, separation of charge variants, increased purity and recovery of the desired antibody. SUMMARY OF THE INVENTION

The present invention describes a process for the purification of antibodies by anion exchange chromatography performed in flow-through mode in the neutral pH range.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is an illustration of a chromatogram from the procedure as described in Example 1 . The line marked "Cond" represents the increase in conductivity in mS/cm. Peak A, represents the eluate obtained from protein A chromatography resin.

Fig. 2 is an illustration of a chromatogram from the procedure as described in Example 2. Peak A and B represent the eluate obtained from cation exchange chromatography. Peak A and B are charge variants of the anti-CD20 antibody.

Fig. 3 and 4 are illustrations of a chromatogram from the procedure as described in Example 3. Figures represent the flow-through fraction of the anion exchange chromatography.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a process for purification of antibodies by anion-exchange chromatography. The process avoids use of very low or high pH buffers. The neutral pH buffer conditions described in the invention also facilitates easy "switch over" between alternate chromatographic steps without need for significant buffer exchange or neutralization steps. The conditions described in the present invention results in effective separation of charge variants, removal of impurities such as HCP, HCD and viruses and results in optimum yield of the desired antibody.

The term "antibody" as used herein refers to immunoglobulins and can be isolated from various sources, such as murine, human, recombinant etc. In its broadest sense it includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments. It also includes truncated antibodies, chimeric, humanized or pegylated antibodies, isotypes, allotypes and alleles of immunoglobulin genes and fusion proteins, which contain an immunoglobulin moiety.

The term "impurities" as used herein refers to a material that is different from the desired polypeptide. They may be nucleic acids such as host cell DNA, host cell proteins, variants of the desired polypeptide, another polypeptide, viruses, endotoxin etc.

The term "flow-through mode" as used herein refers to a process wherein the desired protein is not bound to a chromatographic resin but is instead obtained in the unbound or "flow-through" fraction during loading or post loading washes of a chromatography support. The desired protein in the flow-through can be collected as various fractions and pooled together or can be collected as a single fraction.

The term neutral pH range refers to the pH range of about 7.0 to about 7.5. In an embodiment, the invention provides a method of the purification of an antibody comprising an anion exchange chromatography operated in flow-through mode, wherein the buffer solution used is in the pH range of about 7.0 to about 7.5.

In one embodiment of the invention, the pH of the buffer is about 7.0.

In another embodiment of the invention, the pH of the buffer is about 7.5.

In yet another embodiment, the invention provides a method for the purification of an antibody wherein a cation exchange chromatography precedes or follows the said anion exchange chromatography.

In a further embodiment of the invention, a protein-A chromatography precedes the cation exchange and anion exchange chromatography.

The embodiments mentioned herein may optionally include one or more tangential flow filtration, concentration, diafiltration or ultra filtration steps.

The embodiments mentioned herein optionally include one or more viral inactivation steps or sterile filtration or nano filtration steps.

The embodiments mentioned herein may include one or more neutralization steps.

The protein A chromatographic resin used may be any protein A or variant or a functional fragment thereof coupled to any chromatographic support. In embodiments, the protein A resin is Mabselect™ (GE-Healthcare Life sciences), an affinity matrix with recombinant Protein-A ligand. The resin is made of highly cross- linked agarose matrix.

Cation exchange chromatographic step mentioned in the embodiments may be carried out using any weak or strong cation exchange chromatographic resin or a membrane, which could function as a weak or a strong cation exchanger. Commercially available cation exchange support include a resin, but are not limited to, 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, S-Ceramic Hyper D, from Pall Corporation or a carboxymethyl based group e.g., CM Sepharose Fast Flow from GE Healthcare, Macro-Prep CM from BioRad, CM-Ceramic Hyper D, from Pall Corporation, Toyopearl CM-650S, CM-650M and CM-650C from Tosoh. In embodiments of the invention, a strong cation exchange resin, such as SP- Sepharose ^® (GE Healthcare Life Sciences) is used. This resin is made using a highly cross-linked, 6 % agarose matrix attached to a sulfopropyl functional group.

