DESC:FIELD OF THE INVENTION
The present invention relates to the field of cell culture. In particular, the present invention discloses a cell culture process for controlling post translational modifications in an antibody composition.
BACKGROUND OF THE INVENTION
Post-translational modifications (PTM) are covalent modification in proteins, which occur, during or after protein biosynthesis and enrich the structural and biophysical diversity of proteins. PTMs may be classified according to the modification involved: the addition of functional groups (e.g., phosphorylation and glycosylation); attachment of other polypeptides (e.g., ubiquitination and SUMOylation); changing of the chemical nature of amino acids (e.g., acetylation, deamidation and oxidation); and cleavage of the backbone by proteolysis. PTMs influence the tertiary and quaternary structure of a protein and thus it is necessary to target appropriate PTM profile/content to ensure efficacy and safety of therapeutic proteins.
Glycosylation is one of the most important PTM associated with eukaryotic proteins. The two major types of glycosylation in eukaryotic cells are N-linked glycosylation and O-linked glycosylation. There is heterogeneity associated with both N and O-linked glycosylation, firstly, because not all potential glycosylation sites are glycosylated and, secondly, due to difference in range of sugar structures that may be attached at a particular glycosylation site. This heterogeneity in glycosylation has been shown to significantly impact efficacy of therapeutic proteins, in particular, effector functions of therapeutic antibodies. For example, several studies have established the role of galactosylated glycans in influencing complement dependent cytotoxicity (CDC) (H. Gazzano-Santoro, P. Ralph, T. Ryskamp, A. Chen, V. Mukku, J. Immunol. Methods 202 (1997) 163 & Boyd, P. N., Lines, A. C. & Patel, A. K.(1995), Mol. Immunol. 32, 1311–1318 & Nature Reviews Drug Discovery 8, 226-234, March 2009). Thus, for a particular therapeutic protein, in which effector function is dictated predominantly by CDC, it is desirable to obtain appropriate galactosylation variants content in the said therapeutic protein.
Another subset of PTM generally associated with biopharmaceuticals 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. Similarly, presence of additional lysine residues, glycine amidation, succinimide formation, amino acid oxidation or removal of sialic acid introduce additional positive charges that may result in considerable basic variants population. However, and in particular, the acidic species has been demonstrated to have low binding response and binding potency as compared to the main or basic peaks in an antibody composition. Thus, it becomes additionally imperative to regulate and achieve target acidic species content in an antibody composition.
Besides maintaining the quality attributes (including glycosylation and variants content) within defined acceptance limits, regulatory agencies require demonstration of documented evidence of ‘consistency’ in the specific process, employed for the production of a therapeutic protein.
Alongside, any cell culture process optimized for the commercial production of therapeutic protein shall also be aimed to result in high cell viability and titer.
Thus a cell culture process for manufacturing a therapeutic protein needs to be qualified in three aspects; firstly, to achieve target PTM values for adequate potency, efficacy or stability, secondly, demonstrate consistency between batches or reduced batch to batch variability and, thirdly, economics of manufacturing process (titer). Thus, the objective of the present invention is to develop a cell culture process that meets the aforementioned requirements.

SUMMARY OF THE INVENTION
The present invention discloses a cell culture process for obtaining an antibody composition comprising target percentage of post-translational modifications, in particular, galactosylated glycans content and/or acidic variants content. The cell culture process is a fed-batch process, wherein the cells are cultured in the presence of controlled concentration of one or more nutrients, followed by harvesting the culture at specific concentration of by-products of the fed nutrients to yield the target PTM values in an antibody composition. The advantage of the cell culture process, whereby, harvest is determined by ‘by-products’ of fed nutrients and not by conventional means such as day or time of harvest, is that it offers less batch to batch variability, particularly in galactosylated glycans content and acidic variants content. Additionally, the process results in an adequate yield of the therapeutic product.
Further, for the aforementioned cell culture process which involves harvesting based on the level of by-products, the measurement equipment for by-products could be integrated with the vessel or bioreactor for cell culture, subsequently be used for on-line and/or real time measurement viz., automated, reducing the chances of manual contamination.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 represents concentration of ammonium in culture medium, at the harvest of cell culture, for different batches, performed as per example-I, and corresponding percentage of galactosylated glycans content in TNFa antibody composition for each batch. The dotted line represents the trendline (linear).
Figure 2 represents concentration of ammonium in culture medium, at the harvest of cell culture, for different batches, performed as per example-I, and corresponding titer of TNFa antibody composition for each batch.
Figure 3 represents concentration of lactate in culture medium, at the harvest of cell culture, for different batches, performed as per example-I, and corresponding percentage of acidic variants content in TNFa antibody composition for each batch. The dotted line represents the trendline (linear).
Figure 4 represents concentration of lactate in culture medium, at the harvest of cell culture, for different batches, performed as per example-I, and corresponding titer of TNFa antibody composition for each batch.
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 medium” refers to a liquid or gel containing nutrients to support the growth of cells. Culture medium may be chemically defined or alternatively may contain undefined components such as hydrolysates. Typically, a cell culture medium 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.
The term “antibody” refers to whole antibody or an antigen binding fragment thereof.
“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. acidic or basic species can be observed when antibodies are analyzed using charge based methods such as ion exchange chromatography (IEX) or isoelectric focusing (IEF). An "acidic variant" is a variant of a protein of interest which is more acidic (e.g. as determined by cation exchange chromatography) than the protein of interest. An example of an acidic variant is a deamidated variant.
“Galactosylated glycans” refers 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).
Detailed description of the embodiments
The present invention discloses a cell culture process, the process comprising culturing cells in the presence of controlled concentration of one and/or more nutrients in the culture medium, followed by harvesting the cell culture at defined concentration range of by-product of one and/or more nutrients to obtain an antibody composition comprising target percentage of post translational modifications.
