DESC:FIELD OF THE INVENTION
The present invention relates to the field of cell culture. In particular, the invention discloses a process for modulating high mannosylated and afucosylated glycans in a glycoprotein composition by supplementing manganese at a specific phase in the growth curve of the cell culture. 5
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 10 acid except proline, and O-linked glycosylation in which glycans are attached to serine or threonine. N-linked glycans further are of two types - high mannose type consisting of two N-acetylglucosamines plus a large number of mannose residues (more than 4), and the complex type that contain more than two N-acetylglucosamines plus any number of other types of sugars (galactose, fucose 15 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 are actually glycosylated. This may be a consequence of the competitive action of diverse enzymes involved in glycosylation and are key to understanding 20 glycoprotein heterogeneity (Mariño, K., (2010) Nature Chemical Biology 6,713-723).
Recombinant monoclonal antibodies (mAbs) represent the largest and fastest growing group of therapeutic glycoproteins. It has been demonstrated that the structure and composition of the glycan moieties on mAbs can have a profound 25 effect on their safety and efficacy. For e.g. the extent and nature of glycosylation in monoclonal antibodies (mAbs) affects its clearance, immunogenicity, and solubility.
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The efficacy of therapeutic antibodies is mediated by two independent mechanisms, (a) the efficacy resulting from target antigen neutralization or apoptosis and (b) biological activities referred to as antibody effector functions mediated via effector cells or complement system known as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) 5 respectively.
Several studies have demonstrated that low fucose levels i.e. higher afucosylated glycoform composition enhance mononuclear cell-mediated antibody-mediated cellular cytotoxicity (ADCC) (Mori K, (2007), Cytotechnology 55(2–3):109–114. and Shields RL. (2002), J Biol Chem 277(30):26733–26740). Additionally low 10 fucosylation evades the inhibitory effect of serum immunoglobulin G (IgG) on ADCC by its high binding to effector cell receptors (Fc?RIIIa) (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 15 effector function in NK cells with the low-affinity Fc?RIIIa 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).
However in contrast, Peipp M. et al showed that polymorphonuclear cells 20 preferentially kill via high-fucosylated antibody composition. Further, the same study concluded that high-fucose antibody induced superior ADCC in blood from granulocyte colony-stimulating factor-primed donors containing higher numbers of activated polymorphonuclear cells. (Peipp M et al., Blood. 2008 Sep 15; 112(6):2390-9. doi: 10.1182/blood-2008-03-144600). 25
Further, the efficacy of therapeutic antibodies is also affected by the serum clearance rate i.e. serum half-life of antibodies. The serum half-life of IgG antibodies is regulated by a number of receptors, including the mannose receptors, which bind both high-mannose-containing pathogens as well as endogenous
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proteins (Stahl PD. 1992, Curr Opin Immunol. 4:49–52 & Lee SJ, Evers S, Roeder D, Parlow AF, Risteli J, Risteli L, Lee YC, Feizi T, Langen H, Nussenzweig MC. 2002. Science. 295:1898–1901). Goetze et al. showed that IgGs containing high-mannose glycans are cleared more rapidly in humans than other glycan forms (Andrew MGoetze et al. Glycobiology vol. 21 no. 7 pp. 949–959, 5 2011). Thus, there may be preferential clearance of glycoform bearing the terminal mannose residues which would impact the pharmokinetic profile of an antibody composition. Hence the reduction of high mannose bearing glycoforms improves half-life of an antibody composition which is a desirable quality attribute. 10
The maintenance and control of specific glycan profile of a glycoprotein is particularly important for the development of a biosimilar. Hence, there is a need for development of cell culture processes for obtaining a ‘specific’ glycan profile.
The components used for modulating a particular glycan content need to be chosen so as to not to affect or significantly alter the amount/level of other glycan 15 content. In addition, a combination of components, when chosen for modulating or obtaining a specific glycoprofile, should be compatible with each other in yielding the targeted glycan profile without necessarily affecting the titer and/or productivity of the process.
Thus, the objective of the invention is to provide a cell culture process for 20 obtaining a specific glycoprotein composition taking into account several discrete, but inter-linked factors that influence the glycan content of the glycoprotein composition for therapeutic use on one hand and the overall yield of the glycoprotein on other.
