FIELD OF THE INVENTION

The present invention relates to a method of purification of anti-VEGF antibody from one or more impurities using cation exchange chromatography. BACKGROUND OF THE INVENTION

Vascular endothelial growth factor (VEGF), also known as vasculotropin, plays a key role in the regulation of normal and abnormal angiogenesis, including that associated with tumor growth (Ferrara N. and Davis-Smyth T., Endocr. Rev. 18: 4-25, 1997). Increased expression of VEGF is seen in most variety of human cancers, including gastrointestinal tumors. (Brown L.F., et.al., Cancer Res. 53(19): 4727-35, 1993 and Uchida S., et.al., Br J Cancer 77(10): 1704-9, 1998). Also, elevated levels of VEGF are reported to cause vascular diseases of the eye specifically in patients with diabetic and other ischemia-related retinopathies.

Furthermore, studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by age-related macular degeneration (Lopez et.al., Invest. Ophthalmol. Vis. Sci. 37: 855-868, 1996) Given the importance and expanding role of VEGF in various pathological conditions, anti-VEGF therapies have become increasingly desirable.

Anti-VEGF antibodies selectively bind with high affinity to human VEGF, neutralize the biological activity of VEGF and thereby suppress the development of a variety of disease conditions. Monoclonal anti-VEGF antibodies such as

bevacizumab (Avastin ^® ) and ranibizumab (Lucentis ^® ) are used for the treatment of certain human cancers, retinal disorders and age-related macular degeneration.

Therapeutic proteins, including monoclonal anti-VEGF 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 and act as impurities when present in inappropriate amounts. The presence of these impurities is a potential health risk, and hence their removal from the final product is a regulatory requirement and creates a significant challenge in the development of methods for the purification of therapeutic monoclonal antibody, including anti-VEGF 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. US 20090148435 describe a method of purifying antibody using cation exchange chromatography wherein a high pH wash step is used prior to elution.

US 7863426 discloses a method of producing a host cell protein reduced antibody preparation comprising steps of cation and anion exchange

chromatography wherein prior to applying the cation exchange eluate to the anion exchange resin, the pH and conductivity of the eluate is adjusted to be substantially similar to the pH and conductivity of the anion exchange resin.

The prior art typically use low or high pH wash buffer conditions, and/or 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 of buffer conditions during or in between the steps may result in considerable reduction in antibody yield and, further, may decrease the stability of the antibody. In particular, frequent buffer changes pose difficulties during scale up or large scale operations. Thus the objective of the current invention is to provide an improved method of purification of anti-VEGF antibody by cation exchange chromatography in which, the pH is kept constant throughout the process and a wash step at specific conductivity range is introduced to remove significant amounts of acidic variant impurity prior to the elution of the antibody. Thus the process requires only minor step increase in conductivity during elution. In addition, the eluate may be collected as a 'whole' without the need for fractionation and further pooling of desired fractions. Another objective of the invention is to elute the antibody from the cation exchange resin using an elution buffer of a specific conductivity range, such that the eluate is suitable for loading onto the next chromatographic step without any further adjustment in conductivity and pH. Thus, this buffer condition obviates a major process complexity i.e., the buffer exchange step in between the chromatographic steps.

SUMMARY OF THE INVENTION The present invention provides a method of purification of anti-VEGF antibody by cation exchange chromatography wherein the process includes wash and elution steps of specific conductivity range to remove the acidic variants and other impurities, thus increasing the recovery and purity of the antibody.

In addition, the process facilitates collection of the cation exchange eluate as 'whole' and eliminates the need for fractionation and "cautious pooling" of desired fractions. Also, the cation exchange eluate may be loaded onto the subsequent anion exchange chromatographic resin without any further adjustments in

conductivity and pH.

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 antibody of interest.

FIG.2 is an illustration of a chromatogram from the procedure as described in example 2. The figure depicts the impact of wash buffer conductivity in removing the acidic variant impurity and in recovery of protein of interest, Chromatograms overlaid on one another represent elution profiles of acidic variant and the antibody of interest, when wash buffers varying in conductivity (represented in mS/cm) are used. Peaks falling under the region marked "AcV" represent the acidic variant. Peaks, under region "A" represent the antibody of interest.

FIG.3 is an illustration of a chromatogram from the procedure as described in example 2. The line marked "Cond" represents the increase in conductivity in mS/cm. Peak "AcV" represents the acidic variant eluted with wash buffer at a conductivity of 5.2 mS/cm. Peak "A" represents the antibody of interest.

FIG.4 is an illustration of a chromatogram from the procedure as described in example 3. The line marked "Cond" represents the increase in conductivity in mS/cm. Region marked "A" represents the antibody of interest obtained as a flow through.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of purification of anti-VEGF antibody from one or more impurities using cation exchange chromatography. In an embodiment, the invention provides a method of purification of an anti-

VEGF antibody from a sample comprising one or more impurities, comprising; a) loading the sample onto a cation exchange chromatographic resin b) washing the cation exchange resin with a wash buffer at a conductivity from about 5.0 mS/cm to about 5.5 mS/cm, and c) eluting the said antibody from the cation exchange resin with an elution buffer.