Anion exchange chromatography mentioned in the embodiments may be carried out using any weak or strong anion exchange chromatographic resin or a membrane, which could function as a weak or a strong anion exchanger. Commercially available anion exchange resins 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 3OQ, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX and FAST Q SEPHAROSE from GE Healthcare, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Q-Ceramic Hyper D, DEAE-Ceramic Hyper D, from Pall Corporation. In embodiments of the invention, a strong anion exchange resin, such as Q- Sepharose Fast Flow ^® (GE Healthcare Life Sciences) is used. This resin is made using a highly cross-linked, 6 % agarose matrix attached to -O-CH ₂ CHOHCH2OCH2CHOHCH ₂ N ⁺ (CH3)3 functional group. Alternatively, the anion exchange chromatography could be carried out using a monolithic column, disk or tubular, that functions as an anion exchanger.

Examples of buffering agents used in the buffer solutions include, but are not limited to, TRIS, phosphate, citrate, acetate, succinate, MES, MOPS, or ammonium and their salts or derivatives thereof.

The invention is more fully understood by reference to the following examples. These examples should not, however, be construed as limiting the scope of the invention.

EXAMPLE 1

Protein A chromatography

An anti-CD20 antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 7,381 ,560, which is incorporated herein by reference. The cell culture broth containing the expressed antibody was harvested, clarified and subjected to protein A affinity chromatography as described below.

The clarified cell culture broth was loaded onto a protein A chromatography column (Mabselect, VL44x250, 205 mL) that was pre-equilibrated with Tris buffer solution (pH 7.0). The column was then washed with equilibration buffer. This was followed by a wash with Tris buffer (pH 7.0) with higher conductivity and a final wash with citrate buffer at pH 5. The bound antibody was eluted using citrate buffer, pH 2.5 - 3.5.

EXAMPLE 2

Cation exchange chromatography

The eluate obtained from the protein A chromatography procedure described in Example 1 was loaded onto a cation exchange resin (SP Sepharose, VL44x250, 304 mL) pre-equilibrated with Tris buffer (pH 7.5). This was followed by washing the resin with a wash buffer of Tris buffer (pH 7.5). A second wash step was performed with wash buffer consisting of citrate buffer, pH 6.5. The bound antibody was eluted using a buffer of citrate buffer, pH 6.5 at conductivity between 9-12 mS/cm.

EXAMPLE 3

Anion exchange chromatography

The eluate obtained from the cation exchange chromatography procedure described in Example 2 was loaded onto an anion exchange resin (Q-Sepharose FF, VL32x250, 80 mL) pre-equilibrated with 5 column volume of an equilibration buffer (40 mM Tris buffer, pH 7.5, at a conductivity value of 3.0 to 6.0 mS/cm). This was followed by a post load wash with the equilibration buffer and the load and wash flow-through was collected.

Table 1

Cation Exchange 0.17 0.3 0.81 81 .1 chromatography

eluate

Anion Exchange 0.08 BDL 0.67 95.6 chromatography

flow through

BDL: Below Detection Limit Table 2:

Basic Basic

Acidic

Sample K0 (%) Variant-1 Variant-2 variants (%)

(%) (%)

Protein A eluate 10.64 18.03 64.09 7.24

Cation Exchange 13.73 23.30 62.97 BDL chromatography

eluate

Anion Exchange 14.03 23.60 62.37 BDL chromatography flow

through

K0: Species devoid of the C-terminal lysine residue

BDL: Below Detection Limit

Example 4

Nano filtration

The flow-through obtained from the anion exchange chromatography procedure described in Example 3, was applied onto a nano filtration unit (Ultipor ^® VF grade DV 20, Pall Corporation) using a buffer of 40 mM Tris, pH 7.5, at a conductivity of 3.0 mS/cm. Nanofiltered filtrate composition may then be collected and used.