WO2015140700A1 - Cell culture process - Google Patents
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- WO2015140700A1 WO2015140700A1 PCT/IB2015/051930 IB2015051930W WO2015140700A1 WO 2015140700 A1 WO2015140700 A1 WO 2015140700A1 IB 2015051930 W IB2015051930 W IB 2015051930W WO 2015140700 A1 WO2015140700 A1 WO 2015140700A1
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- cell culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Definitions
- Protein glycosylation is one of the most important post-translation modifications associated with eukaryotic proteins.
- the structure and composition of the glycan moieties on protein can have a profound effect on the safety and efficacy of therapeutic proteins for e.g. the extent and nature of glycosylation in monoclonal antibodies (imAbs) affects the immunogenicity, solubility and half-life of imAbs
- ADCC antibody-dependent cellular cytotoxicity
- CDC complement-dependent cytotoxicity
- ADCC requires the presence of N-glycans, i.e. oligosaccharides covalently attached at the conserved Asn297 in the Fc region, and is sensitive to change in the oligosaccharide structure. It has been demonstrated that antibodies lacking core fucose residues in the Fc N-glycans exhibit high efficacy due to stronger ADCC even at low concentrations compared to their fucosylated counterparts (R. L. Shields et al., 2002, J. Biol. Chem. 277, 26733-26740). Additionally low fucosylation evades the inhibitory effect of serum immunoglobulin G (IgG) on ADCC by its high binding to effector cell receptors (FcYRIIIa) (S.
- IgG serum immunoglobulin G
- FcYRIIIa effector cell receptors
- non-fucosylated antibodies include achieving therapeutic efficacy at low doses, inducing high efficacy against tumor cells that express low levels of antigen or triggering high effector function in NK cells with the low-affinity FcvRllla allotype for the IgGs (N. Yamane-Ohnuki et al., 2004, Biotechnol Bioeng.;87:614-622 & R. Niwa et al., 2005, Clin Cancer Res.;11.2327-2336 & R. Niwa et al, 2004 Clin Cancer Res. ;10:6248-6255).
- the (G1 F-G1 F) glycoform of rituximab triggered a CDC response twice as large as that triggered by the (G0F- G0F) glycoform (http://www.nature.com/nrd/journal/v8/n3/full/nrd2804.html-accessed on 23/12/201 1 ). Presence of higher mannose glycans has been associated with faster clearance of glycoprotein from serum (Werner, R. G., Kopp, K. and Schlueter, M. (2007), 96: 17-22. doi: 10.1 1 1 1 /j.1651 -2227.2007.00199.x). Hence it becomes imperative to produce a glycoprotein composition comprising specific percentage of each glycoform variants.
- the sugar composition as well as the structural configuration of a glycan structure depends on in-vivo factors (e.g. cell lines) and "external factors" (e.g. medium composition, osmolality, pH, temperature). In fact, small changes in cell culture conditions during production can cause uncharacteristic glycosylation which may lead to potential risks (Dr Daryl Fernandes; European Biopharmaceutical Review. Winter 2004. pp 92 - 97).
- the objective of the invention is to provide a cell culture process for modulating and/or to obtain a specific glycoprofile composition taking into account these numerous separate but inter-linked factors influencing the glycan content and/or productivity.
- the invention describes a cell culture a cell culture process for producing glycoprotein having a glycoform composition comprising increased percentage of afucosylated glycans by culturing recombinant cells producing said glycoprotein in a medium comprising betaine.
- about 40 imM of betaine results in an increase in the afucosylated glycan level by at least about 25 % as compared to the cell culture medium without betaine.
- the cell culture process of the present invention with a medium comprising betaine and manganese and galactose results in a glycoprotein composition comprising specific, high mannosylated, afucosylated and galactosylated glycan content.
- betaine as a cell culture medium besides increasing the afucosylated glycans, serves to act as an osmoprotectant, considering increase in culture medium osmolality during the course of cell culture.