In a further embodiment, the desired antibody is eluted from the cation exchange resin with an elution buffer at conductivity from about 7.0 mS/cm to about 7.5 mS/cm

In another embodiment, the invention provides a method of purification of an anti-VEGF antibody by cation exchange chromatography, wherein the cation exchange eluate is collected as a whole or as a single fraction from the cation exchange resin. In yet another embodiment, the invention provides a method of purification of an anti-VEGF antibody by cation exchange chromatography, and the eluate obtained from the cation exchange resin is then loaded onto an anion exchange resin, wherein the conductivity and pH of the load is same as the conductivity and pH of the cation exchange eluate.

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, anion exchange chromatographic step is performed in the flow-through mode.

The chromatographic steps mentioned in the embodiment 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" 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.

"Anion exchange resin" mentioned in the embodiments refers to a solid phase which has a positively charged ligand such as quaternary amino groups, attached thereto. The anion exchange resin can be 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-

CH2CHOHCH2OCH2CHOHCH2N+(CH3)3 functional group.

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

"Anti-VEGF antibody" as used herein refers to any antibody that is capable of inhibiting one or more of the biological activities of VEGF. The antibody may be isolated 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 terms "purification" or "purifying" as used interchangeably herein, refer to increasing the degree of purity of a protein of interest or the antibody, from a sample comprising the antibody and one or more impurities. Typically, the degree of purity of the target antibody is increased by removing (completely or partially) at least one impurity from the composition or sample.

The term "impurity" as used herein refers to any material other than the desired antibody. The impurity includes, without limitation: host cell materials, such as Chinese Hamster Ovary Proteins (CHOP), leached protein A, nucleic acid, acidic variants, fragment, aggregate or derivative of the desired antibody, another polypeptide, endotoxin, viral contaminant and cell culture media component.

The term "acidic variant" as used herein refers to a variant of the polypeptide or antibody of interest which is more acidic and may have a slightly different pi (e.g. as determined by cation exchange chromatography) than the polypeptide or antibody of interest. A deamidated variant of the polypeptide or antibody of interest is an example but not a limitation of an acidic variant.

The term "sample" as used herein comprises the antibody (i.e., anti-VEGF antibody) and one or more impurities. The sample may be "partially purified" (i.e., having been subjected to one or more purification steps) or may be obtained directly from a host cell or organism producing the antibody, a harvested cell culture fluid or a cell culture supernatant.

The term "load" as used herein refers to the sample loaded onto an ion exchange resin in an appropriate buffer. 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. In some cases, the wash buffer and loading buffer may be the same or different.

The term "elution buffer" as used herein refers to the buffer that is used to elute the antibody from the ion exchange resin.

The term "Flow-through mode" as used herein refers to that process wherein the antibody is not bound to the chromatographic resin but instead obtained in the unbound or "flow-through" fraction during loading or post load wash of the

chromatography resin. The buffering agents used in the buffer solutions include, and are not limited to citrate, phosphate, hydrochloride, acetate, chloride, 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. EXAMPLE 1

Protein A chromatography

Anti-VEGF antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 7,060,269, 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, followed by a wash with Tris buffer (pH 7.0) of high conductivity. 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 (POROS HS 50, VL32X250, 160 ml) pre-equilibrated with 50 mM phosphate buffer, pH 6.2, followed by washing the resin with a wash buffer of 35 mM phosphate buffer, pH 6.2. A second wash step was performed with 15 column volume (CV) of buffer consisting of 60 mM

phosphate, pH 6.2, at a conductivity of 5.0 to 5.5 mS/cm. Further increase in conductivity to 5.8 mS/cm, resulted in loss of recovery of antibody of interest and hence the wash buffer conductivity is maintained below the conductivity value of 5.8 mS/cm. The bound antibody was then eluted using 15 CV 90 mM phosphate, pH 6.2, at conductivity between 7 to 7.5 mS/cm.

Table 1 : Impact of wash buffer conductivity on removal of acidic variant

Wash buffer Amount of Acidic Amount of Acidic

conductivity (mS/cm) variant in load (%) variant in eluate (%)

4.8 18 16.2

5.04 18 1 1 .0

5.2 18 7.6

Table 2: Amount of impurities at the end of each chromatographic step

NA- Not Applicable

BDL - Below Detection Limit

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 CV of equilibration buffer consisting of 90 mM sodium phosphate, pH 6.2, conductivity of 7 to 7.5 mS/cm. This was followed by a 5 CV of post load wash with equilibration buffer and the load and wash flow- through was collected.