DESC:FIELD OF THE INVENTION
The invention describes a cell culture process for obtaining a desired glycoprotein. More specifically, the invention describes a cell culture process wherein cells are maintained at a particular temperature and pH for a specific period of time, after which, temperature and pH are changed to obtain a glycoprotein with a particular glycoform composition.
BACKGROUND OF THE INVENTION
Recombinant glycoproteins and particularly monoclonal antibodies (mAbs) have spearheaded treatment of various diseases such as cancers, inflammatory disorders and autoimmune diseases. The development of these complex glycoproteins for therapeutic applications has been achieved by large scale expression of proteins from recombinant mammalian cell culture systems, which provide optimum glycosylation and protein yield. However, protein production from these cell culture systems pose significant challenges as it requires optimization of several cell culture process parameters such as temperature, pH, osmolality etc.
Temperature and pH are the basic physiological parameters that significantly influence protein production from recombinant cell culture. Temperature, in general, affects cell growth wherein a reduction in temperature arrests cell growth and thus prolongs cell viability (Moore et al., (1997), Cytotechnology. 23:47–54). This is important as cell culture requirements vary over the course of the cell culture process. At the outset improved cell growth is advantageous, however in later stages extended cell survival becomes more important. Hence, it becomes imperative to maintain high viable cell density for a longer period of time to obtain high product titers. In this respect, introducing one or more temperature shifts during cell culture has been suggested (Chen et al., J Biosci Bioeng. 2004; 97 (4):239-43; US 8129145; US 7541164). For this, mammalian cells are cultured at two or more different temperatures, wherein cells are initially cultured at a higher temperature to attain cell growth and then shifted to a lower temperature to improve productivity of cells and quality of product (Weidemann et al., Cytotechnology. 1994, 15(1-3): 111-6).
The pH of cell culture process influences cell growth and hence glycoprotein production in a manner specific to the cell lines, clonal variation and product (Sauer et al. Bioteclmology and Bioengineering 2000, Vol 67, pg. 586-597; Yoon et al., Biotechnology and Bioengineering 2004, Vol 89, pg. 346-356; Kim, S H and Gyun M L, J. Microbiol. Biotechnol. (2007), 17(5), 712–720). A reduction in pH during cell culture has been suggested to enhance cell viability by a hitherto unknown mechanism (Oguchi et al., Cytotechnology (2007) 52:199–207; Borys et al., Biotechnology (NY). 1993 Jun; 11(6):720-4). It has been argued that reducing pH favors cell viability by reducing apoptosis and hence helping to maintain an optimal viable cell density (VCD). The rationale is the fact that amount of externally supplied dissolved CO2 levels can be reduced so as to negate the negative effects of CO2. Furthermore reducing pH has an advantage of requiring less amount of base to negate the effect of lactic acid production by cells (which can have deleterious effects) at the later stages of cell culturing.
The effect of temperature and pH shift on the cell culture process has been reported recently and suggests an improvement in protein yield (US 2013/0130316A1; EP 2563906A2). The process involves culturing cells at first pH for at least two days and a first temperature for at least three days. Subsequently, the pH and temperature are shifted to a lower value and cells are cultured at reduced pH and temperature for a specific time period. Afterwards, cells are cultured at a higher pH and temperature.
Protein glycosylation and factors affecting glycosylation:
Protein glycosylation is one of the most important post-translational 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 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 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 usually a range of glycan structures associated with each site.
The process of N-linked glycosylation begins co-translationally in the Endoplasmic reticulum where a complex set of reactions result in the attachment of Glc3NAc2Man9 (3 glucose, 2 N-acetylglucosamine and 9 mannose) to a carrier molecule called dolichol, that is then transferred to the polypeptide chain as it is translocated into the ER lumen (Schwarz, F. and Aebi M., (2011) Current Opinion in Structural Biology, 21:576–582; Burda, P. & Aebi M., (1999) Biochimica and Biophysica Acta, General Subjects, Volume 1426, Issue 2, Pages 239-257). The glycan complex so formed in the ER lumen is modified by enzymatic actions in the Golgi apparatus. If the saccharide is relatively inaccessible, it will most likely stay in the original high-mannose (HM) form. If it is accessible, then many of the mannose residues will be cleaved off and the saccharide will be further modified, resulting in the complex type N-glycan structure (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).
