FIELD OF INVENTION

The invention deals with purification of proteins. In particular, it describes a two step chromatographic process for purification of proteins.

BACKGROUND OF INVENTION

The design of efficient and economic purification methods for proteins and peptides produced by recombinant DNA technology is a major challenge in bringing new biopharmaceuticals to the market. The challenges of purification vary depending upon the source and location of the product. However, regardless of the source (such as expression system type), a purification process normally follows the capture-intermediate purification-polishing approach to attain a purified product. This often results in a process involving three to five chromatographic purification steps. Purification of antibodies usually involves an affinity chromatography as the first step in downstream processing. Protein A chromatography is one of the most widely used affinity chromatographies for antibody purification due to its high specificity of binding to the Fc region of the antibody (Ab) molecule (Gagnon, P. Purification Tools for Monoclonal Antibodies Validated Biosystems, Tucson, AZ, 1995, Fahrner, L. R et al, (2008), Biotechnology and genetic engineering reviews, 18: 301-327). However, additional steps that constitute the intermediate purification and polishing are considered necessary to eliminate traces of process and product related impurities such as, protein A leachates, host cell protein (HCP) etc.

Prior art discloses several such methods for purification of crude or partially purified samples that comprise of 3-5 chromatographic steps. These include protein A affinity chromatography (Gagnon, P. Purification Tools for Monoclonal Antibodies, Validated Biosystems, Tucson, AZ, 1995), gel filtration for separating uncomplexed antibodies from IgG-protein A complexes (Balint et.al, Cancer Res 1984; 44, 734-743), as well as ion exchange (US4983722, US20070292442 and EP1601697), hydroxyapatite and immobilized metal affinity chromatography (MAC).

Since each chromatographic step requires optimization of several process conditions such as, pH, buffer type, salt type and concentrations etc., purification protocols can be time consuming, expensive and result in significant product losses. Considering the cumbersome nature of the steps involved, alternatives that alleviate the complexity of the process are desirable. The present invention describes a simple two step process wherein protein A chromatography step is followed by a cation exchange step.

The present invention describes a two-step chromatographic process for purification of mAbs, wherein the first step involves capture using protein A chromatography and the second step involves impurity clearance and variant separation on cation exchange chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Protein A chromatography as described in Example 1 of the invention. The eluate as represented by peak A was used for further purification by Cation exchange chromatography.

Figure 2: Cation Exchange chromatography as described in Example 2 of the invention. The fractions were analyzed for percentage reduction of protein A contamination.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a two-step chromatographic process for purification of mAbs, wherein the first step involves capture using Protein A chromatography and the second step involves impurity clearance and variant separation on cation exchange chromatography.

One embodiment of the invention involves a method for the purification of an antibody comprising:

a) A first purification step by protein A chromatography, wherein the antibody is eluted at low pH

b) A second purification step by cation exchange chromatography

The cation exchange chromatography is preferably performed in the bind elute, wherein the desired protein is bound to the chromatography resin when loaded on to the column and subsequently eluted with an elution buffer. The desired protein may be collected as a single fraction or as various fractions which are pooled together.

In another embodiment of the invention, the antibody is eluted in the first purification step at a pH of about 3.3 to 4.5 and loaded on to the cation exchange resin at a pH of about 6.0.

In a preferred embodiment of the invention, the antibody is eluted in the first purification step at a pH of 3.S and loaded on to the cation exchange resin at a pH of 6.0.

The protein A chromatographic resin used may be any protein A or variant or a functional fragment thereof coupled to any chromatographic support. Preferably the protein A resin is Prosep A (Millipore). For the purpose of the invention, fresh (i.e. not used before) protein A chromatographic resin may be used to obtain a feed stream for the second chromatographic step. After washing with loading buffer and intermediate wash, the elution is carried out at low pH.

The cation exchange resin used in the invention may be a weak cation exchanger, preferably CM Ceramic (Paul Corporation). The buffering agents used in the buffer solutions are selected from the group consisting of TRIS, phosphate, citrate or acetate salts or derivatives and is most preferably phosphate.

DEFINITION

The term "antibody" as used herein refers to immunoglobulins that compose of four polypeptide chain consisting of two light and two heavy chains. It includes immunoglobulins isolated from various sources, such as murine, human, recombinant etc, truncated antibodies, chimeric, humanized or pegylated antibodies, isotypes, allotypes and alleles of immunoglobulin genes. The term antibody as used herein also refers to antibody like proteins such as fusion proteins which contain an immunoglobulin moiety.

Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples, being provided only for purposes of illustration.

These examples should not be construed as limiting the scope of the invention in any manner.

EXAMPLES

Example 1

Purification by protein A chromatography

A serum free culture supernatant of CHO cells containing monoclonal antibodies was purified by protein A chromatography. The cell culture supernatant was loaded onto the protein A chromatography column (Prosep A) that was pre-equilibrated with 5 column volumes (CV) of 50 mM Tris, 150 mM NaCl, pH 7.5 buffer. The column was then washed with 5 CV of the equilibration buffer (50 mM Tris, 150 mM NaCl, pH 7.5). This was followed by a wash with 5 CV of 50 mM Tris, 750 mM NaCl, pH 7.5 buffer and a final wash with 25 mM Tris at pH 7.5. The bound antibody was eluted using a low pH buffer such as 200 mM acetate buffer, pH 3.3. The peak A in the chromatogram (Figure 1) shows the protein A chromatography eluate.

Example 2

Purification by Cation exchange chromatography

The protein A chromatography eluate obtained from example 1 was purified by cation exchange chromatography. The protein A eluate obtained was first incubated at pH 3.5, 25 °C for 30 min for viral inactivation and then neutralized with Tris Acetate buffer (pH- 9.0) to achieve a pH of 6. The sample was then filtered through 0.8/0.2i membrane filter and loaded onto a weak cation exchange column, CM ceramic (weak cation) pre-equilibrated with 5 CV of equilibration buffer. This was followed by washing with a wash buffer (approximately 5 CV). After loading and washing, the elution was carried out with a linear gradient from 35mM to 80 - 100 mM phosphate Buffer pH 6.0 + 0.2 over a slope of 10 - 15 CV.

Table I: Impurities levels of Protein A leachae in Cation exchange chromatography eluate

*BDL- below detection limit

CLAIMS

1. A method for the purification of an antibody comprising:

a) A first purification step by protein A chromatography, wherein the antibody is eluted at low pH

b) A second purification step by cation exchange chromatography

2. A method of purification as described in claim 1 wherein the antibody is eluted in the first step at a pH of about 3.3-4.5 and loaded onto the cation exchange resin at a pH of about 6.0.

3. A method of purification as described in claim 1 wherein the antibody is eluted in the first purification step at a pH of 3.5 and loaded on to the cation exchange resin at a pH of 6.0.