DESC:A CELL CULTURE PROCESS
FIELD OF INVENTION
The Biological material used in the invention was not obtained from India.
The present invention relates to cell culture process for culturing mammalian cell producing a recombinant protein, e.g., an antibody. The invention provides a mammalian cell culture process comprising use of cell culture medium, feed and/or additives for producing antibody compositions having a target/predetermined glycosylation profile. In particular, the invention provides a cell culture process comprising use of additives to obtain an antibody composition from mammalian cell culture, wherein the composition comprises low % total afucosylated antibody variants and high % galactosylated variants.
BACKGROUND OF THE INVENTION
Therapeutic monoclonal antibodies (mAbs), a class of recombinant proteins are the fastest growing biotherapeutics products. They find use in the treatment of various diseases like cancer, autoimmune, cardiovascular and inflammatory disorders. Therapeutic mAbs are commonly produced in mammalian expression system such as Chinese hamster ovary (CHO) cells due to the fact that they have post-translational modifications (PTMs) which are integral to their function as therapeutic molecules.
Glycosylation is the major PTM that is of importance in relation to mAbs being successfully used a biotherapeutics. The glycosylation profile of mAbs have a profound impact on the various biological activity such as stability, safety and efficacy of the protein. For example, the absence of fucose in the glycan structure of the Fc region of the mAbs has been associated with high antibody dependent cell mediated cytotoxicity (ADCC) activity (Shields, R.L., et al., Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem, 2002. 277(30): p. 26733-40; Shinkawa, T., et al., The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem, 2003. 278(5): p. 3466-73), and presence of high mannose glycans has been associated with faster clearance of mAbs from serum (Goetze, A.M., et al., High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology, 2011. 21(7): p. 949-59. Removal of terminal galactose residues from alemtuzumab was shown to reduce complement dependent cytotoxicity (CDC), without effecting Fc?R-mediated functions (Boyd, P.N., A.C. Lines, and A.K. Patel, The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-1H. Mol Immunol, 1995. 32(17-18): p. 1311-8). Similarly, removal of terminal galactose in rituximab reduced CDC by approximately half, relative to unmodified (variably galactosylated) control rituximab (Hodoniczky, J., Y.Z. Zheng, and D.C. James, Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog, 2005. 21(6): p. 1644-52).
‘Biosimilars’, sometimes called ‘similar biological medicinal product’ or ‘follow-on biologic’ or ‘subsequent entry biologic’, are biotherapeutic products (including therapeutic mAbs) which are similar to already licensed biotherapeutic product (the ‘reference product’). Regulatory agencies mandate that biosimilars must demonstrate high similarity to the reference product, in terms of quality characteristics, biological activity, safety and efficacy, in order to have marketing approval (Sullivan, P.M. and L.M. DiGrazia, Analytic characterization of biosimilars. Am J Health Syst Pharm, 2017. 74(8): p. 568-579).
Given the importance of glycosylation in regulatory approval of reference products and biosimilars alike, much effort have been undertaken to understand the cell culture process parameters which may have a bearing on the glycosylation pattern of biotherapeutics products. Previous studies have demonstrated that glycosylation of mAbs can be influenced by various factors such as pH, temperature, dissolved oxygen, ammonia, and media supplements such as nucleotide sugar precursors and manganese chloride. These studies focused on individual factors, establishing empirical relationships between the individual factor in question and the specific set of glycan species it affects (Radhakrishnan, D., A.S. Robinson, and B.A. Ogunnaike, Controlling the Glycosylation Profile in mAbs Using Time-Dependent Media Supplementation. Antibodies, 2017. 7(1): p. 1). Hence, cell culture processes that provides methods to control glycosylation pattern of therapeutic mAbs are desirable.
The present invention provides mammalian cell culture process comprising use of cell culture medium, feed or additives for producing antibody compositions having a target/predetermined glycosylation profile. In particular, the invention provides a cell culture process comprising use of additives to obtain an antibody composition from mammalian cell culture, wherein the composition comprises low % total afucosylated antibody variants and high % galactosylated variants.
The present invention provides cell culture process for obtaining an antibody composition comprising low % total afucosylated antibody variants and high % galactosylated antibody variants while maintaining high titre. Further the present invention also provides a cell culture process to obtain a target/predetermined glycosylation profile of antibody while maintaining high titre.
