DESC:The following specification particularly describes the invention and the manner in which it is to be performed:

A CELL CULTURE PROCESS

INTRODUCTION
The present invention relates to the field of cell culture and, in particular, to a cell culture process for modulating percentage of glycoforms in an antibody composition.

BACKGROUND OF INVENTION
Protein glycosylation is one of the most important post-translation modifications associated with eukaryotic proteins. There are two major types of glycosylation in eukaryotic cells, N-linked glycosylation in which glycan moiety is attached to the asparagine residue of the recognition sequence Asn-X-Thr/Ser, where “X” is any amino acid except proline, and O-linked glycosylation in which glycan moiety is attached to serine or threonine present in the glycoprotein. N-linked glycans further are of two types - high mannose type consisting of two N-acetylglucosamines plus a large number of mannose residues, and the complex type that contain more than two N-acetylglucosamines plus any number of other types of sugars (galactose, sialic acid etc.).
Numerous factors can influence the N-/O-glycan site occupancy and the type of glycan on each site. The factors may be intrinsic [e.g. cell type, availability of the glycosylation enzyme etc.] or extrinsic [culture media, temperature etc.] to the cells producing the said glycoprotein composition.
It has been demonstrated that the structure and composition of the glycan moieties, i.e. individual glycoforms and their percentage, can have a profound effect on safety and efficacy of the monoclonal antibodies (mAb) intended for therapeutic use.
Several studies have demonstrated that low fucose levels i.e. higher afucosylated glycoform in an antibody composition enhance the antibody-mediated cellular cytotoxicity (ADCC) (Mori K, (2007), Cytotechnology 55(2–3):109–114. and Shields RL. (2002), J Biol Chem 277(30):26733–26740). A no. of strategies have been employed to increase the afucosylated content, the pronounced ones are knock-in and knock out of glycosyltransferase enzymes or using fucose analogs (Schneider M. Glycobiology, Volume 27, Issue 7, 1 July 2017, Pages 601–618 and US9504702B2).
High-mannose content of therapeutic mAbs is considered an important product quality attribute that can contribute to increased clearance of therapeutic antibodies. However, in certain instances hypermannosylated or high mannose glycoforms are required for target drug delivery as they can potentially elicit immune recognition response (Walsh G. and Jefferis R., (2006), Nat. Biotechnology, 24:1241-1252).
Thus, there is considerable motivation for manufacturers and regulators to understand the factors influencing the glycoforms, characterize the content of glycoform in mAbs composition, and, if necessary, modulate the glycoform distribution in mAbs composition during production, in order to maintain a consistent glycan profile
However, when optimizing the cell culture process for obtaining a specific glycoform profile in a mAb composition, consideration should be taken so as to not to alter the yield and the product titer.
The objective of the invention is to provide a cell culture process for modulating the percentage of glycoforms in a mAb composition without an impact on yield or titer of the mAb, so produced.

SUMMARY OF THE INVENTION
The present invention discloses a cell culture process for modulating percentage of glycoforms in a mAb composition, the process comprising supplementing the cell culture media, used for culturing the cells, with a mixture of two different types of osmolytes. In particular, out of the two different osmolytes supplemented, one is a sugar and the other one is an alkali metal salt. More particularly, the cell culture medium is supplemented with mannose and either KCl or NaCl. The supplementation of mannose and either KCl or NaCl to the culture media, used for culturing the cells, has a synergistic effect on modulation of percentage of glycoforms, in particular on increase in % of high mannose and/or afucosylated and/or total afucosylated glycans.

DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “cell culture process” as used herein refers to a process of culturing cells that are capable of producing recombinant protein or antibody of interest.
The term “cell culture medium” or “culture media” or “media” refers to a solid, liquid or semi-solid composition containing components / 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” or “mAb composition” refers to a population of antibody molecules or fragments thereof. The individual antibody molecules in a composition may have one or several post translational modifications (PTM) (e.g. glycosylation, basic and acidic variants), imparting the antibody molecules a different molecular weight, charge, solubility or combinations thereof. For instance, individual glycofoms, i.e. antibody molecules
The term “glycan” refers to a monosaccharide or polysaccharide moiety.
The term “glycoforms” refer to the glycoproteins with various oligosaccharide entities or moieties linked in their entirety to the Asparagine 297 (as per Kabat numbering) of the human Fc region of the glycoproteins in question, co translationally or post translationally within a host cell. The glycan moieties may be added during protein glycosylation include M3, M4, M5-8, M3NAG etc. Examples of such glycans and their structures are listed in Table 1. However, Table 1 may in no way be considered to limit the scope of this invention to these glycans.
As used in the specification and claims, the term "modulated” or “modulating” or “altered” or “altering” of glycoform" refers to a change in percentage (or relative quantity) of an individual glycoform in a mAb composition by culturing a recombinant cell expressing the antibody in a culture process/media as disclosed in the instant invention as compared to a process/media which is not conducted as per the instant invention. In particular, the "modulated” or “modulating” or “altered” or “altering” of glycoform refers to a change in percentage (or relative quantity) of an individual glycoform in a mAb composition obtained by the process comprising supplementing the cell culture media, used for culturing the cells, with a mixture of two different types of osmolytes as compared to additive effect on % of individual glycoform in a mAb composition obtained by the process comprising supplementing the cell culture media, used for culturing the cells, with each of the osmolytes separately.
The term “osmolality” as used herein is defined as a measure of the milli osmoles of solute per kilogram of solvent (mOsm/kg) and may include ionized or non-ionized molecules and may change during the cell culture process for e.g. by addition of feed, salts, additives or metabolites. Osmolality can be measured by means that are well-known in the art. For example, an osmometer (Fisher Scientific, Pittsburgh, PA) can be used.
The term “osmolyte” as used herein refers to compounds affecting osmosis. The osmolytes as used in the present invention are soluble in the cell culture media.
Table 1 as given below represents the glycan species for reference purpose only and in no way limits the scope of the instant invention.
The term “% of high mannose glycans” refers to: M5, M6, M7, M8 and M9
The term “% of afucosylated glycans” refers to: M3NAG, G0, G1A, G1B, G2, and G2S1.
The term “% of total afucosylated glycans” refers to: M5, M6, M7, M8, M9, M3NAG, G0, G1A, G1B, G2, and G2S1.

Table I: Representative table of various glycans

DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention describes a cell culture process for modulating percentage of glycoforms in an antibody composition, the process comprising supplementing a cell culture media, for culturing the cell producing said antibody, with a mixture of two different types of osmolytes.
In the above mentioned embodiment, out of the two different osmolytes supplemented, one osmolyte is a sugar and the other is an alkali metal salt.
In particular, the sugar supplemented can be selected from a group consisting of mannose, sucrose, fructose, glucose, mannitol, tagatose, raffinose etc., and in particular the alkali metal salt can be sodium chloride (NaCl) or potassium chloride (KCl).
An embodiment of the invention discloses a cell culture process for modulating percentage of glycoforms in an antibody composition, the process comprising supplementing a cell culture media used for culturing the cells with a mixture of mannose and either of KCl or NaCl.
In any of the above mentioned embodiments, the supplementation of the two different osmolyte is in the concentration range such that the osmolality of the culture media, so supplemented, is not substantially changed.
In any of the above mentioned embodiments, the supplementation of two different osmolytes is in the concentration range such that the osmolality of the culture media so supplemented does not increase more than about 30% as compared to the culture media in which there is no supplementation of either osmolyte.
In any of the above mentioned embodiments, the supplementation of two different osmolytes is in the concentration range such that the osmolality of the culture media, so supplemented, remain/maintained in the range of about 400-600 mOsm/kg.
In any of the above mentioned embodiments, the supplementation of two different osmolytes does not impact the titer of the antibody composition so produced.
In any of the above mentioned embodiments, the modulation of percentage of glycoforms, is increase in percentage of high mannose and/or total afucosylated glycans in an antibody composition.
In any of the above mentioned embodiments, the modulation of glycoform is an increase in high mannose glycan in an antibody composition by at least 1.5 times as compared to when mAb composition is obtained by the process comprising supplementing the cell culture media, used for culturing the cells, with each of the osmolytes separately.
In any of the above mentioned embodiments, the modulation of glycoprofile is increase in total afucosylated glycan in an antibody composition by at least 1.2 times as compared to when mAb composition is obtained by the process comprising supplementing the cell culture media, used for culturing the cells, with each of the osmolytes separately.
In any of the above mentioned embodiments, the modulation of glycoprofile in an antibody composition is increase in high mannose glycan by at least 1.5 times and increase in total afucosylated glycan is by at least 1.2 times as compared to when mAb composition is obtained by the process comprising supplementing the cell culture media, used for culturing the cells, with each of the osmolytes separately.
In any of the above mentioned embodiments the antibody so obtained is an anti-IL-6R antibody, more particularly the antibody so obtained is tocilizumab.
In any of the above mentioned embodiments, the cells producing said antibody, are mammalian cells. The preferred mammalian cells are Chinese hamster Ovary (CHO) cells.
In any of the above mentioned embodiments, the supplementation of osmolytes can be anytime during the cell culture process. In particular, the supplementation can during the initiation of the cell culture process or the supplementation can be in single/multiple batches added during the log phase and/or the stationary phase.

