DESC:FIELD OF THE INVENTION
The present invention generally relates to the field of cell culture. In particular, the invention discloses a process for obtaining a glycoprotein with a glycoform composition comprising reduced percentage of high mannosylated and afucosylated glycans. The cell culture process involves distributed feeding of manganese over the course of cell culture.
BACKGROUND OF THE INVENTION
Protein glycosylation is one of the most important post-translation modifications 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. N-linked glycans further are of two types - high mannose type consisting of two N-acetylglucosamines plus a large number of mannose residues (more than 4), and the complex type that contain more than two N-acetylglucosamines plus any number of other types of sugars (galactose, fucose etc). In both N- and O-glycosylation, there is normally a range of glycan structures associated with each site (microheterogeneity). Macroheterogeneity results from the fact that not all N-glycan or O- glycan recognition sequences (Asn-X-Ser/Thr for N-glycan and serine or threonine for O-glycan present in the glycoproteins) are actually glycosylated. This may be consequence of the competitive action of diverse enzymes involved in glycosylation and are key to understanding glycoprotein heterogeneity (Mariño, K., (2010) Nature Chemical Biology 6,713-723).
Recombinant monoclonal antibodies (mAbs) represent the largest and fastest growing group of therapeutic glycoproteins. It has been demonstrated that the structure and composition of the glycan moieties on mAbs can have a profound effect on their safety and efficacy for e.g. the extent and nature of glycosylation in monoclonal antibodies (mAbs) affects its clearence, immunogenicity, and solubility.
The efficacy of therapeutic antibodies is mediated by two independent mechanisms, (a) the efficacy resulting from target antigen neutralization or apoptosis and (b) biological activities referred to as antibody effector functions mediated via effector cells or complement system known as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) respectively. Several studies have demonstrated that low fucose levels i.e. higher afucosylated glycoform composition enhance mononuclear cell-mediated 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). However in contrast, Peipp M. et al showed that polymorphonuclear cells preferentially kill via high-fucosylated antibody composition. Further, the same study concluded that high-fucose antibody induced superior ADCC in blood from granulocyte colony-stimulating factor-primed donors containing higher numbers of activated polymorphonuclear cells. Since, the impact of fucosylation on ADCC activity is primarily dependent on the effector cells employed, it may be desirable to obtain an antibody composition comprising decreased afucosylated content (Peipp M, Blood. 2008 Sep 15;112(6):2390-9. doi: 10.1182/blood-2008-03-144600).
Further, the efficacy of therapeutic antibodies is also affected by the serum clearance rate i.e. serum half life of antibodies. The serum half life of IgG antibodies is regulated by a number of receptors including the mannose receptors, which bind both high-mannose-containing pathogens as well as endogenous proteins (Stahl PD. 1992, Curr Opin Immunol. 4:49–52 & Lee SJ, Evers S, Roeder D, Parlow AF, Risteli J, Risteli L, Lee YC, Feizi T, Langen H, Nussenzweig MC. 2002. Science. 295:1898–1901). Goetze et al. showed that IgGs containing Fc high-mannose glycans are cleared more rapidly in humans than other glycan forms (Andrew MGoetze et al. Glycobiology vol. 21 no. 7 pp. 949–959, 2011). Thus, there may be preferential clearance of glycoforms bearing the terminal mannose residues which would impact the pharmokinetic profile of an antibody composition. Hence the reduction of high mannose bearing glycoforms improves the half life of an antibody composition which is a desirable quality attribute.
As emphasized above, fucosylated and high mannosylated glycoform variants can have marked influence on efficacy of mAbs and their utility as therapeutics, thus there is a critical requirement for developing production processes to achieve the same.
