FIELD OF INVENTION

The invention deals with purification of polypeptides. In particular, it describes a method of purifying a polypeptide by ion exchange chromatography under optimal load conditions.

BACKGROUND OF INVENTION

The design of efficient and economic purification methods for polypeptides 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. 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. An example of a chromatographic capture step is protein A affinity chromatography that is widely used for the purification of antibodies (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 (in the case of protein A chromatography), host cell protein (HCP), host cell DNA (HCD) endotoxins etc.

Ion exchange chromatography is a technique commonly used to purify proteins. In ion exchange chromatography, charged patches on the surface of the solute are attracted by opposite charges attached to a chromatography matrix, provided the ionic strength of the surrounding buffer is low. Elution is generally achieved by increasing the ionic strength (i.e. conductivity) or pH of the buffer to compete with the solute for the charged sites of the ion exchange matrix. The change in conductivity or pH may be gradual (gradient/linear elution) or stepwise (step elution).

Since each chromatographic step requires optimization of several process conditions such as, pH, buffer type, salt type, concentrations etc., optimizing 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. Interestingly, the present invention provides an optimized load condition for ion exchange chromatography that significantly reduces the need for optimizing several process conditions for purification of polypeptides. Loading the polypeptide onto the ion exchange resin in the specified range results in effective separation of various charged variants and the required percentage purity for the polypeptide to function as a therapeutic substance. Use of specified range of load conditions in the ion exchange chromatography, largely reduces the amount of host cell proteins, protein A leachates, host cell DNA, and other high molecular weight impurities from the polypeptide composition.

SUMMARY OF THE INVENTION

The present invention describe a method of purification of polypeptides, using a cation exchange chromatography wherein the amount of the desired polypeptide loaded onto the cation exchange resin is less than 20 mg/ml of the resin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Cation Exchange Chromatogram: Set 1 that has a load of lOmg of protein per ml of cation exchange resin compared with Set 2 that has a load of 20mg of protein per ml cation exchange resin. Cation Exchange chromatogram obtained after performing purification as described in example 2 of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a chromatographic process for purification of polypeptides using ion-exchange chromatography. In particular, the present invention provides an optimal concentration range of polypeptide loaded onto the cation exchange resin. Loading at the specified range results in effective separation of charged variants and removal of impurities such as HCP and Protein A leachates and an optimum yield of the desired polypeptide.

As used herein, "polypeptide" refers generally to peptides and proteins having more than about ten amino acids. Examples of polypeptide would include, but not limited, to antibodies, cytokines, chemokines, enzymes, hormones, growth factors, modified proteins, PEGylated forms of proteins and their derivatives, etc.

Antibody mentioned herein 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.
An embodiment of the invention provides a method for purifying a polypeptide by loading the polypeptide composition comprising the polypeptide and impurities onto a cation exchange resin, wherein the amount of polypeptide loaded onto the cation exchange resin is less than 20 mg of the polypeptide per mL of cation exchange resin.
An impurity is a material that is different from the desired polypeptide. The impurity may be nucleic acids such as host cell DNA, host cell proteins, variants of the desired polypeptide, another polypeptide, endotoxin etc.

In another embodiment, the amount of polypeptide loaded on to the cation exchange resin ranges from about 5 mg to 19 mg of polypeptide per mL of cation exchange resin, more preferably from about 6 mg to about 18 mg of polypeptide per mL of cation exchange resin.

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

The cation exchange chromatographic steps 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.

The cation exchange chromatography mentioned in the embodiment may be preceded by one or more affinity chromatography, ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography or mixed mode chromatography steps.

The cation exchange chromatography may be followed by one or more affinity chromatography, ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography or mixed mode chromatography steps.

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

The embodiments mentioned herein may 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 polypeptide mentioned in the embodiments may be an antibody.

The polypeptide mentioned in the embodiments may be a protein or a derivatized form of the protein such as pegylated protein.

The buffering agents used in the buffer solutions include, and are not limited to phosphate, acetate, citrate, succinate, MES, MOPS, TRIS or ammonium and their salts or derivatives as well as combinations of these.

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.

EXAMPLES

Example 1: Purification of anti- Vascular Endothelial Growth Factor (anti-VEGH antibody

The clarified culture supernatant containing the desired protein was purified by protein A chromatography. The cell culture supernatant was loaded onto the protein A chromatography column (Prosep Ultra A) that was pre-equilibrated with 50 mM Tris buffer, 150 mM NaCl, pH 7.0. The column was then washed with the same buffer followed by washes with 50 mM Tris acetate, 750 mM NaCl, pH 7.0 and 25 mM Tris acetate at pH 7.0. The bound antibody was eluted with 200 mM acetate buffer pH of 3.3.

The protein A eluate was neutralized with Tris CI buffer (pH 9.0) to a pH of 6, and loaded onto a cation exchange column (CM Hyper D), pre-equilibrated with 35 mM Phosphate buffer, pH 6.0, at a load concentration of 16.3 mg of the antibody per ml of the cation exchange resin. The column was washed with 35 mM sodium phosphate, pH 6.0 and the antibody eluted with 80 mM sodium phosphate buffer, pH 6.0. The eluate was analyzed for protein A leachate and host cell protein (HCP) (Table I).

The eluate from cation exchange was loaded onto a Q-Sepharose (anion exchange) column that was pre-equilibrated with 80 mM phosphate buffer, pH 6.0. The desired antibody was obtained in the flow through.