In any of the above mentioned embodiments, the supplementation of osmolytes can be as an individual components or added to basal media or feed.

One skilled in the art will recognize at what temperature or temperature shift and/or pH and pH shift a particular cell line should be cultured.

The cell culture media that are useful in the application include but are not limited to, the commercially available products PF CHO (HyClone®), PowerCHO® 2 (Lonza), Zap-CHO (Invitria), CD CHO, CDOptiCHOTM and CHO-S-SFMII (Invitrogen), ProCHOTM (Lonza), CDM4CHOTM (Hyclone), ActiPro (HyClone), Ex-CELL CDCHO Fusion (Merck, Sigma), Ex-CELL Advanced CHO fed batch medium (Merck, Sigma), Ex-CELL Cell Vento 4 CHO medium (Merck Sigma), Dynamis™ AGT™ Medium (Thermo Fisher Scientific) DMEM (Invitrogen), DMEM/F12 (Invitrogen), Ham’s F10 (Sigma), Minimal Essential Media (Sigma), and RPMI -1640 (Sigma) and IS CHO-CD G10.3 (Irvine scientific).
The feed or feed medium in the present invention may be added in a continuous, profile or a bolus mode. One or more feeds may be added in one manner (e.g. profile mode), and other feeds in second manner (e.g. bolus or continuous mode). Further, the feed may be composed of nutrients or other medium components that have been depleted or metabolized by the cells. The feed may be concentrated form of initial cell culture media itself or may be a different culture media. The components may include hormones, growth factors, ions vitamins, nucleoside, nucleotides, trace elements, amino acids, lipids or glucose. Supplementary components may be added at one time or in series of additions to replenish the depleted components. Thus the feed can be a solution of depleted nutrient(s), mixture of nutrient(s) or a mixture of cell culture medium/feed providing such nutrient(s).
The cell culture feed that are useful in the invention include but are not limited to, the commercially available products Efficien tFeed A+ AGT (Product Code A2502301), EfficientFeed B+ AGT (Product Code A2503004) and EfficientFeed C+ AGT (Product Code A2503101) (Thermo Fisher Scientific) Cell Boost 2 (CB-2, GE Healthcare Bio-Sciences AB, Hyclone, Product Code, SH 30596.03), Cell Boost 4 (CB-4, GE Healthcare Bio-Sciences AB , HyClone, Product Code. SH30928), Cell Boost 7 a (CB7a, GE Healthcare Bio-Sciences AB, Hyclone, Product Code, SH31026) Cell Boost 7b (CB 7b, E Healthcare Bio-Sciences AB, Hyclone, Product Code, SH31027) PF CHO (Thermo Scientific Hyclone, Product Code, SH30333.3).
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
An anti-IL-6R 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 were seeded at a density of ~0.5 million cells/ml in to the culture media.
The cell culture was initiated at a temperature of 37oC and subsequently on day 6 the temperature was lowered to 33 oC, which was maintained till the harvest of the cell culture.
On day 3, 5, and 7 the cell culture was supplemented with the feed media.
The culture was harvested on day 13.
Following the above process, an antibody composition was obtained [Sample I (a)].
In an alternate, during the initiation of the cell culture process, the culture media was supplemented with mannose at concentration of 27.8 mM [Sample I (b)].
In an alternate, during the initiation of the cell culture process, the culture media was supplemented with KCl at concentration of 30 mM [Sample I (c)].
In yet another alternate, during the initiation of the cell culture process, the culture media was supplemented with mannose at concentration of 27.8 mM, and KCl at concentration of 30 mM [Sample I (d) and Sample 1(e)].
The osmolality of the culture media (after all feed additions), titer and the percentage of individual glycoforms in the antibody compositions [Sample I (a)-(e)] were determined and are given in Table II.