SUMMARY OF THE INVENTION
The present invention relates to cell culture process for culturing mammalian cell producing a recombinant therapeutic protein, e.g., an antibody. The invention provides mammalian cell culture process comprising use of cell culture medium, feed and/or additives for producing antibody compositions having a target/predetermined glycosylation profile. In particular, the invention provides a cell culture process comprising use of additives to obtain an antibody composition from mammalian cell culture, wherein the composition comprises low % total afucosylated antibody variants and high % galactosylated variants as compared to antibody composition obtained by the same process but without use of additives. Additionally, the use of additives as provided in the present invention results in high titre of antibody produced as compared to a same process without the use of said additives.
Cell culture additives as described herein may encompass any component added to the cell culture medium/feed which has the ability to change the environment of the cell culture.
Specially, the cell culture additives encompassed include, but not limited to, sugars, amino acids, metals, nucleotide precursors and other chemical or biological compounds. The additives used herein are, but not limiting to, fucose and/or galactose in order to obtain the desired antibody composition.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “about” refers to a range of values that are similar to the stated reference value to a range of values that fall within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference value.
The terms "additive" or "supplement" as used herein refer to any supplementation made to cell culture medium/feed to achieve the goals described in this disclosure. An "additive" or "supplement" can include a single substance, e.g., galactose, insulin, or fucose, or can include multiple substances, e.g., galactose, insulin, and fucose; or galactose and insulin; or galactose and fucose.
The term “antibody” or “monoclonal antibody” refers to an intact antibody or an antigen binding fragment thereof.
The term “antibody composition” refers to a population of antibody molecules or fragments thereof that is produced by mammalian cell culture. 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.
The term “afucosylated antibody variants” refer to the antibodies variants which lack fucose in oligosaccharides bound to the antibody constant region (Fc).
The term “galactosylated antibody variants” refer to antibodies containing terminal galactose residues such as G1A, G1B, G1AF, G1BF, G2, G2F and G2SF.
The terms “cell culture medium”, “culture medium”, "media", "medium", as used herein refer to a solution containing nutrients which are required to support the growth of the cells in cell culture. The term would include basal medium which is typically used to support the cell growth during the initial growth phase of cell culture. It would also include feed medium which is typically used to support the later growth phase and production phase of cell culture.
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 “control” herein the present invention refers to the cell culture process of culturing a population of cells that are capable of producing recombinant protein of interest or antibody without the addition of additives.
The term "biosimilar" refers to a recombinant protein, commonly with identical amino acid sequence to a reference product that contains, similar, very similar to or same post-translational modifications as the reference product yielding no clinically meaningful difference in terms of safety, purity and potency.
The term "reference product" refers to a currently or previously marketed recombinant protein, also described as the "originator product" or "branded product" serving as a comparator in the studies.
The term “target/predetermined glycosylation profile” refers to the glycosylation profile of the ‘reference product’.
The term “temperature shift” refers to the change in temperature during the cell culture process.
The term “viable cell count” or “viable cell concentration” or “VCC” refers to number of live cells in the total cell population.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration of viable cell count (VCC) as described in Examples I, II, III and IV.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention provides mammalian cell culture process comprising use of cell culture medium, feed or additives for producing antibody composition having target/predetermined glycosylation profile. In particular, the invention provides a cell culture process comprising use of additives to obtain an antibody composition from mammalian cell culture, wherein the composition comprises low % total afucosylated antibody variants and high % galactosylated variants. Further, the cell culture process of present invention allows production of antibody composition at high titre wherein the antibody composition comprises low % total afucosylated antibody variants and high % galactosylated antibody variants.
Any mammalian cell or cell type which is suitable for expression of recombinant proteins in a cell culture medium may be used for the present invention. Non-limiting examples of mammalian cells that may be used with the present invention include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK21) cells and murine myeloma cells (NS0 and Sp2/0) human retinoblasts (PER.C6 cell line), human embryonic kidney cell line (HEK-293 cell line) (Dumont, J., et al., Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol, 2016. 36(6): p. 1110-1122). In a preferred embodiment, CHO cell lines expressing recombinant proteins may be used in accordance with the present invention.