Robust stable production of completely non-fucosylated therapeutic antibodies at consistent quality has been achieved by several methods such as (a) the generation of a glycosylation enzyme mutants/knockout host cell line, in which the endogenous a-1,6-fucosyltransferase (FUT8) gene is knocked out. (b) using specific inhibitors for e.g. kifunesine (Shen, A., Ng, D., Joly, J., Snedecor, B., Lu, Y., Meng, G., Nakamura, G., and Krummen, L.(2007) metabolic engineering to control glycosylation,in cell culture and upstream processing (ed M.butler), Taylor and Francis group & J Bischoff, L Liscum and R Kornfeld, (1986), The Journal of Biological Chemistry, 261, 4766-4774). WO 2013114164 A1 claims a cell culture process comprising temperature and pH shifts to increase the percentage of afucosylated glycans. Additionally, WO2013114165 describes a cell culture process for obtaining a glycoprotein with the enhanced afucosylation glycan content, wherein the method involves culturing cells in a medium supplemented with galactose.
Studies by Pacis et al. show a decrease in mannose glycoforms upon supplementing cell culture medium with manganese. (Pacis, E., Yu, M., Autsen, J., Bayer, R. and Li, F. (2011), Biotechnology and Bioengineering, 108: 2348–2358. doi: 10.1002/bit.23200). Likewise, US20110053223 discloses a cell culture process for accumulation of mannose5 glycans by culturing cells in medium comprising manganese at a concentration of 0.25 µM or less. Further, WO2013114245 discloses a process for increasing percentage of Man5 and/or afucosylated glycans by culturing cells expressing said recombinant glycoprotein in culture medium comprising divalent manganese ion or its salts thereof.
Thus prior art provides cell culture process for reducing high mannose (or increasing mannose 5) and/or increasing afucosylated content of a glycoform composition by using manganese in the cell culture process. However the prior art does not provide a process for reducing both the high mannose and afucosylated content of a glycoform composition. For aforementioned reasons, glycoform composition with reduced high mannose and afucosylated glycans can have immense therapeutic and industrial value.
The present invention provides a cell culture process for obtaining a glycoform composition with reduced high mannose and afucosylated glycans by distributing manganese supplementation over the course of cell culture.
SUMMARY OF THE INVENTION
The present invention describes a cell culture process comprising distributed feeding of manganese for obtaining a glycoform composition comprising reduced percentage of high mannosylated and afucosylated glycans.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an illustration of percentage of high mannosylated and afucosylated glycans in a glycoform composition obtained by the process as described in example I and example II
Figure 2 is an illustration of VCC profile obtained by the process as described in example I and example II
Figure 3 is an illustration of % viability profile obtained by the process as described in example I and example II
Figure 4 is an illustration of IVCC profile obtained by the process as described in example I and example II
Figure 5 is an illustration of antibody titer obtained by the process as described in example 1 and example 2.

DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “glycan” refers to a monosaccharide or polysaccharide moiety.
The term “glycoprotein” refers to protein or polypeptide having at least one glycan moiety. Thus, any polypeptide attached to a saccharide moiety is termed as glycoprotein.
The term “glycoform” or “glycovariant” have been used interchangeably herein, and refers to various oligosaccharide entities or moieties linked in their entirety to the Asparagine 297 (as per Kabat numbering) of the Fc region of the antibody in question, co translationally or post translationally within a host cell. The glycan moieties added during the glycosylation process may 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.
The term “Antibody composition” as used herein pertains to the quantity or percentage of different glycoforms present in an antibody preparation.
“Afucosylated glycans” described here, consists of glycan moiety wherein fucose is not linked to the non-reducing end of N-acetlyglucosamine. Without limitation, examples of afucosylated glycans include M3NAG, G0, G1A, G1B etc.
“Fucosylated glycans” described here, consists of glycan moiety wherein fucose which is linked to the non-reducing end of N-acetlyglucosamine. Without limitation, examples of fucosylated glycans include M3NAGF, G1AF, G1BF, G2F, G2SF, G2S2F etc.
“High mannosylated glycans” described here, consists of comprising glycan moiety which comprises 5 or more mannose residues. Without limitation examples of high mannosylated glycans include M5, M6, M7, M8, M9 etc..
