FIELD OF THE INVENTION

The present invention relates to the field of cell culture and in particular to a cell culture process for controlling post translational modifications in an antibody composition.

BACKGROUND OF THE INVENTION

Post-translational modification (PTM) are covalent modification that occur in most eukaryotic proteins during or after their biosynthesis and they 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 has been demonstrated to significantly impact their biological activity and stability.

Hence, it is necessary to control and target appropriate PTM profile/content in a therapeutic protein and is mandated by regulatory agencies to ensure the efficacy and safety of such protein. 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, in which glycans are attached to the asparagine of the recognition sequence Asn-X-Thr/Ser, where "X" is any amino acid except proline, and O-linked glycosylation in which glycans are attached to serine or threonine. Specifically, the structure and composition of the glycan moieties of a protein have a profound effect on its immunogenicity, solubility and half-life, thus influencing its safety and efficacy in a therapeutic use. Studies by Gazzano-Santoro et.al has demonstrated that an increase in GO glycan levels of antibodies is associated with a reduction in biological activity 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). Removal of terminal galactose residues from the chimeric mouse-human lgG1 antibody (alemtuzumab) reduces CDC, without effecting FcyR-mediated functions (Boyd, P. N., Lines, A. C. & Patel, A. K.(1995), Mol. Immunol. 32, 131 1 -1318). Further, it has been reported that G1 F-G1 F 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). Thus, given the role of the galactosylation on the activity and efficacy of a glycoprotein, monitoring and controlling the galactosylation level in a glycoprotein composition becomes critical.

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 imAbs 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. It may thus be desirable to control the acidic species in an antibody population.

As said earlier, all PTMs, in particular, glycosylation and charge variants are critical quality attributes of a product which are controlled by a cell culture process and may need to be modulated to an appropriate amount for establishing the efficacy and stability of a therapeutic glycoprotein. Thus, the objective of the present invention is to develop a process or method to control individual glycan content and/or charge variants content in an antibody composition, particularly in a therapeutic antibody composition. SUMMARY

The invention describes a cell culture process for controlling acidic variant and/or galactosylated glycans content in an antibody composition by modulating the concentration of amino acids in the culture media, used in culturing the cells that produce the aforementioned antibody composition. In particular, the disclosed cell culture process comprises modulating the concentration of charged amino acids and hydrophobic amino acids in the cell culture media, used in culturing the cells that produce an antibody composition comprising desired acidic variant and/or galactosylated glycans content in an antibody composition. Specifically, an increase or decrease of the charged amino acids level relative to the hydrophobic amino acids level in a cell culture media, used in culturing the cells that produce an antibody composition comprising specific / range of charged variant and/or galactosylated glycan content in an antibody composition. In addition, modulating the cumulative concentration of charged and hydrophobic amino acids, relative to each other, results in about 50 % increase in titer, to about 3 g of antibody /L of culture media at harvest.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 represents concentration of charged amino acids and concentration of hydrophobic amino acids in Media l-IV, and their effect on the acidic variants content of TNFa antibody composition, as demonstrated in example-l. Figure 2 represents the average of concentration of charged amino acids and concentration of hydrophobic amino acids in Media l-IV and Media V- VIII, and their effect on the galactosylated glycans content of TNFa antibody composition, as demonstrated in example-ll. Figure 3 represents the average of concentration of charged amino acids and concentration of hydrophobic amino acid in Media l-IV and Media V- VIII, and their effect on the titer (in g/L) of TNFa antibody composition, as demonstrated in example-ll.

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 or antibody of interest.

The term "culture media" refers to a compositions, inclusive of liquid, solid or gel, having nutrients to support the growth of cells. Culture media may be chemically defined or 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 media is usually formulated to a particular osmolality and pH values that are optimal for a particular cell line.

The term "antibody" refers to an intact 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 G1 A, G1 B, G1 AF, G1 BF, 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). "Charged amino acids" are amino acids that have substituents that carry either negative or positive charges in aqueous solution at neutral pH. Charged amino acids are hydrophilic in nature. Out of the 20 amino acids, aspartic acid/aspartate (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg) & histidine (His) represent charged amino acids. "Hydrophobic amino acids" are amino acids with nonpolar substituents. The nine amino acids that have hydrophobic side chains are glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (lie), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).

The term "modulate" with reference to concentration of amino acids in a cell culture media means increasing or decreasing the concentration of either one amino acid or a combination of amino acids, from a first value to a second value, wherein the second value is higher or lower than the first value. Detailed description of the embodiments

The present invention discloses a cell culture process for controlling the post translational modifications, in particular, acidic variants content and galactosylated glycans content in an antibody composition, by modulating the concentration of amino acids in the cell culture media, used in culturing the cells that produce the aforementioned antibody composition.

An embodiment of the invention discloses a cell culture process for controlling the acidic variants content in an antibody composition, by modulating the concentration of charged and hydrophobic amino acids in the cell culture media, used in culturing the cells that produce the aforementioned antibody composition.

One embodiment of the invention discloses a cell culture process for controlling the acidic variants content in an antibody composition, by modulating the cumulative concentration of charged amino acids relative to the cumulative concentration of hydrophobic amino acids in the cell culture media, used in culturing the cells that produce the aforementioned antibody composition.

