INTRODUCTION

The invention describes a method for controlling glycosylation in a glycoprotein by subjecting cells to a temperature and pH shift. Particularly, the invention relates to a process for obtaining a glycoprotein composition containing increased percentage of high mannose glycans and reduced percentage of galactosylated glycans. Protein glycosylation is one of the most important post-translation modifications associated with eukaryotic proteins. This is evidenced by the fact that more than 50% of the eukaryotic proteins are glycosylated (Apweiler et al., 1999, Biochim Biophys Acta 1473(l):4-8.).The structure and composition of the saccharide (glycan) moieties added upon glycosylation can have profound effect on the stability, safety and efficacy of these proteins (Wong CH, 2005, J Org

Chem 70(11): 4219-25). Hence, an understanding of glycosylation and modes of controlling the same are of immense significance.

Broadly, the two major types of glycosylation in eukaryotic cells are - N- linked glycosylation in which glycans are attached to the asparagine residue and O-linked glycosylation in which glycans are attached to serine or threonine residues. Further, N-linked glycans are of two types - 'high mannose' glycans consisting of two N-acetylglucosamines plus a large number of mannose residues (more than 4), and 'complex' glycans 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 usually a range of glycan structures associated with each site.

Among glycoproteins, monoclonal antibodies (mAbs) have emerged as major therapeutic agents against various diseases owing to their higher target specificity, better pharmacological potencies, and lower side effects in comparison to small -molecule drugs (Zhong X. and Somers W., 2012, Recent Advances in Glycosylation Modifications in the Context of Therapeutic

Glycoproteins, Integrative Proteomics, ISBN: 978-953-51-0070-6). The in-vivo physiological activity of mAbs is mediated by two independent mechanisms, (a) target antigen neutralization or apoptosis and (b) antibody effector functions which include antibody-dependent cellular cytotoxicity (ADCC) and

complement-dependent cytotoxicity (CDC). Importantly, the effector functions of antibodies has been shown to correlate with the glycan structures associated with these mAbs. Further, glycosylation of mAbs are also known to improve therapeutic efficacy through its impact on protein pharmacodynamics (PD) and pharmacokinetics (PK) (Mahmood I, Green MD, 2005, Clin Pharmacokinet 44(4): 331-47 ; Tang et al., 2004, J Pharm Sci 93(9):2184-204.). The N- glycosylation in mAbs involves attachment of oligosaccharides at asparagine (Asn)-297 in the CH ₂ domain of Fc region of IgGs. This is a unique feature of IgG, making it a "key player" in functioning of the immune system. However, mAb homogeneity is difficult to achieve, complicated by the fact that nonhuman systems, which are generally used for the large scale production of these therapeutic antibodies, may secrete IgGs with significant variation in the glycan structures. This can potentially result in suboptimal effector functions of these antibodies. Clearly, methods for improving, controlling and modifying glycosylation have immense impact on the production and functionality of therapeutic mAbs.

Several factors affect glycosylation profile of a glycoprotein. These include cell line characteristics, process control parameters and cell culture media components (Andersen et al., 2000, Biotechnol Bioeng 70(1): 25-31.; Butler, 2005, Appl Microbiol Biotechnol. 68(3): 283-291.) A number of strategies exist in the prior art to modulate compositions of different glycoforms. One of the suggested approaches is genetic manipulation of the cell lines for glycosyl transferases, enzymes responsible for glycosylation (Yamane-Ohnuki et al., 2004, Biotechnol Bioeng., 87: 614-622; Shinkawa et al., 2003, J. Biol. Chem.,

278: 3466-73; Mori et al., 2004, Biotechnol Bioeng., 88:901-8; Ferrara et al., 2006, Biotechnol Bioeng., 93: 851-61).

