DESC:FIELD OF THE INVENTION
The invention describes a method for obtaining a glycoprotein with a specific glycoform composition. Particularly, the invention relates to a cell culture process for reducing the galactosylation content and/or increasing the G0F content of a glycoprotein by supplementing the cell culture medium with arginine. 5
BACKGROUND OF THE INVENTION
Protein glycosylation is one of the most important post-translation modifications associated with eukaryotic proteins. This is evidenced by the fact that more than 50% of the eukaryotic proteins are glycosylated (Apweiler et al., 1999, Biochim Biophys Acta 1473(1):4-8.). The structure and composition of the saccharide 10 (glycan) moieties added can have profound effect on the stability, safety and efficacy of these proteins (Wong CH, 2005, J Org Chem 70(11):4219-25). Hence, an understanding of glycosylation and modes of controlling the same gains high significance.
Among glycoproteins, monoclonal antibodies (mAbs) have emerged as major 15 therapeutic agents against various conditions (Zhong X. and Somers W., 2012, Recent Advances in Glycosylation Modifications in the Context of Therapeutic Glycoproteins, Integrative Proteomics, ISBN: 978-953-51-0070-6). The in-vivo physiological activity of mAbs is mediated by two independent mechanisms, (a) target antigen neutralization or apoptosis and (b) antibody effector functions which 20 include antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Importantly, the effector functions of antibodies has been shown to correlate with the glycan structures associated with these mAbs.
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Further, glycosylation of mAbs are also known to improve therapeutic efficacy through its impact on protein pharmacodynamics (PD) and pharmacokinetics (PK) (Mahmood I, Green MD, 2005, Clin Pharmacokinet 44(4):331-47; Tang et al., 2004, J Pharm Sci 93(9):2184-204.). The N-glycosylation in mAbs involves attachment of oligosaccharides at asparagine (Asn)-297 in the CH2 domain of Fc 5 region of IgGs. This is a unique feature of IgG, making it a “key player” in functioning of the immune system. However, mAb homogeneity is difficult to achieve, complicated by the fact that recombinant systems, used for the large scale production of these therapeutic antibodies may secrete IgGs with altered and significant variation in the glycan structures. This can potentially result in 10 suboptimal effector functions of these antibodies. Thus, methods for improving, controlling and modifying glycosylation have immense impact on the production and function of therapeutic mAbs.
Several factors affect glycosylation profile of a glycoprotein. These include cell line characteristics, process control parameters and cell culture media components 15 (Andersen et al., 2000, Biotechnol Bioeng 70(1):25–31.; Butler, 2005, Appl Microbiol Biotechnol. 68(3):283–291.)
Effect of galactosylation /glycosylation on mAbs
Several studies have suggested that the terminal galactose content of IgG improves CDC as a result of increased antibody binding to C1q, without affecting the ADCC 20 activity (Hodoniczky J, Zheng YZ, James DC: Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog
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2005, 21:1644-1652). Further, addition of galactose is a template for addition of sialic acid on mAbs (Mariño, K., (2010) Nature Chemical Biology 6,713-723). This increased terminal sialylation can increase the serum half-life of many glycoproteins (Raju TS: Glycosylation variations with expression systems and their impact on biological activity of therapeutic immunoglobulins. Bioprocess Int 2003, 5 1:44-53). However, in contrast, studies have also demonstrated that increased sialylation of Fc glycans results in decreased ADCC activity of recombinant IgGs. (Scallon BJ, Tam SH, McCarthy SG, Cai AN, Raju TS: Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol Immunol 2007, 44:1524-1534). Without being bound by theory, this evidence 10 suggests that a decreased galactosylation may then result in a decreased sialylation and hence may prevent the decrease in ADCC activity of rIgGs.
Thus, considering the effect of galactosylation on the functional activity of mAbs and hence use of monoclonal antibodies as therapeutics, a cell culture process which produces an antibody composition comprising decreased percentage of 15 galactosylated and/or increased percentage of G0F glycoform is desirable.