An embodiment of the invention discloses a cell culture process for reducing the acidic variants content in antibody composition, wherein the cumulative concentration of charged amino acids is lower relative to the cumulative concentration of hydrophobic amino acids in the culture media, used in culturing the cells that produce the aforementioned antibody composition. Yet another embodiment of the invention discloses a cell culture process for reducing the acidic variants content in antibody composition, wherein the cumulative concentration of charged amino acids is equal to the cumulative concentration of hydrophobic amino acids in the culture media, used in culturing the cells that produce the aforementioned antibody composition.

In any of the above mentioned embodiments, the charged amino acids, that are modulated to control the acidic variants content, are selected from the group consisting of aspartate, arginine and lysine, and the hydrophobic amino acids that are modulated to control the acidic variants content, are selected from the group consisting of proline and valine.

In any of the above embodiments, the acidic variants content in the antibody composition is less than about 17%, preferably less than about 15%.

In any of the above embodiments, the percentage reduction in acidic variants content is at least 10%.

In another embodiment, the invention discloses a cell culture method for controlling galactosylated glycans content in an antibody composition, by modulating the concentration of charged and hydrophobic amino acids in the cell culture media, used in culturing the cells that produce the aforementioned antibody composition.

An embodiment of the invention discloses a cell culture process for increasing galactosylated glycans content in an antibody composition, wherein the concentration of charged amino acids is greater relative to the concentration of hydrophobic amino acids in the culture media, used in culturing the cells that produce the aforementioned antibody composition.

One embodiment of the invention discloses a cell culture process for controlling the galactosylated glycans content in an antibody composition, by modulating the cumulative concentration of charged amino acids relative to the cumulative concentration of hydrophobic amino acids in the cell culture media, used in culturing the cells that produce the aforementioned antibody composition. In an embodiment, the invention discloses a cell culture process for increasing galactosylated glycans content in an antibody composition, wherein the cumulative concentration of charged amino acids is greater relative to the concentration of hydrophobic amino acids in the cell culture media, used in culturing the cells that produce the aforementioned antibody composition.

In any of the above mentioned embodiments, the charged amino acids, that are modulated to control the galactosylated glycans content, are selected from the group consisting of aspartate, arginine and lysine and hydrophobic amino acids, that are modulated to control the galactosylated glycans content, are selected from the group consisting of isoleucine and phenylalanine.

In any of the above embodiments, for controlling galactosylated glvan content in an antibody composition, the cell culture media comprises greater than or equal to 1 .5 g/L of each of charged amino acid concentration, and greater than 1 .0 g/L of each of hydrophobic amino acid concentration. In other words, the concentrations of aspartate, arginine or lysine is > 1 .5 g /L and the concentration of isoleucine or phenylalanine is > 1 .0 g /L in the cell culture media. In any of the above embodiments, the galactosylated glycans content in an antibody composition is greater than about 19%.

In any of the above embodiments, the percentage increase in galactosylated glycans content is about or more than 25%.

In any of the above embodiments, disclosed to control or increase the content of galactosylated glycans in an antibody composition, the titer of the said antibody is more than 2g/L on harvest.

In the above embodiment, the increase in titer is about 50%. 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 concentration of amino acid may be achieved in basal media and/or by supplementation with feed containing amino acid or supplementing each amino acid separately.

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 comprise a temperature shift from 37°C to 34°C.

In any of the above mentioned embodiments, the cell culture process may 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 comprise a pH shift from about 7.2 to about 7.0.

Example-I

TNF-a antibody was cloned and expressed in a fed-batch culture 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 at temperature of about 37°C and pH of about 7.2. Subsequently on day 4, the temperature was reduced to about 34°C and pH was reduced to about 7.0. The culture was harvested on or after 240 hours.

The cells are cultured in either of the culture media l-VIII.

Table I and II represents amino acids in culture media l-IV and culture media V-VIII respectively. The percentage of acidic species in the antibody composition obtained by culturing cells in Media l-IV, as determined by weak-cation exchange chromatography is disclosed in Table -III. The percentage of acidic species in the antibody composition as obtained by culturing cells in Media V-VIII, as determined by weak-cation exchange chromatography is similar to as disclosed in Table-Ill

Example-ll

TNFa antibody was cloned and expressed in fed-batch culture 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 at temperature of about 37°C and pH of about 7.2. Subsequently on day 4, the temperature was reduced to about 34°C and pH was reduced to about 7.0. The culture was harvested on or after 240 hours.

The cells are cultured in either of the culture media l-XVI.

Table IV represents concentration (average) of amino acids in Media l-IV and Media IX-XII.

Table V represents concentration (average) of amino acids in Media V-VIII and Media XIII-XVI.

The average of percentage of galactosylated glycans in the antibody composition and titer of antibody obtained by culturing cells in either Media l-IV or Media IX-XII are as disclosed in Table VI and Table VII respectively.

The average of percentage of galactosylated glycans in the antibody composition and titer of antibody obtained by culturing cells in either Media V-VIII or Media XIII-XVI are similar to as disclosed in Table VI and Table VII respectively. Table I: Amino acids concentration (in g/L) in different cell culture media

Table III: Percentage of acidic species of TNFa antibody composition produced in different cell culture media from a process as described in example I

Table IV: Amino acids concentration (g/L) in cell culture media

Table VI: Percentage of galactosylated glycans of TNFa antibody composition produced from a process as described in example II

Table VII: Titer (g/L) of the TNFa antibody composition produced from process as described in example II