- the increase in afucosylation content and/or the specific glycoform composition obtained using the said process is consistent across batches. Obtaining a consistent glycoform profile is desired and highly significant not only in reducing the batch to batch variations but because the therapeutic efficacy of a glycoprotein molecule is defined by its glycoprofile.
- Figure 1 is an illustration of VCC profile obtained by the process as described in example I (cell culture medium without betaine) and example II (cell culture medium with betaine)
- Figure 2 is an illustration of % viability profile obtained by the process as described in example I (cell culture medium without betaine) and example II (cell culture medium with betaine)
- Figure 3 is an illustration of IVCC profile obtained by the process as described in example I (cell culture medium without betaine) and example II (cell culture medium with betaine)
- Figure 4 is an illustration of percentage of high mannosylated, afucosylated and galactosylated glycans in six batches of glycoform composition obtained by the process as described in example III (cell culture medium with betaine, manganese and galactose)
- FIG. 5 is an illustration of VCC profile obtained by the process as described in example III (cell culture medium with betaine, manganese and galactose)
- Figure 6 is an illustration of % viability obtained by the process as described in example III (cell culture medium with betaine, manganese and galactose)
- Figure 7 is an illustration of IVCC obtained by the process as described in example III (cell culture medium with betaine, manganese and galactose)
- Figure 8 is an illustration of antibody titer obtained by the process as described example III (cell culture medium with betaine, manganese and galactose)
- glycocan refers to a monosaccharide or polysaccharide moiety.
- glycoprotein refers to protein or polypeptide having at least one glycan moiety wherein glycan refers to a monosaccharide or polysaccharide moiety.
- glycoprotein any polypeptide attached to a saccharide moiety is termed as glycoprotein.
- 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.
- Table 1 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.
- 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).
- glycoform composition or “distribution” as used herein pertains to the quantity or percentage of different glycoforms present in a glycoprotein.
- Fucosylated glycans described here, consists of glycan moiety wherein fucose which is linked to the non reducing end of N-acetlyglucosamine.
- examples of fucosylated glycans include M3NAGF, G1 AF, G1 BF, G2F, G2SF, G2S2F etc.
- “Afucosylated glycans” described here consists of glycan moiety wherein fucose is not linked to the non reducing end of N-acetlyglucosamine.
- examples of afucosylated glycans include M3NAG, GO, G1 A, G1 B etc.
- High mannosylated glycans described here, consists of glycan moieties which comprises 5 or more mannose residues. Without limitation, examples of high mannosylated glycans include M5, M6, M7, M8, M9 etc.
- Galactosylated glycans described herein, consists of glycan moieties which comprises galactose residues.
- examples of galactosylated glycans include G1 A, G1 B, G1 AF, G1 BF, G2, G2F, G2SF etc.
- the term "osmolality” as used herein is defined as a measure of the osmoles of solute per kilogram of solvent (osmol/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.
- VCC Viable cell count
- % 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.
- 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.
- Table I Representative table of various glycans
- the invention generally relates to the field of cell culture. More particularly, the invention relates to culturing cells in the presence of betaine to obtain a glycoprotein with a glycoform composition comprising increased percentage of afucosylated glycans.
- the invention discloses a cell culture process for obtaining a glycoprotein with a glycoform composition comprising target values of mannosylated, galactosylated and afucosylated glycans.
- the invention discloses a cell culture process comprises culturing cells in a medium comprising betaine for obtaining a glycoprotein having a glycoprofile comprising increased percentage of afucosylated glycans as compared to the medium without betaine.
- the invention discloses a cell culture process comprises culturing cells in a medium comprising betaine for obtaining a glycoprotein having a glycoprofile comprising increased percentage of afucosylated glycans as compared to the medium without betaine, wherein, the increase in said afucosylated glycans is by at least about 25 %.
- the invention discloses a cell culture process comprises culturing cells in a medium comprising betaine for obtaining a glycoprotein having a glycoprofile comprising afucosylated glycans in the range of about 7% to about 10 %.
- the cell culture medium comprises about 40 imM betaine.
