DESC:FIELD OF THE INVENTION
The present invention generally relates to the field of cell culture. In particular, the invention discloses a cell culture process wherein temperature is shifted at about 20 hours to obtain a glycoprotein with a glycoform composition having increased percentage of high mannosylated glycans.
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 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 a 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. The efficacy of these glycoprotein is mediated by two independent mechanisms, (a) target antigen neutralization or apoptosis and (b) the antibody effector functions mediated via effector cells or complement system known as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) respectively. The efficacy is further modified by the changes in glycosylation profile and by its effect on ADCC and/or CDC. Marcella et al. have demonstrated that high mannose glycans in a glycoform composition enhance ADCC effector functions and increases mAb activity. Further, an increased Fc? receptor binding activity, a representative of ADCC mediated by Natural Killer (NK) cells is obtained with high mannose glycoforms. Additionally, high mannose bearing glycoforms exhibit decreased CDC activity, which might be advantageous for certain patients where CDC activity is linked to undesirable side effect(s) (Marcella Yu et al. MAbs. 2012 July 1; 4(4): 475–487). Thus increased percentage of high mannosylated glycans in a glycoform composition can have a marked influence on their efficacy and utility as therapeutics. Hence, there is a critical requirement for developing the production processes to achieve the same.
Several studies have been performed to understand the effects of cell culture process parameters on mannosylation content of the glycoform composition in a glycoprotein. Pacis et al. have demonstrated modulation of the mannosylation content by 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). Further, Jian Wu disclosed a process for achieving low mannose content (i.e less than about 10% M5 or greater glycoforms in the glycoprotein composition by maintaining the cell culture medium at low osmolality (US 8354105 B2).
Apart from desired percentage of glycan(s) in an antibody composition, an increased protein yield or titer is essential to meet the high dose requirements of mAbs. Temperature shift is a well-known physiological parameter for improving cell culture performance such as prolonging cell viability and improving product titers (US7541164 & EP2563906 A2). Such biphasic cell culture conditions typically consists of an initial phase of rapid cell growth at a first temperature, followed by reduction of the culture temperature thus initiating a production phase characterized by increased productivity and sustained viability. Studies have demonstrated the use of temperature shift to produce more homogeneous proteins (EP 2660328 A1). Further, temperature shift by improving cell viability and in turn increasing overall protein production improves glycosylation (Moore A et al. Cytotechnology. 1997 Jan;23(1-3):47-54. doi: 10.1023/A:1007919921991).
However the timing of this “metabolic shift” i.e. from growth phase to production phase, needs to be optimized in order to allow adequate cell growth, the absence of which could be detrimental to the quality and quantity of product being expressed.
The present invention provides a cell culture process for obtaining a glycoprotein with a glycoform composition having an increased percentage of high mannose glycans by culturing cells at first temperature for about 20 hours followed by reducing the cell culture temperature to obtain the said glycoprotein.
SUMMARY OF THE INVENTION
The present invention describes a cell culture process to obtain a glycoprotein with a glycoform composition with increased percentage of high mannose glycans. The cell culture process involves culturing cells at first temperature for about 20 hours followed by subjecting cells to reduced temperature to obtain increased percentage of high mannose glycans.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an illustration of percentage viability profile obtained by the process as described in example I and example 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 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.
The “glycoform composition” or “distribution” as used herein pertains to the quantity or percentage of different glycoforms present in a glycoprotein.
“Afucosylated glycans” described here, consists of glycan moieties 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.
“High mannosylated glycans” described here, consists of glycan moieties 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.

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 “% Viability” refers to percentage of viable cells out of total number 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.
Detailed description of the embodiments
In one embodiment the invention provides a cell culture process for obtaining a glycoprotein composition wherein the said cell culture process comprises culturing cells at first temperature for about 20 hours from initiation of cell culture i.e post inoculation of cell culture medium, and subsequently subjecting cells to a second temperature.
In a further embodiment the invention provides a cell culture process comprising culturing cells at first temperature for about 20 hours from initiation of cell culture i.e post inoculation of cell culture medium and subsequently subjecting cells to a second temperature for obtaining a glycoprotein with a glycoform composition that comprises increased percentage of high mannosylated glycans, as compared to cell culture not comprising a temperature shift.
In another embodiment, the invention provides a cell culture process for obtaining a glycoprotein with a glycoform composition comprising about 16% high mannosylated glycans, wherein the cell culture process comprises temperature shift.
In yet another embodiment the invention provides a cell culture process comprising a temperature shift for obtaining a glycoprotein with a glycoform composition wherein high mannosylated glycans are increased by about 67 %, as compared to a cell culture process not comprising a temperature shift.
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 temperature of a cell culture is selected based on the temperature range at which cells remain viable, produce glycoprotein of interest in increased quantity and desirable qualitative profiles. In general, mammalian cells grow well and produce desirable glycosylation profiles of glycoprotein 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, cell 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 temperature/temperature range may be selected based on the temperature range at which cells remain viable, produce glycoprotein of interest in increased quantity and desirable qualitative profiles. Those of ordinary skill in the art will be able to choose optimum temperature and/or temperature range depending on the cell type.
In another embodiment, the invention provides a method for obtaining a glycoform composition comprising culturing cells at temperature of about 35-37°C, followed by lowering of temperature by about 2-7°C.
In yet another embodiment, the invention provides a method for obtaining a glycoform composition comprising culturing cells at about 37°C, followed by subjecting cells to about 35°C.
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), 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 are 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 depeleted 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).
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 IS CHO-CD culture media (IS CHO-CD G10.3, Irwine Scientific, Catalog no. 98810) at 35°C and pH 7.2 Subsequently, feed Cell Boost - 4 (CB4, Hyclone, Catalog No.:SH30928.03) was added on day 2, 3, 4, 5, and 6 respectively. Additionally on day 4, manganese and betaine were added at concentration of 10 µM and 40 mM respectively. The culture was harvested on day 12 or at viability less than or equal to 60%, whichever was earlier and percentage of high mannose glycans and titre (I) were determined as represented in table II. Additionally % viability, was determined as represented in figures 1.
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 IS CHO-CD culture media (IS CHO-CD G10.3, Catalog no. 98810) at 37°C and pH 7.2. The cells were cultured for 16-20 hours 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 respectively. Additionally on day 4, 10 µM manganese and 40 mM betaine were added to the cell culture.The culture was harvested on day 12 or at viability less than or equal to 60%, whichever was earlier and percentage of high mannose glycans and titre (II) was determined as represented in table II. Additionally % viability was determined as represented in figures 1-4.

Table II: % High Mannosylated glycans and titer in a glycoform composition
Data points % High Mannose glycans Titer (mg/ L)
I 9.5 987.0
II 16.0 901.5
,CLAIMS:We claim:
1) A cell culture process comprising a temperature shift at about 20 hours from initiation of cell culture i.e. post inoculation of cell culture medium, for obtaining a glycoprotein with a glycoform composition comprising an increased percentage of high mannosylated glycans, as compared to cell culture not comprising a temperature shift at about 20 hours.
2) A cell culture process of claim1, wherein the glycoprotein composition comprises about 16% high mannosylated glycans.
3) A cell culture process according to claim 1, wherein the said increase in high mannosylated glycan is by about 67 %.
4) A cell culture process of claim1, wherein cell culture comprises culturing cells at temperature of about 35-37°C, followed by lowering of temperature by about 2-7°C.