SUMMARY OF THE INVENTION 25
The present invention describes a cell culture process for obtaining a glycoprotein composition comprising high mannosylated and afucosylated glycans, wherein the cell culture process comprises addition of manganese for modulating the amount
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of high mannose and afucosylated glycans. Particularly, modulation of high mannose and afucosylated glycan content is achieved by adding manganese at a specific phase in the growth curve of the cell culture.
The invention describes a cell culture process comprising addition of manganese at/during the phase of a cell culture growth curve described as “death phase”, to 5 achieve a significant reduction in high mannose and afucosylated glycans content in a glycoprotein composition. Delayed addition of manganese viz., at/during death phase or at an IVCD of greater than 26, results in about 50 % reduction in content of high mannose (HM) and afucosylated (AF) glycans compared to the content of HM and AF glycans obtained when manganese not so supplemented. 10
Specifically, the inventive method resulting in reduced levels of HM and AF glycans in a glycoprotein composition consequently results in an increase in the ADCC activity of the said glycoprotein composition.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a general illustration of cell culture phases showing the lag phase, 15 logarithmic (log) phase, Plateau (or stationary) phase and death (or decline) phase.
Figure 2 is an illustration of average of percentage of high mannosylated and afucosylated glycans in a glycoprotein composition obtained in no. of batches representative of example I, II and III. The error bars represent standard deviation for respective data point. 20
Figure 3 represents cell culture progression in terms of viable cell density w.r.t culture age for seven batches illustrating example I. The dotted vertical lines represent the cell culture phase where manganese is supplemented. Each point in the graph represents average of seven batches and standard deviation among batches is represented as error bars. 25
Figure 4 represents cell culture progression in terms of viable cell density w.r.t culture age for four batches illustrating example II. The dotted vertical lines represent the cell culture phase where manganese is supplemented. Each point in
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the graph represents average of four batches and standard deviation among batches is represented as error bars.
Figure 5 represents cell culture progression in terms of viable cell density w.r.t culture age for three batches illustrating example III. The dotted vertical lines represent the cell culture phase where manganese is supplemented. Each point in 5 the graph represents average of three batches and standard deviation among batches is represented as error bars.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “glycan” refers to a monosaccharide or polysaccharide moiety. 10
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 “large-scale production” of glycoprotein composition refers to an amount typically required for the industrial production of recombinant 15 polypeptides, used for preparation of therapeutically active biopharmaceuticals.
The term “glycoform” or “glycovariant” have been used interchangeably herein, and refers to various oligosaccharide entities or moieties linked in their entirety to the Asparagine 297 (as per Kabat numbering) of the Fc region of the antibody in question, co translationally or post translationally within a host cell. 20
The abbreviations used in present invention for denoting the sugars comprising the glycan moieties are: M: Mannose, NAG: N- N-acetylglucosamine, F: Fucose, G: Galactose, S: Sialic Acid. The no. post the letter/letter set indicates no. of respective preceding sugar moiety present in the glycan moiety. For e.g. M3NAG denotes glycan moiety comprising three mannose and one N- N-25 acetylglucosamine
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The glycan moieties added during the glycosylation process may include M3, M4, M5-8, M3NAG etc. Examples of such glycans and their structures are listed in Table 1. However, Table 1 may in no way be considered to limit the scope of this invention to these glycans.
The term “glycoprotein or antibody composition” as used herein pertains to the 5 quantity or percentage of different glycoforms present in a glycoprotein or an antibody preparation.
“Afucosylated glycans” described here, consists of glycan moiety wherein fucose is not linked to the non-reducing end of N-acetlyglucosamine. Without limitation, examples of afucosylated glycans include M3NAG, G0, G1A, G1B etc. 10
“Fucosylated glycans” described here, consists of glycan moiety wherein fucose which is linked to the non-reducing end of N-acetlyglucosamine. Without limitation, examples of fucosylated glycans include M3NAGF, G1AF, G1BF, G2F, G2SF, G2S2F etc.
“High mannosylated glycans” described here, consists of comprising glycan 15 moiety which comprises 5 or more mannose residues. Without limitation, examples of high mannosylated glycans include M5, M6, M7, M8, M9 etc.
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, 20 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).