- the betaine supplementation is done on the 4 th day of the initiation of cell culture i.e., post inoculation of the culture
- the invention discloses cell culture process comprising culturing cells in a medium comprises galactose, manganese and betaine for obtaining a glycoprotein with a glycoprofile composition comprising 8% to about 1 1 % of high mannosylated glycans, about 7% to about 10% of afucosylated glycans and about 33% to about 37% of galactosylated glycans.
- the cell culture medium comprises about 40 imM betaine.
- the cell culture medium comprises about 40 imM betaine and about 33 imM galactose and about 10 - 20 uM manganese.
- the cell culture medium 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 Medium (Sigma), and RPMI -1640 (Sigma).
- 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).
- 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 medium itself or may be a different culture medium.
- 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.
- 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).
- 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.
- betaine, manganese and galactose may be supplemented during the cell culture process. Such supplementation can be performed individually or in combination with or without feed.
- the feed may be added on a single day or may be added on multiple days.
- the cell culture medium comprises betaine, manganese and galactose at the time of seeding of cells.
- betaine, manganese and galactose may be supplemented during the cell culture process. Such supplementation can be performed individually or in combination with or without feed.
- Examples of useful salts of divalent manganese ions include, but are not limited to, manganese sulphate and manganese chloride.
- 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 desired qualitative profiles.
- mammalian cells grow well and produce desirable glycosylation profiles of glycoprotein in commercially viable quantity within temperature range of 25°C to 42°C.
- optimum temperature range for CHO cells is approximately 35°C to 37°C.
- 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.
- 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.
- the other cell culture parameters such as pH and osmolality may be maintained or shifted to one or more subsequent values based on achieving desirable cell viability, cell density, titer or glycosylation profile.
- Those of ordinary skill in the art will be able to choose optimum values/ranges for the same depending on the cell type.
- 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 medium (IS CHO-CD G10.3, Catalog no. 98810) containing 6 g/L galactose at 35°C and pH 7.2. Further, feed Cell Boost 4 (Thermo Scientific HyClone, Catalog No.:SH30928.03) was added on day 2, 3, 4, 5, and 6 respectively. Additionally, 10 ⁇ of manganese chloride was added on day 4. The culture was harvested on day 12 or at viability less than or equal to 60%, whichever was earlier. Viable cell count (VCC), % viability, Integral viable cell count (IVCC), titer and afucosylation content (I) were determined.
- VCC Viable cell count
- IVCC Integral viable cell count
- I afucosylation content
- 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 medium (IS CHO-CD G10.3, Catalog no. 98810) containing 6 g/L galactose at 35°C, pH 7.2.
- feed Cell Boost 4 Thermo Scientific Hyclone, Catalog No.:SH30928.03 was added on day 2, 3, 4, 5, and 6 respectively. Additionally on day 4, 10 ⁇ manganese chloride and 40 imM 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.
- Afucosylation content (Table II), Viable cell count (VCC), % viability, Integral viable cell count (IVCC), titre (II) were determined as represented in figures.
- 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 a culture medium POWER CHO ® 2 (Lonza, Catalog no.:15-771 ) at 35°C and pH of 7.2.
- Cell Boost 4 (Thermo Scientific Hyclone, Catalog No. SH30928.03), betaine and galactose were added on day 2, 3, 4, 5, and 6.
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Abstract
The invention discloses a cell culture process comprising culturing cells in the presence of betaine to obtain a glycoprotein with a glycoform composition comprising increased percentage of afucosylated glycans. Further, the invention 5 discloses a cell culture process comprising manganese, galactose and betaine for obtaining a glycoprotein with a glycoform composition comprising target values of mannosylated, galactosylated and afucosylated glycans.
Description
CELL CULTURE PROCESS RELATED APPLICATION
This application is related to and takes priority from Indian provisional application 1444/CHE/2014 and 1443/CHE/2014 filed on 19th March, 2014 and is herein incorporated in its entirety.