Example 2: Purification of anti- Human Epidermal growth factor Receptor 2 (anti-HER2) antibody

The clarified culture supernatant containing the desired protein was purified by protein A chromatography. The cell culture supernatant was loaded onto the protein A chromatography column (Prosep vA Ultra) that was pre-equilibrated 50 mM Tris buffer, 150 mM NaCl, pH 7.5. The column was then washed with the same buffer followed by washes with 50 mM Tris-Cl, 1 M NaCl, pH 7.5 and 50 mM citrate, pH 6.0. The bound antibody was eluted with 50 mM citrate buffer, pH 2.8. The eluate was analyzed for protein A leachates and host cell protein (HCP) (Table I).

After neutralization using 1 M Tris-Cl, pH 9.0, the protein A was loaded onto a Q-Sepharose column that was pre-equilibrated with citrate buffer, pH 7.0. The desired antibody was obtained in the flow through.

The anion exchange flow through was loaded onto a strong cation exchange column, (POROS HS) at a load concentration of 10 mg of the antibody per ml of the resin. The column was equilibrated with 12.5 mM citrate pH 6.0, followed by washing 12.5 mM citrate buffer, pH 6.0. The antibody was eluted with 50 mM citrate Buffer, pH 6.0.

Example 3: Purification of anti-CD20 antibody

The clarified culture supernatant containing the desired protein was purified by protein A chromatography. The cell culture supernatant was loaded onto the protein A chromatography column (Prosep-A) that was pre-equilibrated 50 mM Tris buffer, 150 mM NaCl, pH 7.5. The column was then washed with same buffer followed by additional washes with 50 mM Tris acetate, 150 mM NaCl, pH 7.5, and 50 mM citrate at pH 6.0.

The bound antibody was eluted using with 50 mM citrate, pH 3.5.

The protein A eluate was loaded onto a Q-Sepharose column that was pre-equilibrated with 50 mM Tris, pH 7.5. The column was washed with the same buffer. The desired product was obtained in the flow through.

The anion exchange flow through was diluted and loaded onto a strong cation exchange column (SP Sepharose), at a load concentration of 10-12 mg of the antibody per ml of the cation exchange resin pre equilibrated with 40 mM Tris, pH 7.5. The column was washed with the same buffer followed by subsequent wash with and 17.5 mM citrate, pH 6.5. The antibody was eluted with 25 mM citrate buffer, 154 mM NaCl, pH 6.5.

Example 4: Purification of Pegylated - Granulocyte Colonv-Stimulating Factor (Peg-GCSF)

A composition containing Pegylated-GCSF was purified by loading 6 mg of the protein per ml of resin on to strong cation exchange column (POROS HS). The column was pre equilibrated with 20 mM sodium acetate buffer, 175 mM NaCl, pH 3, followed by wash with 87 mM sodium acetate, 175 mM NaCl, pH 3.5 followed by elution of the desired protein with 20 mM sodium acetate, 350 mM NaCl, pH 3.5.

Example 5: Purification of anti- Vascular Endothelial Growth Factor (anti-VEGF) antibody

The clarified culture supernatant containing the desired protein was purified by protein A chromatography. The cell culture supernatant was loaded onto the protein A chromatography column (Prosep Ultra A) that was pre-equilibrated with 50 mM Tris buffer, 150 mM NaCl, pH 7.0. The column was then washed with the same buffer followed by washes with 50 mM Tris acetate, 750 mM NaCl, pH 7.0 and 25 mM Tris acetate at pH 7.0. The bound antibody was eluted with 200 mM acetate buffer pH of 3.3.

The protein A eluate was sterile filtered and loaded onto a cation exchange column (CM Hyper D), pre-equilibrated with Tris-Acetate buffer, pH 3.3 at a load concentration of 15 mg of the antibody per ml of the cation exchange resin. The column was washed with 35 mM sodium phosphate, pH 6.2 and the antibody eluted with 80 mM sodium phosphate buffer, pH 6.2. The eluate from cation exchange was loaded onto a Q-Sepharose (anion exchange) column that was pre-equilibrated with 80 mM phosphate buffer, pH 6.0. The desired antibody was obtained in the flow through.

Table I: Levels of Protein A leachates and HCP in Cation exchange load and Cation exchange eluate.

Table II: Levels of HCP and HCD in the cation exchange eluate containing Pegylated-GCSF (example 4)

CLAIMS

1) A method for purifying a polypeptide by loading the polypeptide composition comprising the polypeptide and impurities onto a cation exchange resin, wherein the amount of polypeptide loaded onto the cation exchange resin is less than 20 mg of the polypeptide per mL of cation exchange resin.

2) A method of purification as described in claim 1 wherein the polypeptide composition comprising the polypeptide and impurities is loaded on to the cation exchange column at Ph about 4.0 to pH about 7.0.

2) A method of purification as described in claim 1 wherein amount of polypeptide loaded on to the cation exchange resin ranges from about 5 mg to 19 mg of polypeptide per mL of cation exchange resin.

3) A method of purification as described in claim 1 wherein amount of polypeptide loaded on to the cation exchange resin ranges from about 6 mg to about 18 mg of polypeptide per mL of cation exchange resin.

4) A method of purification as described in claim 1 wherein the cation exchange chromatography may be preceded by one or more affinity chromatography.

5) A method of purification as described in claim 1 wherein the cation exchange chromatography may be preceded by or followed by one or more ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, mixed mode chromatography

6) A method of purification as described in claim 1 wherein the cation exchange chromatography may be preceded by or followed by one or more tangential flow filtration, concentration, diafiltration , ultrafiltration, viral inactivation, sterile filtration, nano filtration or neutralization steps.