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Table I: Representative table of various glycans
Glycan Structure Code Glycan Structure Code
M3
G1AF
M3NAG
G1BF
M3NAGF
M6
G0
G2F
G0F
M7
M5
G2SF
G1A
M8
G1B
G2S2F
Mannose
N-Acetyl Glucosamine
Galactose
2-AB Label
Fucose
Sialic acid
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The growth curve of mammalian cells in suspension culture is a sigmoidal plot of cell density as a function of time. The growth curve (refer Figure 1) comprises 4 phases, lag, exponential or log, stationary or plateau, and death or decline phase.
Lag Phase- This is the 1st phase in the growth curve, characterized by little or no cell division. 5
Exponential or Log phase- It is a period of active cell proliferation hence the cell density increases exponentially.
Stationary or Plateau Phase- This is a post-log phase period characterized by slowdown in the division rate, during which cell proliferation is balanced by cell death. As a result there is no resultant increase in the density of the cell culture. 10
Death or Decline Phase- This phase is characterized by excess of cell death over cell proliferation resulting in decline in cell density.
The term “% Viability” refers to percentage of viable cells out of total no. of cells including the viable and non-viable cells at a particular time point in cell culture.
The term “Viable cell density (VCD)” refers to the no. of viable cell per unit 15 volume at a particular time point in cell culture. The unit usually used for its measurement is million viable cells/ ml. Graphically, the “VCD profile” represents viable cell count (VCC) over the duration/age of cell culture.
As used herein, “IVCD” or “Integral viable cell density” refers to area under the viable cell density curve i.e. viable cell density plotted against culture age. 20
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a cell-mediated immune defense mechanism whereby a target cell whose membrane-surface antigens have been bound by specific antibodies is lysed by an effector cell of the immune system. In-vitro ADCC assays have been abundantly described in prior art. One such method is modified ADCC method. (Mark Barok et al. Mol Cancer 25 Ther. 2007 Jul; 6(7):2065-72).
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Detailed description of the embodiments
The present invention discloses a cell culture process for modulating high mannosylated and afucosylated glycans in a glycoprotein composition by supplementing manganese at a specific phase in the growth curve of the cell culture. 5
One embodiment of the invention discloses a cell culture process comprising addition of manganese at a specific phase in the growth curve of the cell culture to obtain a glycoprotein composition comprising 5-12% of high mannosylated glycans and 4-8 % of afucosylated glycans.
Another embodiment of the invention discloses a cell culture process comprising 10 culturing cells in culture medium wherein manganese is supplemented at or during the death phase of cell culture to obtain a glycoprotein composition comprising reduced percentage of mannosylated and afucosylated glycans compared to a cell culture process wherein manganese not so supplemented.
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In any of the above mentioned embodiments, the cells used are Chinese Hamster Ovary (CHO) cells and the glycoprotein composition obtained is Her-2 antibody composition.
In any of the above mentioned embodiments, the said reduction in high mannosylated glycans is in the range of about 35 % to about 50 % and the said 20 reduction in afucosylated glycans is in the range of about 30 % to about 45 %.
Another embodiment of the invention discloses a cell culture process comprising culturing cells in a culture medium wherein manganese is supplemented at or during the death phase of cell culture to obtain a glycoprotein composition comprising 5-8% of high mannosylated glycans and 4-5 % of afucosylated 25 glycans.
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In any of the above mentioned embodiments, the cell culture process comprises culturing cells in a culture medium wherein the cells are inoculated/seeded at a density of 0.4 million cells/ml to 0.6 million cells/ml.
In any of the above mentioned embodiments, the cell culture process comprises of culturing cells in a culture medium whereby manganese is supplemented at an 5 IVCD greater than 26, preferably greater than 26 and less than 35.
In any of the above mentioned embodiments of the invention, the said cell culture process comprises supplementation with about 20 µM of manganese.
The supplementation of manganese to the cell culture medium can be done either by feeding the cells with a feed comprising manganese or adding manganese 10 separately.
Examples of useful salts of divalent manganese ions include, but are not limited to, manganese sulphate and manganese chloride.
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 15 (Lo1nza), Zap-CHO (Invitria), CD CHO, CDOptiCHOTM and CHO-S-SFMII (Invitrogen), ProCHOTM (Lonza), CDM4CHOTM (Hyclone), DMEM (Invitrogen), DMEM/F12 (Invitrogen), Ham’s F10 (Sigma), Minimal Essential Media (Sigma), and RPMI -1640 (Sigma) and IS CHO-CD G10.3 (Irvine scientific).