BACKGROUND OF INVENTION
Protein glycosylation is one of the most important post-translation modifications associated with eukaryotic proteins. The structure and composition of the glycan moieties on protein can have a profound effect on the safety and efficacy of therapeutic proteins for e.g. the extent and nature of glycosylation in monoclonal antibodies (imAbs) affects the immunogenicity, solubility and half-life of imAbs
The in-vivo physiological activity 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, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Further, in oncology, ADCC is considered a particularly relevant mechanism of action for therapeutic antibodies. ADCC is a mechanism whereby, a target cell (cancer cell) that has been recognized by specific antibodies is actively lysed by an effector cell of the immune system (natural killer (NK), monocyte, macrophage, eosinophil).
ADCC requires the presence of N-glycans, i.e. oligosaccharides covalently attached at the conserved Asn297 in the Fc region, and is sensitive to change in the oligosaccharide structure. It has been demonstrated that antibodies lacking core fucose residues in the Fc N-glycans exhibit high efficacy due to stronger ADCC even at low concentrations compared to their fucosylated counterparts (R. L. Shields et al., 2002, J. Biol. Chem. 277, 26733-26740). Additionally low fucosylation evades the inhibitory effect of serum immunoglobulin G (IgG) on ADCC by its high binding to effector cell receptors (FcYRIIIa) (S. lida et al., 2006, Clin Cancer Res.;12:2879- 2887). Thus, the advantages of non-fucosylated antibodies include achieving therapeutic efficacy at low doses, inducing high efficacy against tumor cells that
express low levels of antigen or triggering high effector function in NK cells with the low-affinity FcvRllla allotype for the IgGs (N. Yamane-Ohnuki et al., 2004, Biotechnol Bioeng.;87:614-622 & R. Niwa et al., 2005, Clin Cancer Res.;11.2327-2336 & R. Niwa et al, 2004 Clin Cancer Res. ;10:6248-6255). Beside the afucosylated antibodies, levels of other glycoform variants can have marked influence on efficacy of imAbs and their utility as therapeutics. For example, removal of terminal galactose residues from the chimeric mouse-human lgG1 antibody (alemtuzumab) results in reduction of complement dependent cytotoxicity (CDC), without affecting FcyR-mediated functions (Boyd, P. N., Lines, A. C. & Patel, A. K.(1995), Mol. Immunol. 32, 131 1 -1318). Further, the (G1 F-G1 F) glycoform of rituximab triggered a CDC response twice as large as that triggered by the (G0F- G0F) glycoform (http://www.nature.com/nrd/journal/v8/n3/full/nrd2804.html-accessed on 23/12/201 1 ). Presence of higher mannose glycans has been associated with faster clearance of glycoprotein from serum (Werner, R. G., Kopp, K. and Schlueter, M. (2007), 96: 17-22. doi: 10.1 1 1 1 /j.1651 -2227.2007.00199.x). Hence it becomes imperative to produce a glycoprotein composition comprising specific percentage of each glycoform variants.
The sugar composition as well as the structural configuration of a glycan structure depends on in-vivo factors (e.g. cell lines) and "external factors" (e.g. medium composition, osmolality, pH, temperature). In fact, small changes in cell culture conditions during production can cause uncharacteristic glycosylation which may lead to potential risks (Dr Daryl Fernandes; European Biopharmaceutical Review. Winter 2004. pp 92 - 97).
Several studies have been performed to obtain relatively high non-fucosylated glycoproteins such as (1 ) engineering the intrinsic glycosylation pathways of host cells by gene inactivation etc. (N Yamane-Ohnuki et al., 2004, Biotechnol Bioeng.
;87 '.61 '4-622) (2) in vitro chemical synthesis of non-fucosylated N-glycoprotein or enzymatic modification of N-glycans to non-fucosylated forms e.g. by using enzymes such as Endo-M, fucosidase etc. (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 & S. Yazawa et al., 1986, Biochem
Biophys Res Commun. 1986;136:563-569).