Thus the sugar composition as well as the structural configuration of a glycan structure depends on the protein being glycosylated, the cells/cell lines, the glycosylation machinery in the Endoplasmic Reticulum and the Golgi apparatus, the accessibility of the enzyme machinery to the glycan structure, the order of action of each enzyme and the stage at which the protein is released from the glycosylation machinery.
In addition to the in vivo factors listed above, “external factors” also affect the glycan structure and hence composition of a glycoprotein. These include the conditions in which the cells, expressing the protein, are cultured, such as pH, temperature, the medium composition, the addition of feed(s), osmolality etc. For example, A temperature shift or specifically decrease in temperature causes a reduction in protein sialylation or alter overall glycosylation (Kaufman et al., (1999) Biotechnol Bioeng. 63, 573-578; Trummer et.al., (2006) Biotechnol Bioeng. 94 1045-1052); Yoon et al., (2003) Biotechnol Bioeng., 82: 289-298; US 2003/0190710 A1; EP 1373547 A1; US 2004/0214289 A1). Further, reducing temperature can increase overall protein production by prolonging cell viability, which may enhance glycosylation. (Moore et al., 1997, Cytotechnology. 23:47–54). Similarly, high pH results in better glycoprotein yields while low pH provide better glycan quality (Borys et al., (1993), Biotechnology 11 720-724).
Glycosylation in monoclonal antibodies
Glycosylation is important for the effectiveness as well as stability of monoclonal antibodies, including its immunogenicity, solubility and half-life. For instance, the absence of fucose in the glycan structure of the Fc region of the antibodies has been associated with higher antibody dependent cell mediated cytotoxicity (ADCC) activity, and presence of higher mannose glycans has been associated with faster clearance of glycoprotein from serum (Werner et al., 2007, Acta Paediatr Suppl. Apr; 96(455):17-22.). Removal of terminal galactose residues from the chimeric mouse–human IgG1 antibody (alemtuzumab) was shown to reduce complement dependent cytotoxicity (CDC), without affecting Fc?R-mediated functions (Boyd et al., 1995, Mol. Immunol. 32, 1311–1318). Similarly, the (G1F–G1F) glycoform of rituximab triggered a CDC response twice as large as that triggered by the (G0F–G0F) glycoform (Jefferis R., Nature Reviews Drug Discovery, 2009, 8, 226-234).
Considering the role of the glycan structure and its composition in the activity and efficacy of a glycoprotein, manufacturing methodologies that can control the glycan composition of glycoproteins without compromising their production would be beneficial.
Several studies have been performed using temperature and/or pH shift(s) for obtaining desired protein quality or quantity. A reduction in overall glycosylation has been obtained by lowering of temperature during the cell culture (US 2003/0190710 A1; EP 1373547 A1; US 2004/0214289 A1). However, these patent applications do not describe a process for obtaining glycoform composition with defined glycosylation, afucosylation or mannosylation content. Further, Borys et. al. describes increase in glycosylation at a low culture pH and a good yield at a high culture pH. However, it does not provide an optimum pH (or pH shift) for obtaining both, good yield and glycosylation.
Similarly, studies on the use of temperature shift for obtaining desired protein quantity have been described (US7541164, EP 2563906A2). However, these teachings either lack in description of the timing of temperature shift, or describe multiple temperature shifts to obtain the desired protein quantity. A combination of temperature and pH shifts has been described to improve protein yields (EP 2563906A2). However, the method refers to temperature and pH at two different time points which is operationally inconvenient for large scale cell culture processes. Further, the process does not describe the effect of temperature and pH shift on protein glycosylation or change in glycosylation.
Thus, given limitations in the prior art, a cell culture process that provides optimum protein yield and desired protein glycosylation will be of immense industrial value.
The present invention provides a cell culture process for obtaining high yield of a glycoprotein with specific glycoform composition by culturing cells at a first temperature and pH for a specified time followed by culturing cells at a second temperature and pH.