The term “osmolality” as used herein is defined as a measure of the osmoles of solute per kilogram of solvent (Osm/kg) and may include ionized or non-ionized molecules. The osmolality may change during the cell culture process for e.g. by addition of feed, salts, additives or metabolites.
The term “Distributed” with respect to feeding of Manganese or supplementation of Manganese refers to the mode of addition of Manganese. It refers to addition of Manganese in a manner such that the cumulative amount of Manganese to be added to the cell culture is distributed either at different time points or different days over the course of cell culture.

Table I: Representative table of various glycans



The term “temperature shift” as used herein is defined as the change in temperature during the cell culture process. For the purpose of this invention, the initial temperature of the cell culture process is higher than the final temperature i.e. cells are subjected to a temperature downshift wherein cells are first cultured at a higher temperature for certain time period after which temperature is reduced, and cells are cultured at this lower temperature for a fixed period of time
The term “Viable cell count (VCC)” refers to the no. of viable cell per unit volume at a particular time point in cell culture. The unit usually used for its measurement is million viable cells/ ml. The “VCC profile” represents graphically the VCC over the entire duration of cell culture.
The term “% Viability” refers to percentage of viable cells out of total no. of cells including the viable and non-viable cells at a particular time point in cell culture. The “% viability profile” represents graphically the % viability over the entire duration of cell culture.
As used herein, “IVCC” or “Integral viable cell concentration” refers to cell growth over time or integral of viable cells with respect to culture time that is used for calibration of specific protein production. The integral of viable cell concentration can be increased either by increasing the viable cell concentration or by lengthening the process time. The “IVCC profile” represents graphically the IVCC over the entire duration of cell culture.
Detailed description of the embodiments
The present invention discloses a cell culture process for obtaining a glycoprotein with a glycoform composition comprising reduced percentage of high mannosylated and afucosylated glycans.
In one embodiment the invention provides a cell culture process comprising manganese supplementation distributed over the course of cell culture, wherein the said supplementations are performed at an interval of about one day from the previous supplementation, for obtaining a glycoprotein with a glycoform composition comprising reduced percentage of high mannosylated and afucosylated glycans, as compared to cell culture process having a bolus addition of manganese.
In further embodiment, the present invention provides a process for obtaining a glycoprotein with a glycoform composition comprising about 11.7 % of high mannosylated glycans and about 5.4 % of afucosylated glycans.
In yet another embodiment, the present invention provides a process for obtaining a glycoprotein with a glycoform composition wherein high mannosylated glycans are reduced by about 43% and afucosylated glycans are reduced by about 29%, as compared to cell culture process having a bolus addition of manganese.
In one embodiment of the invention, the present invention provides a process for obtaining an increase in titer of a glycoprotein, as compared to cell culture process not comprising distributed manganese supplementation,
In further embodiment of the invention, the present invention provides a process for obtaining an increase in antibody titer by about 53%, as compared to cell culture process having a bolus addition of manganese.
In another embodiment of the invention, the cell culture is supplemented with manganese wherein said supplementation is performed in multiple steps during the cell culture process.
In yet another embodiment of the invention manganese is supplemented at least thrice during the cell culture process.
In an embodiment of the invention, the cell culture process comprises supplementation with about 10 µM of manganese.
Examples of useful salts of divalent manganese ions include, but are not limited to, manganese sulphate and manganese chloride.
The supplementation of manganese to the cell culture medium can be done by feeding the cells with a feed comprising manganese or adding manganese separately.
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 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), 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 Cell Boost 2 (CB-2, Thermo Scientific Hyclone, Catalogue no SH 30596.03), Cell Boost 4 (CB-4, Thermo Scientific HyClone, Catalog no. SH30928), PF CHO (Thermo Scientific Hyclone, Catalog no. SH30333.3).