Similarly, studies have been performed by varying cell culture process parameters for modulating afucosylation content of the glycoprotein composition. Yoshinobu et al. have demonstrated that, for the imAbs 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). Ahmed et al. have disclosed use of temperature and pH shift to increase afucosylation content in a glycoprotein composition { WO2013114164 A 1). It has been demonstrated that galactose feeding impacts galactosylation levels (Davies, J.; Jiang, L; Pan, L.Z.; LaBarre, M.J.; Anderson, D.; Reff, M. Biotechnol. Bioeng. 2001 , 74, 288-294). Satakarni et. al. describes a cell culture process for obtaining about 14% to about 18% total afucosylated glycans by culturing cells for a specific time period in a medium supplemented with galactose and at a specific osmolality { WO2013114165 A 1). Further, Ramasamy et al. have demonstrated the role of manganese in increasing Man5 glycans and/or afucosylated glycans in a glycoprotein composition (WO2013114245 A1). \an Wu et al. have demonstrated use of a cell culture process for reducing the high mannose content in a glycoprotein composition, so that less than 10% of the glycoproteins in the composition have more than 4 mannose residues per N-linked oligosaccharide. As said above and, for the reason that a 'specific' amount of glycan in a glycoprotein composition critically influences the efficacy and bio-availability of a therapeutic glycoprotein, it is thus mandated by a regulatory agency to maintain the specific amount. In particular, such maintenance and control of specific glycan profile acquires utmost significance during a biosimilar molecule's approval. Hence there always exists a necessity for a cell culture process in obtaining a specific glycan profile, more importantly, the components used for modulating the glycan content is chosen to not to affect or significantly alter the amount/level of other glycan profile. In addition, a combination of components if chosen for modulating or obtaining a specific glycoprofile should be compatible with each other in yielding the target/specifc glycan profile without necessarily affecting the titre and/or productivity of the process. Thus the objective of the invention is to provide a cell culture process for modulating and/or to obtain a specific glycoprofile composition taking into account these numerous separate but inter-linked factors influencing the glycan content and/or productivity.
SUMMARY OF THE INVENTION
The invention describes a cell culture a cell culture process for producing glycoprotein having a glycoform composition comprising increased percentage of afucosylated glycans by culturing recombinant cells producing said glycoprotein in a medium comprising betaine. In particular, about 40 imM of betaine results in an increase in the afucosylated glycan level by at least about 25 % as compared to the cell culture medium without betaine.
In addition, use of betaine along with manganese and galactose in the said cell culture process results in a specific glycoprofile composition along with a further increase in the afucose glycan level resulting in about 7 % to about 1 0 %. Thus, the cell culture process of the present invention with a medium comprising betaine and manganese and galactose results in a glycoprotein composition comprising specific, high mannosylated, afucosylated and galactosylated glycan content.
Use of betaine as a cell culture medium besides increasing the afucosylated glycans, serves to act as an osmoprotectant, considering increase in culture medium osmolality during the course of cell culture.
Further, the increase in afucosylation content and/or the specific glycoform composition obtained using the said process is consistent across batches. Obtaining a consistent glycoform profile is desired and highly significant not only in reducing the batch to batch variations but because the therapeutic efficacy of a glycoprotein molecule is defined by its glycoprofile.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : is an illustration of VCC profile obtained by the process as described in example I (cell culture medium without betaine) and example II (cell culture medium with betaine)
Figure 2: is an illustration of % viability profile obtained by the process as described in example I (cell culture medium without betaine) and example II (cell culture medium with betaine) Figure 3: is an illustration of IVCC profile obtained by the process as described in example I (cell culture medium without betaine) and example II (cell culture medium with betaine)
Figure 4: is an illustration of percentage of high mannosylated, afucosylated and galactosylated glycans in six batches of glycoform composition obtained by the process as described in example III (cell culture medium with betaine, manganese and galactose)
Figure 5: is an illustration of VCC profile obtained by the process as described in example III (cell culture medium with betaine, manganese and galactose)
Figure 6: is an illustration of % viability obtained by the process as described in example III (cell culture medium with betaine, manganese and galactose) Figure 7: is an illustration of IVCC obtained by the process as described in example III (cell culture medium with betaine, manganese and galactose)
Figure 8: is an illustration of antibody titer obtained by the process as described example III (cell culture medium with betaine, manganese and galactose)
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 wherein glycan refers to a monosaccharide or polysaccharide 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. 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 "glycoform composition" or "distribution" as used herein pertains to the quantity or percentage of different glycoforms present in a glycoprotein.