SUMMARY OF THE INVENTION
The invention describes a cell culture process for obtaining a glycoprotein with a particular glycoform composition. The process involves culturing cells at a particular temperature and pH for 48-70 hours after which temperature and pH are shifted to obtain the desired glycoprotein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an illustration of effect of temperature and pH shift on antibody titer as described in Examples 1-3. Y-axis shows the antibody titer obtained when the temperature and pH shift was performed at indicated time points. X-axis denotes the age of the culture.
Figure 2 is an illustration of effect of temperature and pH shift on viable cell count (VCC) as described in Examples 1-3. Y-axis shows the number of viable cells (*106) when the temperature and pH shift was performed at indicated time points. X-axis denotes the age of the culture.
Figure 3 is an illustration of IVCC profile of cells in culture upon temperature and pH shift as described in Examples 1-3.
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 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 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.
As used herein, “high mannose glycovariant” consists of glycan moieties comprising two N-acetylglucosamines and more than 4 mannose residues i.e. M5, M6, M7, and M8.
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-acetlyglucosamine (for e.g. M3NAG, G0, G1A, G1B, G2).
G0, as used herein refers to protein glycan not containing galactose at the terminal end of the glycan chain.
The term “temperature shift” as used herein refers to any change in temperature during the cell culture process.
The term “pH shift” as used herein refers to a 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.
The “viable cell concentration (VCC)” or “cell viability” is defined as number of live cells in the total cell population.
Table I: Representative table of various glycans
Glycan structure Code Glycan structure Code
M3 M6
M3NAG G2F
M3NAGF M7
G0 G2SF
G0F M8
M5 G2S2F

G1A G1AF
G1B G1BF

Detailed description of the embodiments
The present invention provides a method for obtaining a glycoprotein composition with a specific glycoform profile. In particular, the invention provides a cell culture process wherein cells are maintained at a particular temperature and pH to attain optimum growth, after which, temperature and pH are reduced simultaneously such that high yield of glycoprotein is obtained.
In a first embodiment the present invention provides, a process for obtaining a glycoprotein composition comprising,
a) culturing cells at a first temperature and a first pH for about 48 to about 70 hours
b) subjecting cells to a second temperature and a second pH
In a second embodiment the present invention provides, a process for obtaining a glycoprotein composition comprising about 1.9% to about 3% high mannose glycans, about 1.2% to about 2.3% afucosylated glycans, about 57% to about 65 % of G0F glycans and about 29% to about 33% galactose glycans comprising,
a) culturing cells at a first temperature and a first pH, for about 48 to about 70 hours
b) subjecting cells to a second temperature and a second pH
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. 397:3457-3481and Geoffrey, R. G. et. al. Analytical Biochemistry 1996, Vol. 240, pages 210-226).
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 method for production of glycoproteins with a particular glycoform composition by first culturing cells at temperature of about 35-37°C, followed by lowering of temperature by about 2-7°C.
In further embodiment, the invention provides 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 yet another embodiment, the invention provides method for production of glycoproteins with a particular glycoform composition by first culturing cells at a pH of about 7.1 followed by culturing cells at a pH reduced by about 0.1 to about 0.5 unit.
In further embodiment, the invention provides method for production of glycoproteins with a particular glycoform composition by first culturing cells at a pH of about 7.1 followed by culturing cells at a pH of about 6.9.
In another embodiment the cells are first grown at about 37°C and a pH of about 7.1, followed by subjecting cells to about 35°C and a pH of about 6.9.
In an embodiment, the invention provides a cell culture process comprising maintaining cells at a first temperature and pH to attain optimum growth, after which, temperature and pH are shifted such that high yield of glycoprotein composition with a desired glycoform profile is obtained.
In another embodiment, the invention provides a cell culture process comprising maintaining cells at first temperature and pH for about 48 hours and then subjecting cells to a temperature and pH shift.
In another embodiment, the invention provides a cell culture process comprising maintaining cells at first temperature and pH for about 64 hours and then subjecting cells to a temperature and pH shift.
In another embodiment, the invention provides a cell culture process comprising maintaining cells at first temperature and pH for about 70 hours and then subjecting cells to a temperature and pH shift.