The glycosylation profile of a therapeutic monoclonal antibody has a bearing on its stability, safety and efficacy. Certain glycosylation profile of the therapeutic mAb are desirable based on its mechanism of action. In an embodiment, the cell culture process of the present invention may be used to produce therapeutic mAbs composition in which a low % total afucosylated antibody variants and high % galactosylated antibody variants would be desirable.
Cell culture medium is understood by those skilled in the art to refer to a nutrient solution in which cells, such as animal or mammalian cells, are grown. A cell culture medium generally includes one or more of the following components: an energy source (e.g., a carbohydrate such as glucose); amino acids; vitamins; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements in the micromolar range. Cell culture medium can also contain additional components, such as hormones and other growth factors (e.g., insulin, transferrin, epidermal growth factor, serum, and the like); salts (e.g., calcium, magnesium and phosphate); sugars (e.g. mannose, galactose, fucose); amino acids (glutamine); buffers (e.g., HEPES); nucleosides and bases (e.g., adenosine, thymidine, hypoxanthine ); antibiotics ( e.g., gentamycin); and cell protective agents ( e.g., a Pluronic polyol (Pluronic F68). Commercially available media can be utilized in accordance with the present invention, for example, Dulbecco's Modified Eagles Medium (DMEM, Sigma-Aldrich); RPMI-1640 Medium (Sigma-Aldrich); EX-CELL® Advanced CHO Fed-batch Medium (Sigma-Aldrich); Cell Boost™ 7a and 7b (GE Healthcare Bio-Sciences AB). One skilled in the art would appreciate that some cell culture media are suited to support cells through their initial growth phase (basal medium) while some sustain cells through the later growth phase and production phase of cell culture (feed medium), and would be able to choose appropriate culture medium.
The methods described in the present invention are in recognition of the fact that various parameters of the cell culture process may be used to obtain antibody composition of desired glycosylation profile. In an embodiment, the cell culture process envisages supplementation of cell culture medium with “additives”. In an embodiment, the additives used as disclosed in the invention comprise galactose and/or fucose.
In an embodiment, the cell culture process of the present invention encompasses supplementation of culture medium with different “additives” at the same time as well as different times. For example, in a cell culture process using galactose and/or fucose as additives, addition of galactose and fucose can be done at same time or at different times. Further, in an embodiment, the supplementation of the additive in cell culture takes place at particular time in the cell culture process e.g. the production phase of the cell culture or attainment of a particular viable cell density. Further, in an embodiment, target amount of a particular additive may be achieved by a single bolus addition or through multiple additions. For example 10g/L of an additive in the cell culture may be achieved a single bolus administration, or two additions of 5g/L each, or 4 additions of 2.5g/L each.
An aim in the development of cell culture process of present invention is to have high titre of product while maintaining the correct product profile. In an embodiment, the cell culture process of present invention would comprise use of a temperature shift, wherein the subsequent temperature would be lesser than the previous temperature. In another embodiment, the cell culture process may employ more than one temperature shift. In a particular embodiment, the cell culture process comprises use of a temperature shift from about 37°C to 32°C, which results in high titre of antibody produced.
A person of ordinary skill in the art would be able to determine the glycosylation profile of the antibody composition produced by the cell culture process described herein using the state of the art techniques (Ruhaak, L.R., et al., Glycan labeling strategies and their use in identification and quantification. Anal Bioanal Chem, 2010. 397(8): p. 3457-81; Wuhrer, M., A.M. Deelder, and C.H. Hokke, Protein glycosylation analysis by liquid chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 2005. 825(2): p. 124-33; Guile, G.R., et al., A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal Biochem, 1996. 240(2): p. 210-26).