Other methods include in vitro modification of proteins post protein synthesis to obtain desired glycoprotein (Inazu T., 2007, Research in construction of the complex system for functional oligosaccharides. Proceeding of the Institute of Glycotechnology of Tokai University; 2:42-45; Yazawa et al., 1986, Biochem Biophys Res Commun. 1986; 136: 563-569; Yamamoto et al., 2008, J. Am. Chem. Soc, 130: 501-10). Modifications in cell culture conditions and media

compositions have also been proposed to modulate glycosylation content of glycoproteins. For example, it has been demonstrated that, for the mAbs produced in the rat hybridoma cell line YB2/0 there is a direct correlation between osmolality of the culture medium and afucosylation (Yoshinobu et al., 2010, Animal Cell Technology: Basic & Applied Aspects Volume 16, pp 121 - 125). Makkapati et al. have described a cell culture process for obtaining a glycoprotein composition comprising about 14% to about 18% total afucosylated glycans by culturing cells in a medium supplemented with galactose, at a specific osmolality, and harvesting on about 12th day or at about 50% viability

(WO2013114165 Al). Ramasamy et al. have demonstrated the role of manganese in increasing Mans glycans and/or afucosylated glycans in a glycoprotein composition (WO2013114245 Al). Further, US 2012/0276631 Al teaches about the use of Mn ion and/or D-galactose in the culture media to modulate galactosylation.

Several studies have suggested that the terminal Galactose content of IgG improves CDC as a result of increased antibody binding to Clq, without impacting the ADCC activity (Hodoniczky J, Zheng YZ, James DC: Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog 2005, 21 : 1644-1652). Further, addition of galactose is a template for addition of sialic acid on mAbs (Marino, K., (2010) Nature

Chemical Biology 6,713-723). This increased terminal sialylation can increase the serum half-life of many glycoproteins (Raju TS: Glycosylation variations with expression systems and their impact on biological activity of therapeutic immunoglobulins. Bioprocess Int 2003, 1:44-53). However, in contrast, studies have also demonstrated that increased sialylation of Fc glycans results in decreased ADCC activity of rlgGs. (Scallon BJ, Tarn SH, McCarthy SG, Cai AN, Raju TS: Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol Immunol 2007, 44: 1524-1534). Without being bound by theory a decreased galactosylation will result in a decreased sialylation and hence may prevent the decrease in ADCC activity of recombinant IgGs.

Another important component of glycoform composition of a

glycoprotein is its high mannose (HM) content. Increased HM content has been shown to lead to potential enhancement of its biological activity in terms of higher ADCC activity and greater affinity to FcyRIIIA (Zhou et al., 2008, Biotechnol Bioeng 99(3): 652-665.). Though concerns have been raised on increasing HM content as it might lead to high clearance rate of mAbs, there are no confirmatory evidence to prove it. In fact, Chen et al. have argued against it and have shown that increasing HM glycan can at best support enhancement of glycan cleavage without having any effect on antibody clearance (Chen et al., Glycobiology 19(3):240-249). Hence a process ensuring the control over a particular glycoform, specifically HM and galactose in a glycoprotein

composition may have immense industrial value. However, no teachings exist in the prior art to control both high mannose and galactosylated content of a glycoprotein composition in a single process. The present invention provides a process for obtaining a glycoprotein composition with modified high mannose and galactosylated glycans by subjecting cells to temperature and pH shift. SUMMARY

The invention describes a cell culture process for modifying high mannose and galactosylated content of a glycoprotein composition by subjecting cells to a temperature and pH shift during cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an illustration of antibody titer as described in Examples I and II.

Figure 2 is an illustration of viable cell count as described in Examples I and II.

Figure 3 is an illustration of IVCC as described in Examples I and II. Figure 4 is an illustration of percentage of high mannose and galacotsylated glycans as described in Examples I and II.

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 human Fc region of the glycoprotein in question, co translationally or post-translationally within a host cell. The glycan moieties may be added during protein glycosylation include M ₃ , M ₄ , 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 "glycoform composition" or distribution as used herein pertains to the quantity or percentage of different glycoforms present in a glycoprotein.

