WO2014118685A2

WO2014118685A2 - Method of altering the acidic variant content of antibody

Info

Publication number: WO2014118685A2
Application number: PCT/IB2014/058553
Authority: WO
Inventors: Samir R. KULKARNI; Raghavendra YAMMANURU
Original assignee: Dr. Reddy's Laboratories Limited
Priority date: 2013-01-29
Filing date: 2014-01-26
Publication date: 2014-08-07
Also published as: WO2014118685A3

Abstract

The present invention is related to a method of altering the amount of acidic variants in an antibody composition. In particular, the invention employs a cation exchange chromatography with varying amounts of wash buffer column volume to alter the acidic variant content of the antibody.

Description

METHOD OF ALTERING THE ACIDIC VARIANT CONTENT OF ANTIBODY

RELATED APPLICATION

This application is related to and takes priority from Indian Provisional

Application 381 /CHE/2013 filed 29 January, 2013 and is herein incorporated in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method of altering the acidic variant content of an antibody composition by optimizing the post load wash conditions in a cation exchange chromatography.

BACKGROUND OF THE INVENTION

Overexpression and/ or amplification of human epidermal growth factor receptor type 2 (HER2), a 185-kDa transmembrane tyrosine kinase receptor protein, have been detected in 10%-34% of patients with invasive breast cancer, which is the most common cancer to affect adult females notably associated with decreased overall survival. Recent studies indicate a role of HER2 in the development of numerous types of other human cancers, such as colon, bladder, ovarian,

endometrial, lung, uterine cervix, head and neck, esophageal and gastric carcinomas (C. Gravalos; A. Jimeno, Annals of Oncology. 2008; 19(9):1523-1529). Hence, strategies to target HER2 appear to be important in treating HER2 related

carcinomas and anti-HER2 therapy is one of the first successful examples of a personalized healthcare.

One such anti-HER2 medication is trastuzumab, (Herceptin™), an anti-HER2 monoclonal antibody, which specifically targets and binds HER2 protein, and inhibits the effects of HER2 over expressionvia HER2 protein down regulation, prevention of HER 2 dimerization, inhibition of angiogenesis and induction of immune response. (J. Baselga and J.AIbanell, Annals of Oncology. 2001; 12:S35-S41) And so, trastuzumab is an approved and widely used molecule for the treatment of primary and metastatic HER2-positive breast cancer patients. Recently, FDA has approved trastuzumab for the treatment of patients with HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma. Given the therapeutic l importance of anti-HER2 antibody or trastuzumab, it is important to obtain the protein in highly pure form ready for human clinical use.

However, therapeutic proteins, including monoclonal anti-HER2 antibodies are produced by recombinant DNA technology. Proteins expressed by recombinant DNA methods are typically associated with impurities such as host cell proteins (HCP), host cell DNA (HCD), aggregates, viruses, etc. In addition, in case of monoclonal antibodies, charge variants namely "acidic" and "basic", are frequently formed as a result of glycosylation, deamidation, oxidation, reduction and other post- translational modifications, as well as factors such as the temperature, pH at which cells expressing the antibody are cultured. Presence of these variants in an appropriate amount is critical since any of these changes may potentially affect the potency, immunogenicity or pharmacokinetics of the therapeutic molecule and therefore critical to the product quality. Furthermore, these variants act as impurities when present in excess amounts. As these impurities are a potential health risk, their removal from the final product is a regulatory requirement to ensure the safety and efficacy of a therapeutic antibody. Thus removal or obtaining the variants in an optimum amount in a therapeutic antibody composition creates a significant challenge in the development of methods for the purification of a therapeutic monoclonal antibody, including anti-HER2 antibody.

The prior art discloses various methods for purification of immunoglobulin or antibodies.

WO 89/05157 teaches purification of immunoglobulins by cation-exchange chromatography using varying pH and salt concentrations in the wash and elution steps. 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. US 51 10913 claims purification of murine antibodies using low pH and at least three different pH conditions in the ion- exchange chromatographic step.