The feed or feed medium in the present invention may be added in a continuous, 20 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 25 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
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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 5 Hyclone, Catalogue no SH 30596.03), Cell Boost 4 (CB-4, Thermo Scientific HyClone, Catalog no. SH30928), PF CHO (Thermo Scientific Hyclone, Catalog no. SH30333.3).
The temperature of a cell culture is selected based on the temperature range at which cells remain viable, produce glycoprotein of interest in desirable quantity 10 and qualitative profiles. In general, mammalian cells grow well and produce desirable glycosylation profiles in commercially viable quantity within temperature range of 25oC to 42oC. For example, optimum temperature range for CHO cells is at approximately 35oC to 37oC. Those of ordinary skill in the art will be able to choose optimum temperature and/or temperature range depending on 15 the cell type.
Optionally, cells may be subjected to temperature shift at any time during the course of the cell culture. The temperature shift may be gradual or abrupt. The subsequent temperature may be higher or lower than the initial temperature value. Additionally, the cells may be exposed to more than one such temperature shifts. 20 As with the initial temperature the subsequent temperatures may be selected based on the temperature range at which cells remain viable, produce glycoprotein of interest in desirable quantity and quantitative profiles. Those of ordinary skill in the art will be able to choose optimum temperature and/or temperature range depending on the cell type. 25
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.
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EXAMPLES
HER-2 antibody was cloned and expressed on a large-scale in fed-batch culture using a CHO cell line as detailed in Molecular Cloning: A laboratory Manual by Green and Sambrook. rCHO cells expressing antibody were seeded at a density of ~0.5 million cells/ml in culture media POWER CHO® 2 (Lonza, Catalog no.:15-5 771) at 35°C and pH of 7.2. Subsequently, feed was added on day 2, 3, 4, 5, and 6. The feed comprises CB4 (CB4, Hyclone, Catalog No.:SH30928.03), betaine and galactose. The culture medium was supplemented with 20 µM manganese added at varying phases of cell culture i.e at IVCD values of 17-20 (Example I), 26-29 (Example II) and 29-31 (Example III). IVCD range in Example I is representative 10 of stationary phase and latter two IVCD ranges (Example II and III) represent the death phase of the cell culture.
The culture was harvested on day 12 or at viability less than or equal to 60%, whichever was earlier. The average percentage of high mannosylated and afucosylated glycans in each of the glycoprotein composition were determined as 15 given in Table II and represented in Figure 1. “n” represents the no. of batches performed for a specific experiment /Example. Except for the phase of the cell culture or the IVCD range of manganese addition, the rest of the cell culture conditions including concentration of manganese were kept the same across the batches. 20
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Table II: Average Percentage glycans in a glycoprotein composition
Example
IVCD values
% High Mannosylated Glycans
% Afucosylated Glycans
I (n=7)
17-20
11.4
7.3
II (n=4)
26-29
7.3
4.9
III (n=3)
29-31
5.4
4.0 ,CLAIMS:We claim:
1. A cell culture process comprising addition of manganese to culture media for modulating high mannose and/or afucosylated glycans in a glycoprotein composition. 5
2. The process according to claim 1, wherein the addition of manganese is during death phase of the cell culture.
3. The process according to claim 1, wherein the addition of manganese is at an IVCD value greater than 26.
4. The process according to claim 1, wherein manganese is added at a concentration 10 of about 20 µM.
5. The process according to claim 1, wherein the seeding density for cell culture is about 0.4 million cells/ml to 0.6 million cells/ml.
6. The process according to claim 1, wherein the glycoprotein composition comprises 5-8% of high mannosylated glycans and 4-5 % of afucosylated glycans. 15
7. The process according to claim 1, wherein the reduction in high mannosylated glycans is in the range of about 35 % to about 50 % and the reduction in afucosylated glycans is in the range of about 30 % to about 45 % in the glycoprotein composition as compared to glycoprotein composition produced otherwise.
8. The process according to claim 1, wherein the cell are CHO cells. 20
9. The process according to claim 1, wherein the cell culture process comprises temperature shift and/or pH shift.
10. The process according to claim 1, wherein the glycoprotein composition is an anti-HER2 antibody composition.