“Cell Culture Process”

DESC: FORM 2 THE PATENTS ACT 1970 (39 of 1970) & THE PATENTS RULES, 2003 PROVISIONAL / COMPLETE SPECIFICATION (See section 10 and rule 13) 1. TITLE OF THE INVENTION: “Cell culture process” 2. APPLICANT(S)

(a) NAME: Dr. Reddy’s Laboratories Limited (b) NATIONALITY: Indian (c) ADDRESS: 8-2-337, Road No. 3, Banjara Hills, Hyderabad, Telangana, India-500 034 3. PREAMBLE TO THE DESCRIPTION PROVISIONAL SPECIFICATION The following specification describes the invention. COMPLETE SPECIFICATION The following specification particularly describes the invention and the manner in which it is to be performed. 4. DESCRIPTION (Description shall start from next page)

Description starts from next page.

5. CLAIMS Claims are enclosed in a separate page

6. DATE AND SIGNATURE Given at the end of last page of specification 7. ABSTRACT OF THE INVENTION

Given at a separate page.

FIELD OF INVENTION The present invention relates to the field of cell culture disclosing a process for controlling the post translational modifications, in particular, galactosylated glycans and acidic variants content in an antibody composition. BACKGROUND OF INVENTION Biological molecules, in particular monoclonal antibodies (mAbs) have emerged as important therapeutic agents owing to their high specificity for key targets of inflammatory, oncological and auto immune diseases. Therapeutic mAbs are produced in cells which have been engineered to incorporate the gene encoding the antibody of interest. Mammalian cells are currently the production system of choice for producing the therapeutic mAbs as they can deliver high productivity with suitable product quality attributes e.g. post translational modifications (PTMs). Post translational modifications (PTM) are covalent modifications of antibody molecules, which occur during or after biosynthesis, and enrich the structural and biophysical diversity of an antibody. PTM’s may be classified according to the modification involved: the addition of functional groups (e.g., phosphorylation and glycosylation); attachment of other proteins or peptides (e.g., Ubiquitination, SUMOylation); changing of the chemical nature of amino acids (e.g., acetylation, deamidation and oxidation) or cleavage of the backbone by proteolysis. Glycosylation is one of the most important PTM which involves addition of sugars/glycans to the antibodies and is of two major types - N-linked glycosylation and O-linked glycosylation. As represented in Table – I, for N-linked glycosylation, there are a range of glycan moieties and structures associated with each glycosylation site. This glycan heterogeneity has profound effect on immunogenicity, solubility and half-life, thus, influencing the safety and efficacy of therapeutic antibodies. In particular, several studies have demonstrated that an increase in galactosylated glycan levels in antibodies is associated with an increase in it’s biological effector function as measured by complement dependent activity (CDC) assay (H. Gazzano-Santoro, P. Ralph, T. Ryskamp, A. Chen, V. Mukku, J. Immunol, Methods 202 (1997) 163 & Boyd, PN,, Lines, AC, & Patel, A.K. (1995), Mol. Immunol, 32, 1311-1318). Furthermore, it has also been reported that G1F-G1F glycoform of rituximab triggered a CDC response twice as large as that triggered by the G0F-G0F glycoform (Nature Reviews Drug Discovery 8, 226-234, March 2009). Another subset of PTM generally associated with mAbs are charge variants. Modifications such as deamidation, sialylation, formation of various types of covalent adducts, e.g., glycation, and C-terminal lysine cleavage, result in an increase in the net negative charge on the mAbs causing a decrease in pI values, thereby leading to formation of acidic variants. Basic variants are due to no cleavage/ presence of C-terminal lysine or glycine amidation, succinimide formation, amino acid oxidation or removal of sialic acid, which introduce additional positive charges or removal of negative charges. In particular, the acidic species has been demonstrated to have low binding response and binding potency as compared to the main or basic peak. Hence, it may be desirable to control the acidic species in an antibody composition. Thus, the objective of the present invention is to develop a cell culture process to control galactosylated glycan content and/or charge variants content in an antibody composition, particularly in a therapeutic antibody composition.

SUMMARY OF THE INVENTION The invention discloses a cell culture process for producing an antibody composition comprising culturing cells in a culture media comprising amino acids and/or one or more metal ions to yield increased galactosylated glycan content and decreased acidic variant content in the aforementioned antibody composition. Particularly, the cell culture process for producing an antibody composition comprises culturing cells in a culture media comprising hydrophobic amino acid, in particular tryptophan, and/or one or more divalent metal ions such as copper and/or manganese in the culture media to yield increased galactosylated glycan content and decreased acidic variant content in the aforementioned antibody composition. DETAILED DESCRIPTION OF THE INVENTION Definitions The term “cell culture process” as used herein refers to a process of culturing a population of cells that are capable of producing recombinant protein of interest or antibody. The term “culture media” refers to a solid, liquid or semi-solid containing nutrients to support the growth of cells. Culture media may be chemically defined or alternatively may contain undefined components such as hydrolysates. Typically, a cell culture media contains amino acids, energy sources, lipids, vitamins, growth factors, metal ions and other trace elements etc. The culture medium is usually formulated to a particular osmolality and pH values that are optimal for a particular cell line. “Antibody composition” refers to a population of antibody molecules or fragments thereof. The population of antibody molecules may have one or several post translational modifications (PTM), imparting the antibody molecules a different molecular weight, charge, solubility or combinations thereof. “Charge variants” i.e. variant species having acidic or basic charge due to various post translational modification can be detected by Ion exchange chromatography (IEX) or Isoelectric focusing (IEF). The term ‘main peak’ refers to the peak that elutes in abundance (major peak) during ion exchange chromatography. In particular, during cation exchange chromatography (CEX), the peak that elutes earlier than the main peak has charge that is acidic relative to main peak and is termed as acidic variant peak. The peak that elutes later than the main peak has charge that is basic relative to the main peak and is termed as basic variant peak. “Galactosylated glycans” refer to glycans containing terminal galactose residues such as G1A, G1B, G1AF, G1BF, G2, G2F and G2SF. Various methods described in the art such as Wuhrer et. Al., Ruhaak L.R., and Geoffrey et al., can be used for assessing glycovariants present in a glycoprotein composition (Wuhrer M. et al., Journal of Chromatography B, 2005, Vol.825, Issue 2, pages 124-133, Ruhaak L.R., Anal Bioanal Chem, 2010, Vol.397:3457-3481, Geoffrey, R.G.et.al. Analytical Biochemistry 1996, Vol.240, pages 210-226). The diversity in glycan structure in an immunoglobulin is shown in the accompanying Table-I.