Example – II
An anti-IL-6R 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 were seeded at a density of ~0.5 million cells/ml in to the culture media.
The cell culture was initiated at a temperature of 37oC and subsequently on day 6 the temperature was lowered to 33 oC, which was maintained till the harvest of the cell culture.
On day 3, 5, and 7 the cell culture was supplemented with the feed media.
The culture was harvested on day 10.
Following the above process, an antibody composition was obtained [Sample II (a) - (b)].
In an alternate, along with the feed media, on day 3, 5, and 7 the culture media was supplemented with mannose in a cumulative concentration of 11 mM [Sample II (c)].
In an alternate, along with the feed media, on day 3, 5, and 7, the culture media was supplemented with KCl in a cumulative concentration of 40 mM [Sample II (d)]
In yet another alternate, along with the feed media, on day 3, 5, and 7, the culture media was supplemented with mannose in a cumulative concentration of 11 mM and KCl in a cumulative concentration of 40 mM [Sample II (e)-(f)].
The osmolality of the culture media (after all additions), titer and the percentage of individual glycoforms in the antibody compositions [Sample II (a)-(f)] were determined and are given in Table III.

Example – III
An anti-IL-6R 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 ~0.5 million cells/ml in culture media.
The cell culture was initiated at a temperature of 37 oC and subsequently on day 6 the temperature was lowered to 33 oC, which was maintained till the harvest of the cell culture.
On day 3, 5, 7, 9 and 11 the cell culture was supplemented with feed media.
The culture was harvested on day 13.
Following the above process, an antibody composition was obtained (Sample III (a)).
In an alternate, during the initiation of the cell culture process, the culture media was supplemented with mannose at concentration of 28 mM [Sample III (b)].
In another alternate, the culture media was supplemented with NaCl at concentration of 97.5 mM on day 9 which is the late exponential/early stationary phase of the culture [Sample III (c)].
In yet another alternate, during the initiation of the cell culture process, the culture media was supplemented with mannose at concentration of 28 mM, and the culture media was supplemented with NaCl at concentration of 97.5 mM on day 9 which is the late exponential/early stationary phase of the culture [Sample III (d)].
The osmolality of the culture media (after all additions), titer and the percentage of individual glycoforms in the antibody compositions [Sample III (a)-(d)] were determined and are given in Table IV.