The shift in temperature and pH may be optionally 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 OptiCHOTM 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) or combination thereof. Further, the cell culture medium can be a combination of aforementioned cell culture medium and feed.
The feeds 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 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 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 cell culture media (PowerCHO®2 (Lonza, Catalog no: 12-771Q) and CB-4 feed in 95:5 ratio) at 37 °C and pH 7.1. The cells were cultured for 48 hours, subsequently pH was reduced to 6.9 and temperature was reduced to 35 °C and CB-4 feed was added. The culture was harvested at 244 - 288 hours or at greater than 50% viability. The average antibody yield, viability and IVCC of at least two batches are shown in figures 1-3 respectively. Table II represents the glycoform profile for the same.
Example II
An anti-VEGF antibody was cloned and expressed in a 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 cell culture media (PowerCHO®2 (Lonza, Catalog no: 12-771Q) and CB-4 feed in 95:5 ratio) at 37 °C and pH 7.1. The cells were cultured for 64 hours, subsequently pH was reduced to 6.9 and temperature was reduced to 35 °C and CB-4 feed was added. The culture was harvested at 244 - 288 hours or at greater than 50% viability. The average antibody yield, viability and IVCC of at least two batches are shown in figures 1-3 respectively. Table II represents the glycoform profile for the same.
Example III
An anti-VEGF antibody was cloned and expressed in a 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 cell culture media (PowerCHO®2 (Lonza, Catalog no: 12-771Q) and CB-4 feed in 95:5 ratio) at 37 °C and pH 7.1. The cells were cultured for 70 hours, subsequently pH was reduced to 6.9 and temperature was reduced to 35 °C and CB-4 feed was added. The culture was harvested at 244 - 288 hours or at greater than 50% viability. The average antibody yield, viability and IVCC of at least two batches are shown in figures 1-3 respectively. Table II represents the glycoform profile for the same.
Table II: Glycoform profile of antibodies
Example Temp & pH shift done at % High Mannose
mean (range) % Afucosylation
mean (range) %G0F
mean (range) % Galactose
mean (range)
I 48 2.3 (2.1-2.5) 1.8 (1.7-1.9) 61.1 (60.2-61.9) 31.5 (30.2-32.8)
II 64 2.2 (1.9-2.6) 1.7 (1.2-2.3) 61.9 (58.8-64.6) 31.1
(29.2-32.8)
III 70 2.6 (2.2-2.9) 2.0 (1.9-2.1) 59.1 (57.8-60.4) 32.1 (31.9-32.4)
,CLAIMS:What is claimed is,
1. A cell culture process for obtaining desired recombinant polypeptide comprising the steps of,
culturing cells at first temperature and pH,
shifting the temperature to a lower value and
shifting the pH to a lower value,
wherein the change in temperature and pH is done simultaneously at about 2 days to about 3 days.
2. A cell culture process for obtaining desired glycoprotein wherein the glycosylation comprises of about 57% to about 61% G0F, about 2% to about 3% high mannose, about 1% to about 1.5% of afucose and about 30% to about 33% of galactose, comprising the steps of,
culturing cells at first temperature and pH,
shifting the temperature to a lower value,
shifting the pH to a lower value,
wherein the change in temperature and pH is done simultaneously at about 2 days to about 3 days.

3. The process according to claim 1or claim 2, wherein the difference in the first and the second temperature is about 2°C to about 7°C.
4. The process according to claim 1 or claim 2, wherein the first temperature is in the range of about 35 o C to about 37°C.
5. The process according to claim 1 or claim 2, wherein the second temperature is in the range of about 31° C to about 36° C.
6. The process according to claim 1 or claim 2, wherein the difference in the first and second pH is about 0.1 to about 0.5 units of pH.
7. The process according to claim 1 or claim 2, wherein the first pH value is in the range of about pH 7.0 to about pH 7.2.
8. The process according to claim 1 or claim 2, wherein the second pH value is in the range of about pH 6.5 to about pH 7.0.
9. The process according to claim 1 or claim 2, wherein said second pH is maintained until harvest of the culture.