As used herein, "high mannose glycovariant" consists of glycan moieties comprising two N-acetylglucosamines and more than 4 mannose residues i.e. Ms, M ₆ , M ₇ , and Ms.

"Galactosylated glycans" refers to glycans containing terminal galactose residues such as GIA, GIB, GIAF, GIBF, G ₂ , G ₂ F and G ₂ SF. The "complex glycovariant" as used herein consists of glycan moieties comprising any number of sugars. "Afucosylated glycovariants or glycoforms" described here, consists of glycan moieties wherein fucose is not linked to the non-reducing end of N- acetlyglucos amine (for e.g. M3NAG, Go, GIA, GIB, G ₂ ).

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 term "temperature shift" as used herein is defined as the change in temperature during the cell culture process.

The term "pH shift" used herein is defined as the change in pH during the cell culture process.

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. Table I: Representative table of various glycans

Mannose ♦ 2-AB Labei N-Aeetyi GiticasamSne · Fucose Galactose Sisiic acit ^, Detailed description of the embodiments

The present invention discloses a cell culture process for obtaining a glycoprotein composition with increased percentage of high mannose glycoforms and reduced percentage of galactosylated glycoforms.

In a first embodiment, the present invention provides a cell culture process for obtaining a glycoprotein composition with increased percentage of high mannose glycans comprising culturing cells at a first temperature and a first pH for a first period of time, culturing cells at a second temperature and a second pH for a second period of time, culturing cells at a third pH for a third period of time and recovering the protein from the cell culture.

In another embodiment, the present invention provides a process for obtaining a glycoprotein composition comprising about 4% to about 7% high mannose glycans.

In yet another embodiment, the present invention provides a process for obtaining a glycoprotein composition wherein the high mannose glycans are increased by about 40% to about 145%.

In a second embodiment, the present invention provides a cell culture process for obtaining a glycoprotein composition with decreased percentage of galactosylated glycans comprising culturing cells at a first temperature and a first pH for a first period of time, culturing cells at a second temperature and a second pH for a second period of time, culturing cells at a third pH for a third period of time and recovering the protein from the cell culture

In another embodiment, the present invention provides, a process for obtaining glycoprotein composition comprising about 20% to about 22.5% galactosylated glycans.

In yet another embodiment, the present invention provides a process for obtaining glycoprotein composition wherein galactosylated glycans are reduced by about 24% to about 32%.

In a third embodiment, the present invention provides a cell culture process for obtaining a glycoprotein composition with an increased percentage of high mannose glycans and a decreased percentage of galactosylated glycans comprising culturing cells at a first temperature and a first pH for a first period of time, culturing cells at a second temperature and a second pH for a second period of time, culturing cells at a third pH for a third period of time and recovering the protein from the cell culture.

In another embodiment, the present invention provides a process for obtaining glycoprotein composition comprising about 4% to about 7% high mannose glycans and about 20% to about 22.5% galactosylated glycans.

In yet another embodiment, the present invention provides a process for obtaining a glycoprotein composition wherein the high mannose glycans are increased by about 40% to about 145% and galactosylated glycans are reduced by about 24% to about 32%.

In another embodiment, the invention provides method for production of glycoproteins with a particular glycoform composition wherein the cells are subjected to temperature shift(s). The temperature shift may be a temperature upshift wherein the later temperature is at a higher value than the earlier value or a temperature downshift wherein the later temperature is at a lower value than the earlier value.

In yet another embodiment, the invention provides a method for production of glycoproteins with a particular glycoform composition by first culturing cells at temperature of about 35°C - about 37°C, followed by lowering of temperature by about 2°C - about 7°C.

In further embodiment, the invention provides a method for expression of protein with particular glycoform composition by growing cells at about 37°C, followed by subjecting cells to about 35°C.