Table-II- Osmolality of the culture media (after all feed additions), titer and the percentage of individual glycoforms in the antibody compositions [Sample I (a)-(e)] as obtained in Example-I
Sample Name Conditions Osmolality
(mOsm/kg) % High mannose % Total Afucosylated Titer
(g/L)
Sample I (a) Man(-), KCl (-) 378 1.3 4.8 3
Sample I (b) Man (+), KCl(-) 395 2.4 7.1 2.8
Sample I (c) Man (-), KCl(+) 438 1.9 6.2 2.9
Sample I (d) Man (+), KCl(+) 430 6.3 13.6 2.7
Sample I (e) Man (+), KCl(+) 452 6.0 13.0 2.7
Effect of Mannose alone (A) (I (b)-I (a)) 17 1.1 2.3 -0.2
Effect of KCl alone (B)
(I (c)-I (a)) 60 0.6 1.4 -0.1
A+B 77 1.7 3.7 -0.3
Effect of Mannose and KCl (Avg. of I (d) and I (e))-I (a) 63 4.9 8.5 -0.3

Table-III- osmolality of the culture media (after all additions), titer and the percentage of individual glycoforms in the antibody compositions [Sample II (a)-(f)] as obtained in Example-II
Sample Name Conditions Osmolality
(mOsm/kg) % High mannose % Total Afucosylated Titer
(g/L)
Sample II (a) Man(-), KCl (-) NA 1.3 4.8 3
Sample II (b) Man(-), KCl (-) 351 1.2 4.5 3.0
Sample II (c) Man (-), KCl(+) 426 1.8 7.1 2.7
Sample II (d) Man (+),KCl(-) 340 1.5 5.5 2.8
Sample II (e) Man (+), KCl(+) 421 2.4 8.6 2.7
Sample II (f) Man (+), KCl(+) 420 2.9 9.5 2.5
Effect of Mannose alone (A) (II (d)-(Average II (a) & II (b)) -11 0.3 0.9 -0.2

Effect of KCl alone (B) (II (c)-(Average II (a) & II (b)) 75 0.6 2.5 -0.3

A+B 64 0.9 3.4 -0.5
Effect of Mannose and KCl (Average of II (e) & II(f))- (Average II (a)) & II (b)) 70 1.4 4.4 -0.4

Table-IV-Osmolality of the culture media (after all additions), titer and the percentage of individual glycoforms in the antibody compositions [Sample III (a)-(d)] as obtained in Example-III
Sample Name Conditions Osmolality
(mOsm/kg) % High mannose % Total Afucosylated Titer
(g/L)
Sample III (a) Man(-), NaCl (-) 423 0.9 4.1 2.8
Sample III (b) Man (-), NaCl(+) 575 2.7 7.0 2.4
Sample III (c) Man (+), NaCl(-) 429 1.8 6.8 2.7
Sample III (d) Man (+), NaCl(+) 589 5.1 10.7 2.3
Effect of Mannose alone (A) (III (c)- III (a)) 6 0.9 2.7 -0.1
Effect of NaCl alone (B) (III (b)- III (a)) 152 1.8 2.9 -0.4
A+B 158 2.7 5.6 -0.5
Effect of Mannose and NaCl (III (d)- III (a)) 166 4.2 6.6 -0.5
,CLAIMS:We claim:
1. A method of increasing percentage of high mannose and total afucosylated glycan variants in an antibody composition produced by a mammalian cell comprising, culturing the said mammalian cells in a cell culture medium, supplementing the culture medium with a sugar and alkali metal salt,
wherein, the increase is in comparison to an antibody composition produced by an identical method without the supplementation of sugar and alkali metal salt,
wherein, the method further comprising a temperature shift.
2. The method of claim 1, wherein supplementation with sugar and alkali metal salt synergistically increase the percentage of glycan variants in the antibody composition.
3. The sugar used in claim 1 can be selected from a group consisting of mannose, sucrose, fructose, glucose, mannitol, tagatose, raffinose.
4. The alkali metal salt used in claim 1 can be selected from sodium chloride or potassium chloride.
5. The method of claim 1, wherein the osmolality of cell culture medium is about 400-600 mOsm/kg.
6. The supplementation of sugar and alkali metal salt as claimed in claim 1 can be during the initiation of the cell culture process or during the log phase and/or the stationary phase.
7. The supplementation of sugar and alkali metal salt as claimed in claim 1 can be in a single or multiple batches.
8. The antibody produced in the preceding claims is an anti-IL-6R antibody.