"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, G1 AF, G1 BF, G2F, G2SF, G2S2F etc.
"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, GO, G1 A, G1 B etc.
"High mannosylated glycans" described here, consists of glycan moieties which comprises 5 or more mannose residues. Without limitation, examples of high mannosylated glycans include M5, M6, M7, M8, M9 etc.
"Galactosylated glycans" described herein, consists of glycan moieties which comprises galactose residues. Without limitation, examples of galactosylated glycans include G1 A, G1 B, G1 AF, G1 BF, G2, G2F, G2SF etc.
The term "osmolality" as used herein is defined as a measure of the osmoles of solute per kilogram of solvent (osmol/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 "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 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.
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.
Table I: Representative table of various glycans
Mannose
N-Acetyl Glucosamine
Galactose
2-AB Label
Fucose
Sialic acid
Detailed description of the embodiments
The invention generally relates to the field of cell culture. More particularly, the invention relates to culturing cells in the presence of betaine to obtain a glycoprotein with a glycoform composition comprising increased percentage of afucosylated glycans. In particular, the invention discloses a cell culture process for obtaining a glycoprotein with a glycoform composition comprising target values of mannosylated, galactosylated and afucosylated glycans. In one embodiment, the invention discloses a cell culture process comprises culturing cells in a medium comprising betaine for obtaining a glycoprotein having a glycoprofile comprising increased percentage of afucosylated glycans as compared to the medium without betaine. In another embodiment, the invention discloses a cell culture process comprises culturing cells in a medium comprising betaine for obtaining a glycoprotein having a glycoprofile comprising increased percentage of afucosylated glycans as compared to the medium without betaine, wherein, the increase in said afucosylated glycans is by at least about 25 %.
In yet another embodiment, the invention discloses a cell culture process comprises culturing cells in a medium comprising betaine for obtaining a glycoprotein having a glycoprofile comprising afucosylated glycans in the range of about 7% to about 10 %.
In any of the above embodiments, the cell culture medium comprises about 40 imM betaine.
Further, in any of the above embodiment, the betaine supplementation is done on the 4th day of the initiation of cell culture i.e., post inoculation of the culture
In an embodiment, the invention discloses cell culture process comprising culturing cells in a medium comprises galactose, manganese and betaine for obtaining a glycoprotein with a glycoprofile composition comprising 8% to about 1 1 % of high
mannosylated glycans, about 7% to about 10% of afucosylated glycans and about 33% to about 37% of galactosylated glycans.
In any of the above mentioned embodiments, the cell culture medium comprises about 40 imM betaine.
In any of the above mentioned embodiments, the cell culture medium comprises about 40 imM betaine and about 33 imM galactose and about 10 - 20 uM manganese.
The cell culture medium 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 Medium (Sigma), and RPMI -1640 (Sigma).
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 medium itself or may be a different culture medium. 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).
In one embodiment of the invention, betaine, manganese and galactose may be supplemented during the cell culture process. Such supplementation can be performed individually or in combination with or without feed.
In an embodiment of the invention, the feed may be added on a single day or may be added on multiple days.
In a further embodiment of the invention, the cell culture medium comprises betaine, manganese and galactose at the time of seeding of cells.
In another of the invention, betaine, manganese and galactose may be supplemented during the cell culture process. Such supplementation can be performed individually or in combination with or without feed.
Examples of useful salts of divalent manganese ions include, but are not limited to, manganese sulphate and manganese chloride.