The temperature of a cell culture is selected based on the temperature range at which cells remain viable, produce glycoprotein of interest in desirable quantity and qualitative profiles. In general, mammalian cells grow well and produce desirable glycosylation profiles in commercially viable quantity within temperature range of 25oC to 42oC. For example, optimum temperature range for CHO cells is at approximately 35oC to 37oC. Those of ordinary skill in the art will be able to choose optimum temperature and/or temperature range depending on the cell type.
Optionally, cells may be subjected to temperature shift at any time during the course of the cell culture. The temperature shift may be gradual or abrupt. The subsequent temperature may be higher or lower than the initial temperature value. Additionally, the cells may be exposed to more than one such temperature shifts. As with the initial temperature the subsequent temperatures may be selected based on the temperature range at which cells remain viable, produce glycoprotein of interest in desirable quantity and quantitative profiles. Those of ordinary skill in the art will be able to choose optimum temperature and/or temperature range depending on the cell type.
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
A HER-2 antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 5821337 which is incorporated herein by reference. rCHO cells expressing antibody were seeded at a density of ~0.5 million cells/ml in culture media (IS CHO-CD G10.3, Catalog no. 98810) containing galactose (6 g/L) at 37°C and pH of 7.2. The cells were cultured for one day after which the the temperature was shifted to 35oC. Subsequently, cell boost 4 (CB4, Hyclone, Catalog No.:SH30928.03) was added on day 2, 3, 4, 5, and 6 respectively. Additionally, 10 µM Manganese and 15 mM betaine were added to the cell culture medium on day 4. The culture was harvested on day 12 or at viability less than or equal to 60%, whichever was earlier. The percentage of high mannosylated and afucosylated glycans in a glycoform composition (I) were determined as given in Table II and represented in Figure 1. Additionally viable cell count (VCC), % viability, Integral viable cell count (IVCC) and titer were determined as represented in figures 2-5.

Example II
A HER-2 antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 5821337 which is incorporated herein by reference. rCHO cells expressing antibody were seeded at a density of ~0.5 million cells/ml in culture media (IS CHO-CD G10.3, Catalog no. 98810) containing galactose ( 6 g/L) at 37°C and pH of 7.2. The cells were cultured for one day after which the temperature was shifted to 35oC. Subsequently, feed Cell Boost - 4 (CB4, Hyclone, Catalog No.:SH30928.03) was added on day 2, 3, 4, 5, and 6. Further, 15 mM betaine was added on day 4. Also, cell culture medium was supplemented with 10 µM Manganese, distributed over three days (day 4, 5, and 6). The culture was harvested on day 12 or at viability less than or equal to 60%, whichever was earlier. The percentage of high mannosylated and afucosylated glycans in a glycoform composition (II) were determined and the values as given in Table II and represented in figure 1. Additionally viable cell count (VCC), % viability, Integral viable cell count (IVCC) and titer were determined as represented in figures 2-5.

Table II: Percentage glycans in a glycoform composition
Antibody composition % High Mannosylated Glycans % Afucosylated Glycans
I 20.6 7.6
II 11.7 5.4
,CLAIMS:We claim:
1. A cell culture process comprising manganese supplementation distributed over the course of cell culture, wherein the said supplementations are performed at an interval of about one day from the previous supplementation, for obtaining a glycoprotein with a glycoform composition comprising reduced percentage of high mannosylated and afucosylated glycans, as compared to cell culture process having a bolus addition of manganese.
2. A cell culture of claim 1, wherein the said glycoprotein comprises about 11 % of high mannosylated glycans and about 5 % of afucosylated glycans.
3. A cell culture process of claim 1, wherein the said glycoprotein comprises a reduction by about 43% in high mannosylated glycans and reduction by about 28% in afucosylated glycans.
4. A cell culture process of claim 1, wherein the said cell culture process further results in an increase in titer of the glycoprotein.
5. A cell culture process of claim 5, wherein the said antibody titer is increased by about 53%.