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with one or more additives
d) recovering the recombinant protein/antibody from the cell culture
thereby obtaining an antibody composition comprising low % total afucosylated antibody variants and high % galactosylated antibody variants as compared to antibody composition obtained by the same process but without use of step (c) enumerated above.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose
d) recovering the recombinant protein/antibody from the cell culture
thereby, obtaining an antibody composition comprising low % total afucosylated antibody variants and high % galactosylated antibody variants as compared to antibody composition obtained by the same process but without use of step (c) enumerated above.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose
d) recovering the recombinant protein/antibody from the cell culture
thereby, obtaining a high titre of an antibody composition comprising low % total afucosylated antibody variants and high % galactosylated antibody variants as compared to antibody composition obtained by the same process but without use of step (c) enumerated above.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose, wherein the cumulative amounts of each is at least about 4g/L
d) recovering the recombinant protein/antibody from the cell culture
thereby, obtaining a high titre of an antibody composition comprising low % total afucosylated antibody variants and high % galactosylated antibody variants as compared to antibody composition obtained by the same process but without use of step (c) enumerated above.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose when the viable cell count is at least about 8 X 106 cells/mL, wherein the cumulative amounts of each additive added is at least about 4g/L
d) recovering the recombinant protein/antibody from the cell culture
wherein the first temperature in step (a) is about 36.5°C, the second temperature of about 32°C in step (b), and the temperature shift in step (b) is applied on day 6 of cell culture,
thereby obtaining a high titre of an antibody composition comprising low % total afucosylated antibody variants and high % galactosylated antibody variants as compared to antibody composition obtained by the same process but without use of step (c) enumerated above.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with one or more additives
d) recovering the recombinant protein/antibody from the cell culture
thereby obtaining an antibody composition comprising about 2.7 to 3.1 % total afucosylated antibody variants and about 45 to 49 % galactosylated antibody variants.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose
d) recovering the recombinant protein/antibody from the cell culture
thereby obtaining an antibody composition comprising about 2.7 to 3.1 % total afucosylated antibody variants and about 45 to 49 % galactosylated antibody variants.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose, wherein the cumulative amounts of each is at least about 4g/L
d) recovering the recombinant protein/antibody from the cell culture
thereby obtaining an antibody composition comprising about 2.7 to 3.1 % total afucosylated antibody variants and about 45 to 49 % galactosylated antibody variants.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose when the viable cell count is at least about 8 X 106 cells/mL, wherein the cumulative amounts of each is at least about 4g/L
d) recovering the recombinant protein/antibody from the cell culture
thereby obtaining an antibody composition comprising about 2.7 to 3.1 % total afucosylated antibody variants and about 45 to 49 % galactosylated antibody variants.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose when the viable cell count is at least about 8 X 106 cells/mL, wherein the cumulative amounts of each is at least about 4g/L
d) recovering the recombinant protein/antibody from the cell culture
wherein the first temperature in step (a) is about 36.5°C, the second temperature of about 32°C in step (b), and the temperature shift in step (b) is applied on day 6 of cell culture,
thereby obtaining an antibody composition at comprising about 2.7 to 3.1 % total afucosylated antibody variants and about 45 to 49 % galactosylated antibody variants.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a biosimilar monoclonal antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with one or more additives
d) recovering the recombinant antibody from the cell culture
wherein the biosimilar antibody composition obtained has a target/predetermined glycosylation profile based on the glycosylation profile of the reference product monoclonal antibody.
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a biosimilar monoclonal antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose
d) recovering the recombinant protein/antibody from the cell culture
wherein the biosimilar antibody composition obtained has a target/predetermined glycosylation profile based on the glycosylation profile of the reference product monoclonal antibody
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a biosimilar monoclonal antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose, wherein the cumulative amounts of each is at least about 4g/L
d) recovering the recombinant protein/antibody from the cell culture
wherein the biosimilar antibody composition obtained has a target/predetermined glycosylation profile based on the glycosylation profile of the reference product monoclonal antibody
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a biosimilar monoclonal antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose when the viable cell count is at least about 8 X 106 cells/mL, wherein the cumulative amounts of each is at least about 4g/L
d) recovering the recombinant protein/antibody from the cell culture
wherein the biosimilar antibody composition obtained has a target/predetermined glycosylation profile based on the glycosylation profile of the reference product monoclonal antibody
In an embodiment, the present invention provides a cell culture process for culturing mammalian cell producing a biosimilar monoclonal antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with galactose and/or fucose when the viable cell count is at least about 8 X 106 cells/mL, wherein the cumulative amounts of each is at least about 4g/L
d) recovering the recombinant protein/antibody from the cell culture
wherein the first temperature in step (a) is about 36.5°C, the second temperature of about 32°C in step (b), and the temperature shift in step (b) is applied on day 6 of cell culture,
thereby obtaining a biosimilar antibody that has a target/predetermined glycosylation profile based on the glycosylation profile of the reference product monoclonal antibody.