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 desired qualitative profiles. In general, mammalian cells grow well and produce desirable glycosylation profiles of glycoprotein in commercially viable quantity within temperature range of 25°C to 42°C. For example, optimum temperature range for CHO cells is approximately 35°C to 37°C. 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.
Similarly, in accordance with the teaching of the present invention, the other cell culture parameters such as pH and osmolality may be maintained or shifted to one or more subsequent values based on achieving desirable cell viability, cell density, titer or glycosylation profile. Those of ordinary skill in the art will be able to choose optimum values/ranges for the same 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 medium (IS CHO-CD G10.3, Catalog no. 98810) containing 6 g/L galactose at 35°C and pH 7.2. Further, feed Cell Boost 4 (Thermo Scientific HyClone, Catalog No.:SH30928.03) was added on day 2, 3, 4, 5, and 6 respectively. Additionally, 10 μΜ of manganese chloride was added on day 4. The culture was harvested on day 12 or at viability less than or equal to 60%, whichever was earlier. Viable cell count (VCC), % viability, Integral viable cell count (IVCC), titer and afucosylation content (I) were determined.
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 medium (IS CHO-CD G10.3, Catalog no. 98810) containing 6 g/L galactose at 35°C, pH 7.2. Further, feed Cell Boost 4 (Thermo Scientific Hyclone, Catalog No.:SH30928.03) was added on day 2, 3, 4, 5, and 6 respectively. Additionally on day 4, 10 μΜ manganese chloride and 40 imM 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. Afucosylation content (Table II), Viable cell count (VCC), % viability, Integral viable cell count (IVCC), titre (II) were determined as represented in figures.
Example III
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 a culture medium POWER CHO® 2 (Lonza, Catalog no.:15-771 ) at 35°C and pH of 7.2.
Subsequently, Cell Boost 4 (Thermo Scientific Hyclone, Catalog No. SH30928.03), betaine and galactose were added on day 2, 3, 4, 5, and 6. The cumulative concentration of Betaine and galactose in the feed that was added to the cell culture medium from day 2 to day 6 was 40 imM and 33 imM respectively. Additionally, 20 μΜ of Manganese was added on day 6. The culture was harvested on day 12 or at viability less than or equal to 60%, whichever was earlier.
Six identical batches (N=6, represented as I, II, III, IV, V, VI) were run with the aforementioned process and the % high mannosylated, % afucosylated and % galactosylated glycans for each glycoform composition were determined (Table III) and are represented in Figure 1 . Additionally viable cell count (VCC), % viability, Integral viable cell count (IVCC) and final titer were determined and are represented in Figures 4-8.
Table II: % Afucosylated glycans in a glycoform composition
Claims
We claim:
1 ) A cell culture process comprising culturing cells for obtaining a glycoprotein with a glycoform composition comprising increased percentage of afucosylated glycans wherein the said process comprises culturing cells in a cell culture medium comprising betaine.
2) A cell culture process of claim 1 , wherein process comprises culturing cells in a medium comprising betaine for obtaining a glycoprotein having a glycoprofile comprising increased percentage of afucosylated glycans as compared to the medium without betaine, wherein, the increase in said afucosylated glycans is by at least about 25 %.
3) A cell culture process of claim 1 cell culture process comprises culturing cells in a medium comprising betaine for obtaining a glycoprotein having a glycoprofile comprising afucosylated glycans in the range of about 7% to about 10 %.
4) A cell culture process comprising culturing cells in a medium comprises galactose, manganese and betaine for obtaining a glycoprotein with a glycoprofile composition comprising 8% to about 1 1 % of high mannosylated glycans, about 7% to about 10% of afucosylated glycans and about 33% to about 37% of galactosylated glycans.
5) A cell culture process according to claim 1 or 4, wherein the cell culture medium comprises about 40 imM betaine.
6) A cell culture process of claim 4, wherein the said cell culture medium comprises about 33 imM galactose and about 10 - 20 uM manganese.
7) A cell culture process of claim 1 , the betaine supplementation is done on the 4th day of the initiation of cell culture i.e., post inoculation of the culture.
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