In another embodiment, the invention provides method for production of glycoproteins with a particular glycoform composition wherein the cells are subjected to pH shift(s). The pH shift may be a pH upshift wherein the later pH is at a higher value than the earlier value or a pH downshift wherein the later pH is at a lower value than the earlier value.

In another embodiment, the second pH shift is done when the cells in the culture have reached a stationary phase or when the growth of the culture is arrested.

In the embodiment mentioned above, the pH is shifted at about 9 days after inoculation of the culture.

In yet another embodiment, the invention provides a method for production of glycoproteins with a particular glycoform composition by first culturing cells at a pH of about 7.2 followed by culturing cells at a pH reduced by about 0.1 to about 0.5 unit and further culturing cells at a pH increased by about 0.1 to about 0.5 pH unit.

In a further embodiment, the invention provides a method for production of glycoproteins with a particular glycoform composition by first culturing cells at a pH of about 7.2 followed by culturing cells at a pH of about 6.8 and further followed by culturing cells at a pH of about 7.0.

In another embodiment the shift in temperature and pH may be accompanied by addition of nutrient feed. The cell culture media that are useful in the invention include but are not limited to, the commercially available products PF-CHO (HyClone ^® ), PowerCHO ^® 2 (Lonza), Zap-CHO (Invitria), CD CHO, CD OptiCHO™ and CHO-S-SFMII (Invitrogen), ProCHO™ (Lonza), CDM4CHO™ (Hyclone), DMEM

(Invitrogen), DMEM/F12 (Invitrogen), Ham's F10 (Sigma), Minimal Essential Media (Sigma), and RPMI -1640 (Sigma) or combinations thereof.

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 depleted nutrients. 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).

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-VEGF antibody was cloned and expressed in a recombinant CHO cell line as described in U.S. Patent No. 7,060,269, which is incorporated herein by reference. rCHO cells expressing antibody at a seeding density of 0.2-0.6 million cells/ml were seeded in PowerCHO ^® 2 (Lonza, Catalog no: 12-771Q) medium at 37 °C and pH 7.2. The cells were cultured for about 64 hours (2-3 days after inoculation of culture), subsequently pH was reduced to 6.8 and the temperature was reduced to 35 °C. The CB-4 feed was added on day 1, 3, 5 and 7. The culture was finally harvested after 240 hours to 288 hours or at greater than 50% viability. The experiment was run in two separate batches. The average values (I) for antibody titer, VCC, IVCC and percentage of high mannose and galactosylated glycans are shown in Figure 1-4. The percentage of high mannose and galactosylated glycan values are depicted in Table II. Example II

An anti-VEGF antibody was cloned and expressed in a recombinant CHO cell line as described in U.S. Patent No. 7,060,269, which is incorporated herein by reference. rCHO cells expressing antibody at a seeding density of 0.2-0.6 million cells/ml were seeded in PowerCHO ^® 2 (Lonza, Catalog no: 12-771Q) at 37 °C and pH 7.2. The cells were cultured for about 64 hours (2-3 days after inoculation of culture), subsequently pH was reduced to 6.8 and the temperature was reduced to 35 °C. The CB-4 feed was added on day 1, 3, 5 and 7, while pH of cell culture was changed to 7.0 on day 9 (when growth of the culture has been arrested). The culture was finally harvested after 240 hours - 288 hours or at greater than 50% viability. The experiment was run in at least four separate batches. The average values (II) for antibody titer, viable cell counts, IVCC and the percentage of high mannose and galactosylated glycans are depicted in Figure 1-4. The percentage of high mannose and galactosylated glycan values are depicted in Table II.

Table II: Glycoform compsition

%

Example % HM

Galactosylation

(range)

(range)

2.9+0.3 29.6±0.5

I (2.7-3.2) (29.3-30.0)

5.3+0.9 21.6±0.9

II (4.5-6.6) (20.4-22.3)