One embodiment of the invention discloses a cell culture process, the process comprising culturing cells in the presence of glutamine, controlled at or below a concentration of 5mM, followed by harvesting the cell culture when the ammonium concentration is between about 16mM/L to about 23mM/L to obtain an antibody composition comprising about 19% to about 25% of galactosylated glycans content.
One embodiment of the invention discloses a cell culture process, the process comprising culturing cells in the presence of glucose, controlled at or below a concentration of 5g/L, followed by harvesting the cell culture when the lactate concentration is between about 2.4g/L to about 7.2g/L, to obtain an antibody composition comprising about 13% to about 21% of acidic variants content.
In any of the above mentioned embodiments, the titer of the antibody obtained is greater than 1g/L, more preferably greater than 1.75g/L.
In any of the above mentioned embodiments, the antibody so obtained is TNFa antibody, in particular a human TNFa antibody
In any of the above mentioned embodiments, the said human TNFa antibody is adalimumab.
In any of the above mentioned embodiments, the cell culture process is operated in a fed batch mode.
In any of the above mentioned embodiments, the concentration of one and/or more nutrients, in particular, the concentration of glucose and/or glutamine is controlled in protein production phase, in particular after day 3, more particularly after day 7.
In any of the above mentioned embodiments, the cell culture process may comprise a temperature shift, preferably a shift from a higher temperature value to a lower temperature value.
In any of the above mentioned embodiments, the cell culture process may comprise a temperature shift from 37oC to 34oC.
In any of the above mentioned embodiments, the cell culture process may comprise a pH shift, preferably a shift from a higher pH value to a lower pH value.
In any of the above mentioned embodiments, the cell culture process may comprise a pH shift from 7.2 to 7.0, more preferably a pH shift from 7.2 to 6.9.
Cell culture media that are useful in the present invention include but are not limited to, commercially available products ActiPro (Hyclone), PF-CHO (HyClone®), PowerCHO®2 (Lonza), Zap-CHO (Invitria), CD CHO, CD OptiCHOTM and CHO-S-SFMII (Invitrogen), ActiPro (HyClone®), ProCHOTM (Lonza), CDM4CHOTM (Hyclone), DMEM (Invitrogen), DMEM/F12 (Invitrogen), Ham’s F10 (Sigma), Minimal Essential Media (Sigma), and RPMI -1640 (Sigma) or combinations thereof.
Cell culture feed that are useful in the invention include but are not limited to, commercially available products Cell Boost 2 (CB-2, GE Healthcare Hyclone, Catalogue no SH 30596.03), Cell Boost 4 (CB-4, GE Healthcare HyClone, Catalog no. SH30928), Cell Boost 7A (CB-7A, GE Healthcare HyClone, Catalog no. SH31026) and Cell Boost 7B (CB-7B, GE Healthcare HyClone, Catalog. No. SH31027).
Certain aspects and embodiments of the invention are more fully defined by reference to the following examples. These examples should not, however, be construed as limiting the scope of the invention.
EXAMPLES
Example I
TNFa antibody was cloned and expressed in a recombinant CHO (rCHO) cell line as described in detail in Molecular Cloning: A laboratory Manual by Green and Sambrook, which is incorporated herein by reference. rCHO cells expressing antibody at a seeding density of about 0.3 million cells/mL were seeded in culture medium at 37°C and pH 7.2. Subsequently, on day 4, temperature was reduced to 34 °C and the pH was reduced to 6.9. Feed was added to the cell culture medium on day 3, 5 and 7. From day 3 onwards, glucose concentration was controlled at or less than 5g/L and/or glutamine concentration was controlled at or below 5mM. Consequently, culture is harvested at defined ammonium and/or lactate concentration to obtain target percentage acidic variants content and/or galactosylated glycans content.
Concentration of ammonium in culture medium at harvest of cell culture for different batches and the corresponding percentage of galactosylated glycans content in TNFa antibody composition for each batch is as shown in Figure-I and titer of TNFa antibody composition for each of these batches is shown in Figure-II. Concentration of lactate in culture medium at harvest of cell culture for different batches and the corresponding percentage of acidic variants content in TNFa antibody composition for each batch is as shown in Figure-III and titer of TNFa antibody composition for each of these batches is shown in Figure-IV.
,CLAIMS:We claim:
1. A cell culture process comprising culturing cells in the presence of controlled concentration of one and/or more nutrients in the culture medium, followed by harvesting the cell culture at defined concentration range of by-product of one and/or more nutrients to obtain an antibody composition comprising target percentage of post translational modifications.
2. The process of claim 1, wherein the nutrients controlled is glutamine at or below a concentration of 5mM followed by harvesting the cell culture when the ammonium concentration is between about 16mM/L to about 23mM/L.
3. The process of claim 1, wherein the antibody composition so produced comprises about 19% to about 25% of galactosylated glycans content.
4. The process of claim 1, wherein the nutrients controlled is glucose at or below a concentration of 5g/L followed by harvesting the cell culture when the lactate concentration is between about 2.4g/L to about 7.2g/L.
5. The process of claim 1, wherein the antibody composition so produced comprises about 13% to about 21% of acidic variants content.
6. The process of claim 1 wherein the titer of the antibody obtained is greater than 1g/L, more preferably greater than 1.75g/L.
7. The process of claim 1 wherein the antibody so obtained is TNFa antibody, in particular a human TNFa antibody.
8. The process of claim 1 wherein the said human TNFa antibody is adalimumab.
9. The process of claim 1 wherein the cell culture process is operated in a fed batch mode.
10. The process of claim 2 or 4 wherein the concentration of glucose and/or glutamine is controlled in protein production phase, in particular after day 3, more particularly after day 7.