WO 1999/057134 describes the use of ion exchange chromatography for purification of polypeptides by changing the conductivity and/or pH, wherein the change in conductivity and/or pH from load to wash steps is in opposite direction to the change in conductivity and/or pH from wash to elution step. US6471335 teaches a method of purifying an antibody, specifically an anti-HER2 antibody, from its contaminants by loading about 20 mg to 35 mg of antibody per ml of cation exchange resin.

US 20090148435 describe a method of purifying antibody using cation exchange chromatography wherein a high pH wash step is used prior to elution.

Prior arts are accompanied by frequent and significant changes in pH or conductivity of the buffer during a chromatography step and in between the chromatographic steps. These changes in buffer conditions are required to remove the acidic variants and/or other impurities such as host cell proteins from the antibody preparation.

However frequent alterations and/or use of high conductivity in buffers, during or in between the steps, may decrease the stability of the antibody, particularly anti- HER2 antibody, which is susceptible to asparagine deamidation resulting in increased content of acidic variants. Furthermore, prior-art describes methods to obtain a particular or fixed amount of acidic variant content in an antibody

composition. However need for specific amounts of acidic variants differ from one antibody to other antibody molecule.

In addition, prior-art's recommendation and claim of using a low antibody load (onto the cation exchange resin) for purification of antibody, specifically for anti- HER2 antibody, may lead to underutilization of the binding capacity of the resin, in turn increasing the process time and cost.

Thus there is a need for improved methods of purifying recombinant antibodies for pharmaceutical use and the present invention addresses this need.

SUMMARY OF THE INVENTION

The present invention provides a method of altering acidic variant content in an anti-HER2 antibody composition by cation exchange chromatography.

Interestingly, the invention has identified that the acidic variant content in anti- HER2 antibody can be altered by altering the column volumes of post load wash buffer that is passed over the cation exchange chromatographic resin. There is a decrease in the acidic variant content when the post load wash buffer column volume is increased and there is an increase in the acidic variant content when the post load wash buffer column volume is decreased. These variations in the acidic variant content brought by altering i.e. increasing or decreasing, number of wash buffer column volume, is consistent over a particular conductivity range of the wash buffer.

Surprisingly, the results achieved in this study also show that the conditions described in the invention facilitates higher antibody load per ml of the resin thus permitting binding of the resin to its optimum capacity. This, in turn, combined with the said advantages of achieving a particular acidic variant content of anti-HER2 antibody composition, increases the throughput of the process.

BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 : Impact of post load wash buffer column volume on acidic variant content of anti-HER2 antibody at a wash buffer concentration of 100 imM Tris acetate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of obtaining an antibody

composition using cation exchange chromatography.

In an embodiment, the invention provides a method of altering the acidic variant content of an antibody composition comprising,

a) loading the said composition onto a cation exchange chromatography

support,

b) washing the cation exchange support with a wash buffer and

c) eluting the antibody from the said support using an elution buffer and wherein the said acidic variant content is increased by decreasing the wash buffer volume passed over the column and/or

wherein the acidic variant content is decreased by increasing the wash buffer volume passed over the column.

The present invention provides a method of obtaining an anti-HER2 antibody composition using cation exchange chromatography.

In another embodiment, the invention provides a method of altering the acidic variant content of an anti-HER2 antibody composition comprising,

a) loading the said composition onto a cation exchange chromatography

support,

b) washing the cation exchange support with a wash buffer and c) eluting the anti-HER2 antibody from the said support using an elution buffer and

wherein the said acidic variant content is increased by decreasing the wash buffer volume passed over the column and/or

In an embodiment, the invention provides a method wherein the said wash buffer conductivity is less than or equal to 5 mS/cm.

In an embodiment, the invention provides a method wherein the said wash buffer conductivity is from about 3 mS/ to about 5 mS/cm.

In an embodiment, the invention provides a method wherein the said wash buffer concentration is about 80 imM to about 100 imM.

In an embodiment, the invention provides a method wherein the amount of antibody loaded onto the cation exchange resin is equal to or greater than 70 % of the dynamic binding capacity of the resin.

In an embodiment, the invention provides a method wherein the basic variant content is reduced or removed in the eluted anti-HER2 antibody composition.

In an embodiment, the cation exchange chromatographic step may include one or more wash steps prior to the elution of the antibody.

In an embodiment, the cation exchange chromatographic step may be preceded by an affinity chromatography e.g., Protein-A affinity chromatography.