At the same time, alteration in other glycoform composition specific to an antibody such as afucose (AF) and/or high mannose (HM) content may not be desirable as these AF and HM glycoforms are found to differentially impact the activity of an antibody. It is known in the art, that the absence of core fucose residues in the Fc 20 glycans substantially increases the ADCC activity of IgG and, at the same time the enhanced ADCC of afucosylated (AF) forms of therapeutic antibodies through improved Fc gamma RIIIa binding is shown to be inhibited by the fucosylated
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counterparts. On the contrary, Peipp M. et al have shown that polymorphonuclear cells preferentially kill via high-fucosylated antibody composition and induced superior ADCC in blood from granulocyte colony-stimulating factor-primed donors containing higher numbers of activated polymorphonuclear cells (Peipp M, Blood. 2008 Sep 15;112(6):2390-9. doi: 10.1182/blood-2008-03-144600). 5
Another important component of glycoform composition of a glycoprotein is its high mannose (HM) content. Increased HM content has been shown to lead to potential enhancement of its biological activity in terms of higher ADCC activity and greater affinity to Fc?RIIIA (Zhou et al., 2008, Biotechnol Bioeng 99(3):652–665). Results also demonstrate that therapeutic IgGs containing increased HM 10 glycans are cleared more rapidly in humans than other glycan forms. (Yu M. et al., 2012 July 1; 4(4): 475-487). An increase or decrease in the composition or modification in content of glycoforms thus crticially impacts the ADCC activity of an antibody. Accordingly, it is necessary that a method employed to alter a particular glycoform composition shall not alter the content of other Fc 15 glycosylations.
A number of strategies exist in the prior art to modulate compositions of different glycoforms in an antibody composition.
One of the suggested approaches is genetic manipulation of the cell lines for glycosyl transferases, enzymes responsible for glycosylation (Yamane-Ohnuki et 20 al., 2004, Biotechnol Bioeng., 87:614–622; Shinkawa et al., 2003, J. Biol. Chem., 278:3466-73; Mori et al., 2004, Biotechnol Bioeng., 88:901-8; Ferrara et al., 2006, Biotechnol Bioeng., 93:851-61).
Other methods include in vitro modification of proteins post protein synthesis to obtain desired glycoprotein (Inazu T., 2007, Research in construction of the complex system for functional oligosaccharides. Proceeding of the Institute of Glycotechnology of Tokai University; 2:42–45; Yazawa et al., 1986, Biochem Biophys Res Commun. 1986;136:563–569; Yamamoto et al., 2008, J. Am. Chem. 5 Soc.,130:501-10). Modifications in cell culture conditions and media compositions have also been proposed to modulate glycosylation content of glycoproteins. (Yoshinobu et al., 2010, Animal Cell Technology: Basic & Applied Aspects Volume 16, pp 121-125, WO2013114165 A1, WO2013114245 A1, US 2012/0276631 A1)
As stated earlier, a decreased galactosylation is desirable as it may prevent the 10 decrease in ADCC activity of recombinant IgGs. Further and in addition, the specific composition of HM and AF be maintained despite the requirement of decreased galactosylation content. Thus, there is a need for a process which would selectively alter the galactose content without affecting the content or composition of other glycosylated components. 15
SUMMARY OF THE INVENTION
The present invention explores the role of an amino acid, arginine in modifying the glycan composition of a glycoprotein. The invention describes a cell culture process for modifying galactosylation and/or G0F glycoform composition in a 20 glycoprotein preparation wherein the cell culture process is supplemented with an arginine comprising growth media. In addition, the component is chosen such that
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it decreases the galactosylation content and increases the GoF content without significantly affecting the high mannose or afucosylation content of the glycoprotein composition.
In addtition, supplementation of cell culture medium with arginine results in a glycoprotein titer that is relatively higher than the titer obtained from a process that 5 was non-supplemented with arginine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an illustration of antibody titer as described in Examples I and II.
Figure 2 is an illustration of viable cell count as described in Examples I and II. 10
Figure 3 is an illustration of IVCC as described in Examples I and II.