Table – I: Representative of the various N-linked glycan structures in an immunoglobulin

DETAILED DESCRIPTION OF THE EMBODIMENTS The present invention discloses a cell culture process comprising culturing cells in a culture media comprising an amino acid and/or one or more divalent metal ions, thereby controlling the post translational modifications, in particular, galactosylated glycan content and acidic variant content in the produced antibody composition. One embodiment of the invention discloses a cell culture process comprising culturing cells in a culture media comprising a hydrophobic amino acid and one or more divalent metal ions to increase galactosylated glycan content and decrease acidic variants content in the produced antibody composition. In the above mentioned embodiment the amino acid is tryptophan. In the above mentioned embodiment, divalent metal ions are selected from a group consisting of copper and manganese. In the above mentioned embodiments, the percentage increase in galactosylated glycan content in the antibody composition is at least 21%. In any of the above embodiments, if any, the percentage decrease in acidic variant content in an antibody composition is at least 18 %. In any of the above embodiments, the galactosylated glycan content in the antibody composition is not less than about 33%. In any of the above embodiments, the acidic variant content in an antibody composition is less than 30%, more particularly less than 27%. In any of the above mentioned embodiments, the said antibody is TNFa antibody, in particular a human TNFa antibody. In the above embodiments, the said human TNFa antibody is adalimumab. In any of the above mentioned embodiments, the amino acid and metal ions may be in basal media or part of feed or added as individual supplement. Another embodiment of the invention discloses a cell culture process, for producing an adalimumab composition, the process comprising culturing cells in a culture media comprising tryptophan at a concentration of 2 g/L and copper and manganese ions at concentrations of 0.25 µM and 2 µM respectively to obtain 33% of galactosylated glycans and 26% acidic variants in the adalimumab composition. In any of the above mentioned embodiments, the cells are mammalian cell, more preferably CHO cells. In any of the above mentioned embodiments, the cell culture process may comprise a temperature shift, preferably from a higher value to a lower temperature value. In any of the above mentioned embodiments, the cell culture process may further comprise a temperature shift from 37ºC to 34ºC. In any of the above mentioned embodiments, the cell culture process may further comprise a pH shift, preferably from a higher value to a lower pH value. In any of the above mentioned embodiments, the cell culture process may further comprise a pH shift from about 7.2 to about 6.9. EXAMPLES Example – I An anti-TNF-a antibody was cloned and expressed in a culture media using a CHO cell line as detailed in Molecular Cloning: A laboratory Manual by Green and Sambrook. rCHO cells expressing antibody were seeded at a density of ~0.3 million cells/ml in Media - I. The cell culture was maintained at a temperature of about 37ºC and pH of about 7.2. On day 4, the temperature was reduced to about 34ºC and pH was reduced to about 6.9. The antibody, produced by this process [Example IA] was harvested and analyzed. The results of galactosylated glycans and acidic variants for the same are reflected in Table-II. In an alternate condition viz., Example Ib, CHO cells were cultured as aforementioned in Media – I which further comprised of copper (0.25µM) and manganese (2µM) and was further supplemented on Day 6 with tryptophan (2g/L). The antibody (IB) was harvested and analyzed. The results of galactosylated glycan and acidic variant content is mentioned in Table-II. Example no. % Galactosylated glycans % Acidic variants Example-IA 27.3 32.4 Example-IB 33.0 26.5 Table - II: Percentage of galactosylated glycan and acidic variant content ,CLAIMS:We claim: 1. A cell culture process comprising culturing cells in a culture media to obtain a glycoprotein composition wherein the culture media comprises amino acids and one or more metal ions for increasing galactosylated glycan content and for decreasing acidic variant content in the said glycoprotein composition. 2. The process according to claim 1, wherein the amino acid is a hydrophobic amino acid and the metal ions are divalent metal ions. 3. The process according to claim 1, wherein the hydrophobic amino acid is tryptophan and the divalent metal ions are selected from a group consisting of copper and manganese. 4. The process according to claim 1, wherein the percentage increase in galactosylated glycan content in the antibody composition is at least 21%. 5. The process according to claim 1, wherein the percentage decrease in acidic variant content in an antibody composition is at least 18 %. 6. The process according to claim 1, wherein the galactosylated glycan content in the antibody composition is not less than 33%. 7. The process according to claim 1, wherein the acidic variant content in an antibody composition is less than 27%. 8. The process according to claim 1, wherein the cells producing the said glycoprotein are Chinese Hamster Ovary (CHO) cells. 9. The process according to claim 1, wherein the glycoprotein so produced is an anti-TNF-a antibody. 10. The process according to claim 1, wherein culture media comprises of tryptophan at a concentration of 2 g/L and copper and manganese ions at concentrations of 0.25 µM and 2 µM respectively to obtain 33% of galactosylated glycans and 26% acidic variants in the adalimumab composition.