In another embodiment, the cation exchange chromatography step may be followed by an anion exchange chromatographic step. The conditions described in the invention also allow the cation exchange eluate to be directly loaded onto the anion exchange chromatography without any adjustment in pH and/or conductivity of the buffer i.e., does not require any buffer exchange step in between the two ion- exchange chromatographic steps, an added advantage to the inventive process.

The invention also provides a method wherein the basic variant content in the final eluate of the antibody composition is reduced to less than 1 %.

The chromatographic steps mentioned in the embodiments may 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.

A "cation exchange resin" or "cation exchange support" mentioned in the embodiments refers to a solid phase which has a negatively charged ligand such as a carboxylate or sulfonate attached thereto. The cation exchange resin can be any weak or strong cation exchange resin or a membrane which could function as a weak or a strong cation exchanger. Commercially available cation exchange resins include, 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 POROS HS® (Applied Biosystems) is used; the resin is made up of cross- linked poly(styrene-divinylbenzene) flow-through particles surface coated with a polyhydroxylated polymer functionalized with sulfopropyl. Alternatively, the support could be a monolithic column, disk or tubular, that performs the function of a cation exchanger.

A "mixed mode ion exchange resin" which could function as a cation exchanger may also be used for carrying out the embodiments.

"Anti-HER2 antibody" as used herein refers to any antibody that is capable of inhibiting one or more of the biological activities of HER 2 (Human Epidermal Growth Factor Receptor 2). The antibody mentioned herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. The antibody may be from various sources, such as murine, human or recombinant and includes chimeric, humanized, fully human or pegylated forms and truncated antibodies and antibody fragments.

The term "acidic variant" as used herein refers to a variant or isoform of the antibody of interest which is more acidic and has a lower pi value than the antibody of interest. The term "basic variant" as used herein refers to a variant or isoform of the antibody of interest which is more basic and has a higher pi value than the antibody of interest.

The antibody of interest mentioned herein can be anti-HER2 antibody or anti- VEGF antibody.

Anti-HER2 antibody as mentioned herein has a pi value in the range of 8.8 to

9.3

The "dynamic binding capacity" (DBC) or 'break-through capacity' of a chromatographic resin is the amount of target protein the resin will bind under actual flow conditions before significant breakthrough of unbound protein occurs.

The break-through capacity of a resin can be obtained experimentally by passing a solution of a particular solute through the column and by directly

monitoring UV absorbance. The point at which some protein passing through the resin is first detected in the eluate (as is no longer able to find an available binding site in the packed resin) is the break through point and is indicated by an increase in the UV absorbance of the process stream leaving the column. The DBC or breakthrough capacity may be expressed in millimoles or milligrams taken up per gram of dry ion exchanger or per cm³ of bed volume.

The term "wash buffer" as used herein, refers to the buffer that is passed over the chromatographic resin following loading of a sample and prior to elution of the antibody.

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-HER2 antibody was cloned and expressed in a Chinese Hamster Ovary cell line and 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 (Prosep vA ultra, VL44x250, 205 imL) that was pre-equilibrated with sodium acetate, NaCI, pH 7.0 buffer. The column was then washed with a buffer containing sodium acetate and EDTA. The bound antibody was eluted using a buffer containing ~ 200 mM acetic acid, pH 2.8.

EXAMPLE 2

Cation exchange chromatography

To begin with, the DBC or break through capacity was determined for the cation exchange chromatographic resin (POROS HS) based on the breakthrough point or breakthrough curve analysis.

The break-through capacity of a resin can be obtained experimentally by passing a solution of a particular solute through the column and by directly monitoring UV absorbance. The point at which some monoclonal antibody passing through the resin is first detected in the eluate (as is no longer able to find an available binding site in the packed resin) is the break through point and is indicated by an increase in the UV absorbance of the process stream leaving the column.

DBC was calculated from the volume of protein solution that has been applied up to a specific break through point (usually 5 or 10%). The volume applied at 5% breakthrough (V5%) was defined from the fraction with 5% of the peak area in the start material.

DBC at 5% = (V (5%) - Vo) Co/Vc

where Co = antibody concentration (mg/ml),

Vc = geometric total volume (ml)

Vo = void volume (ml).