Figure 4 is an illustration of percentage of galacotsylated (Fig. 4a) and G0F (Fig. 4b) glycans as described in Examples I and II.
DETAILED DESCRIPTION OF THE INVENTION 15
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. 20
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The term “glycoform” or “glycovariant” have been used interchangeably herein, and refers to various oligosaccharide entities or moieties linked in their entirety to the protein or polypeptide.
The “glycoform composition” or distribution as used herein pertains to the quantity or percentage of a specific and/or different glycoforms present in a glycoprotein. 5
As used herein, “high mannose glycovariant” consists of glycan moieties comprising two N-acetylglucosamines and more than 4 mannose residues and shall include without limitation M5, M6, M7, and M8.
“Galactosylated glycans” refers to glycans containing terminal galactose residues and include without limitation G1A, G1B, G1AF, G1BF, G2, G2F and G2SF. 10
G0 as used herein refers to protein glycan not containing galactose at the terminal end of the glycan chain.
G0F as described here consists of glycan moieties wherein fucose is linked to the non-reducing end of N-acetylglucosamine.
Various methods described in the art such as Wuhrer et. al., Ruhaak L.R., and 15 Geoffrey et. al. can be used for assessing glycovariants present in a glycoprotein composition (Wuhrer M. et al., Journal of Chromatography B, 2005, Vol.825, Issue 2, pages 124-133, Ruhaak L.R., Anal Bioanal Chem, 2010, Vol. 397:3457-3481, Geoffrey, R. G. et. al. Analytical Biochemistry 1996, Vol. 240, pages 210-226).
The term “temperature shift” as used herein is defined as the change in temperature 20 during the cell culture process.
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The term “pH shift” as used herein is defined as the change in pH during the cell culture process.
As used herein, “IVCC” or “Integral viable cell concentration” refers to cell growth over time or integral of viable cells with respect to culture time that is used for calibration of specific protein production. The integral of viable cell concentration 5 can be increased either by increasing the viable cell concentration or by lengthening the process time.
Table I: Representative table of various glycans
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Detailed description of the embodiments
The present invention discloses a cell culture process for production of a glycoprotein composition containing increased percentage of G0F glycans and/or reduced percentage of galactosylated glycans.
In an embodiment, the present invention provides a cell culture process for 5 obtaining a glycoprotein composition with an increased percentage of G0F glycans comprising steps of;
a) culturing cells at a first temperature and a first pH for a first period of time
b) culturing cells at a second temperature and a second pH for a second period of time 10
c) supplementing said cell culture with arginine
d) recovering the said glycoprotein from the cell culture.
In another embodiment, the present invention provides a cell culture process for obtaining a glycoprotein composition with a reduced percentage of galactosylated glycans of the glycoprotein comprising steps of; 15
a) culturing cells at a first temperature and a first pH for a first period of time
b) culturing cells at a second temperature and a second pH for a second period of time
c) supplementing said cell culture with arginine
d) recovering the said glycoprotein from the cell culture. 20
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In a further embodiment, the present invention provides a cell culture process for obtaining a glycoprotein composition with an increased percentage of G0F glycans and/or reduced percentage of galactosylated glycans comprising steps of;
a) culturing cells at a first temperature and a first pH for a first period of time
b) culturing cells at a second temperature and a second pH for a second period 5 of time
c) supplementing said cell culture with arginine
d) recovering the said glycoprotein from the cell culture.
In another embodiment, the present invention provides a process for obtaining a glycoprotein composition comprising about 25% galactosylated glycans. 10
In yet another embodiment, the present invention provides a process for obtaining a glycoprotein composition comprising about 66% of G0F glycans.
In a further embodiment, the present invention provides a process for obtaining a glycoprotein composition comprising about 66% of G0F glycans and about 25% galactosylated glycans. 15
In another embodiment, the present invention provides a method for obtaining glycoprotein composition wherein the galactosylated glycan percentage is decreased by about 18%.