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLES
Example I
An anti-a4ß7 integrin antibody was cloned and expressed in a recombinant CHO (rCHO) cell line using techniques described in detail in “Molecular Cloning: A Laboratory Manual (Fourth Edition)”. rCHO cells expressing the antibody were seeded at a density of about 0.49 million cells/mL in basal cell culture medium and cultured at a temperature of about 37°C and pH at a range of about 6.7 to 7.22. The cell culture temperature was reduced to about 32°C on day 6. Feed medium was added on day 3, 5, 7, 9 and 11. The culture was harvested on day 13 or at cell viability greater than 50%. The basal medium and feed medium used did not have galactose or fucose as components. The experiment was run in three different batches. The % total afucosylated variants and % galactosylated variants in the antibody composition obtained are depicted in Table 1.
Example II
The cell culture process described in Example I was carried with following modifications. Galactose (2g/L) was added during the cell culture process on day 6 and 10, thereby having galactose at cumulative concentration of 4g/L. The experiment was run in three different batches. The % total afucosylated variants and % galactosylated variants in the antibody composition obtained are depicted in Table 1.
Example III
The cell culture process described in Example I was carried with following modifications. Fucose (4g/L) was added to culture medium on day 9. The experiment was run in two different batches. The % total afucosylated variants and % galactosylated variants in the antibody composition obtained are depicted in Table 1.
Example IV
The cell culture process described in Example I was carried with following modifications. In addition to the basal and feed medium provided in Example I, galactose and fucose were used in the cell culture process. Galactose (2g/L) was added to the culture medium on day 6 and 10 of the cell culture process, thereby having galactose at cumulative concentration of 4g/L, whereas fucose (4g/L) was added to the culture medium on day 9 of the cell culture process. The experiment was run in twenty different batches. The % total afucosylated variants and % galactosylated variants in the antibody composition obtained are depicted in Table 1.
Table 1. Antibody composition

Example Galactose (g/L) Fucose
(g/L) % Total afucosylated antibody variant
(% TAF) % Galactosylated antibody variant
(% Gal) Titer
(mg/L)
Reference Product NA NA 2.5 ± 0.2 48.19 ± 2.21 NA
I - - 3.5 ± 0.5 35 ± 3 3.3 ± 0.3
II 4 - 3.5 ± 0.2 44 ± 2 4.0 ± 0.2
III - 4 2.8 ± 0.2 46 ± 1 2.8 ± 0.2
IV 4 4 2.9 ± 0.2 47 ± 2 3.9 ± 0.2

,CLAIMS:We claim:
1. A cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with one or more additives selected from galactose and fucose
d) recovering the recombinant antibody from the cell culture
thereby obtaining an antibody composition comprising low % total afucosylated antibody variants and/or high % galactosylated antibody variants as compared to antibody composition obtained by the same process but without use of step (c) enumerated above.
2. A cell culture process for culturing mammalian cell producing a recombinant antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with one or more additives selected from galactose and fucose
d) recovering the recombinant antibody from the cell culture
thereby obtaining an antibody composition comprising about 2.7 to 3.1 % total afucosylated antibody variants and about 45 to 49 % galactosylated antibody variants.
3. A cell culture process for culturing mammalian cell producing a biosimilar monoclonal antibody, the said process comprising steps of
a) culturing cells at a first temperature for a first period of time
b) performing a temperature shift and culturing the cells at the second temperature for a second period of time
c) supplementing cell culture medium with one or more additives selected from galactose and fucose
d) recovering the recombinant antibody from the cell culture
wherein the biosimilar antibody composition obtained has a target/predetermined glycosylation profile based on the glycosylation profile of the reference product monoclonal antibody
4. The temperature shift in the preceding claims is performed on day 6 of the cell culture, wherein the first temperature is about 36.5°C and the second temperature of about 32°C.
5. The additives supplemented in the cell culture medium of the preceding claims, wherein the cumulative amounts of each is at least about 4g/L.
6. The supplementation of the additives in the preceding claims can be done when the viable cell count is at least about 8 X 106 cells/mL.
7. The antibody produced using the cell culture of the preceding claims is anti-a4ß7 integrin antibody.
8. The mammalian cells used in the preceding claims is recombinant CHO cells.