Based on the above, the DBC for POROS HS was estimated to be 58 mg per imL of the resin.

The eluate obtained from the protein-A chromatography procedure described in Example 1 was neutralized and loaded onto the cation exchange resin (POROS HS 50, VL 1 1 X 20) pre-equilibrated with 5 column volume (CV) of equilibration buffer (60 mM Tris acetate buffer, pH 6.0, at a conductivity of 3.0 mS/cm). The mobile phase with a load concentration of about 40 mg of antibody per ml of the cation exchange resin was loaded onto the resin.

This was followed by a wash step that was performed by passing varying amounts of CV of wash buffer consisting of about 100 mM Tris acetate, pH 6.0, at conductivity of about 5 mS/cm. Impact of post load wash buffer column volume on acidic variant content of anti-HER2 antibody at a wash buffer concentration of 100 mM tris acetate is depicted in figure 1 (FIG.1 ).

Alternatively, passing a wash buffer consisting of 80 mM Tris acetate, pH 6.0 at conductivity about 4 mS/cm gave similar results.

The bound antibody was then eluted using approximately 10 CV of elution buffer containing 140 mM to 150 mM acetate buffer, pH 6.0 at a conductivity value of about 6.8 mS/cm to about 7.2 mS/cm.

Experiments with an antibody load of 45 mg/ml or 50 mg/ml of the resin using identical post load wash, and elution conditions, demonstrated similar outcomes. These amounts translate to about 78 % to 86 % of the DBC of the resin respectively and in turn enable optimal utilization of the resin capacity, remarkably increasing the throughput of the process. Interestingly, antibody load as high as 50 mg/ ml of the resin achieved using the present invention, also presents an important advantage over the prior-art that claims a maximum load of only about 35 mg of anti-HER2 antibody per ml of the cation exchange resin.

Similar experimental conditions can be repeated using anti-VEGF antibody to obtain similar outcome in the alteration of the acidic variant content which may increase or decrease by altering the number of post load wash column passed over the column.

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 imL) pre-equilibrated with 5 CV of equilibration buffer comprising 140 mM to 150 mM of Tris acetate pH 6.0. This was followed by a 5 CV of post load wash with equilibration buffer consisting of Tris acetate, pH 6.0 and a conductivity of about 6.8 mS/cm to about 7.2 mS/cm.

The flow-through was collected and subjected to a pre-filter and/or a nano filtration step to obtain an anti-HER2 antibody composition of therapeutic use.

Claims

CLAIMS We Claim:

1 . A method of altering the acidic variant content of an antibody composition comprising,

a) loading the said composition onto a cation exchange chromatography support,

b) washing the cation exchange support with a wash buffer and c) eluting the antibody from the said support using an elution buffer and wherein the said acidic variant content obtained in the eluate is increased by decreasing the wash buffer volume passed over the column and/or

wherein the acidic variant content obtained in the eluate is decreased by increasing the wash buffer volume passed over the column.

2. A method according to claim 1 , wherein the antibody composition is anti-HER2 composition

3. A method according to claim 1 , wherein the conductivity of the said wash buffer is less than or equal to 5 mS/cm.

4. A method according to claim 1 , wherein the conductivity of the said wash buffer is about 3 mS/cm to about 5 mS/cm.

5. A method according to claim 1 , wherein the concentration of the said wash buffer is about 80 imM to about 100 imM.

6. A method according to claim 1 , wherein the elution buffer conductivity is maintained at or below 10 mS/cm.

7. A method according to claim 1 , wherein the amount of antibody loaded onto the cation exchange resin is equal to or greater than 70 % of the dynamic binding capacity of the resin.

8. A method according to claim 1 , wherein the amount of basic variant content in the said eluate of the antibody composition is less than 1 %.

9. A method according to claim 1 , wherein the cation exchange chromatographic step may be preceded by an affinity chromatography.

10. A method according to claim 1 , wherein the cation exchange chromatographic step may be followed by an anion exchange chromatography.

1 1 . A method according to claim 10 wherein the cation exchange eluate may be directly loaded onto the anion exchange chromatography without any adjustment in pH and/or conductivity of the buffer.