In yet another embodiment, the present invention provides a method for obtaining glycoprotein composition wherein the percentage of G0F glycans is increased by 20 about 6% as compared to the control.
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In a further embodiment, the present invention provides a cell culture process for obtaining a glycoprotein composition with increased percentage of G0F glycans and/or reduced percentage of galactosylated glycans comprising steps of;
a) culturing cells at a first temperature and a first pH for a first period of time
b) culturing cells at a second temperature and a second pH for a second period 5 of time
c) supplementing said cell culture with arginine
d) recovering the said glycoprotein from the cell culture,
wherein, the said process does not alter significantly the HM and AF content of the glycoprotein. 10
In yet another embodiment the cell culture medium is supplemented with about 8 mM of arginine.
In yet another embodiment, supplementation of arginine in cell culture media leads to an increase in the titer of the glycoprotein. The increase in titer of the cell culture due to arginine supplementation in the cell culture media is more consistent 15 compared to the titer obtained without arginine supplementation as evident from table II and Figure 1.
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Table II
Experiment
Conc. of Arginine (mM)
Titer (mg/L)
At 260h
At 280h
Without arginine supplementation
0
1238±74
1470±93
8 mM arginine supplementation
8
1343±8
1562±15
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. 5
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 a further embodiment, the invention provides method for expression of protein 10 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 increased percentage of G0F glycans and/or reduced percentage of galactosylated glycans wherein the cells are subjected to pH shift(s). The pH 15 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 5 0.5 unit.
In a 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 shift in temperature and pH may be accompanied by 10 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), 15 DMEM/F12 (Invitrogen), Ham’s F10 (Sigma), Minimal Essential Media (Sigma), and RPMI -1640 (Sigma) or combinations thereof. Further, the cell culture medium can be a combination of any aforementioned cell culture medium and a feed.
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 20 Hyclone, Catalogue no SH 30596.03), Cell Boost 4 (CB-4, Thermo Scientific
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HyClone, Catalog no. SH30928), PF CHO (Thermo Scientific Hyclone, Catalog no. SH30333.3).
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). 5 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 10 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).
Certain aspects and embodiments of the invention are more fully defined by reference to the following examples. These examples should not, however, be 15 construed as limiting the scope of the invention.
EXAMPLES
Example I
An anti-VEGF antibody was cloned and expressed in a recombinant CHO cell line as described in U.S. Patent No. 7,060,269, which is incorporated herein by reference. rCHO cells expressing antibody at a seeding density of 0.2-0.6 million 5 cells/ml were seeded in cell culture media (PowerCHO®2 (Lonza, Catalog no: 12-771Q) and CB4 feed, 95:5) at 37 °C and pH 7.1. The cells were cultured for 64 hrs, subsequently pH was reduced to 6.9 and temperature was reduced to 35 °C followed by addition of the feed CB-4. The culture was finally harvested after 240 hrs to 288 hrs or at greater than 50% viability. The experiment was run in thirteen 10 separate batches and the average values for antibody titer, VCC, IVCC and percentage of G0F, galactosylated glycans, HM and AF are shown in Figure 1-5. The percentage of G0F, galactosylated glycans, HM and AF glycan values are depicted in Table III.
Example II 15
An anti-VEGF antibody was cloned and expressed in a recombinant CHO cell line as described in U.S. Patent No. 7,060,269, which is incorporated herein by reference. rCHO cells expressing antibody at a seeding density of 0.2-0.6 million cells/ml were seeded in cell culture media (PowerCHO®2 (Lonza, Catalog no: 12-771Q) and CB-4 feed, 95:5) at 37 °C and pH 7.1. The cells were cultured for 64 20 hrs, subsequently pH was reduced to 6.9 and temperature was reduced to 35 °C followed by addition of the feed CB-4. On day 4 of seeding, 8 mM of arginine was ,CLAIMS:1. A cell culture process comprising supplementation of culture medium with arginine for increasing the percentage of G0F glycans and/or decreasing the percentage of galactosylated glycans in the glycoprotein composition as compared 5 to cell culture process wherein there is no arginine supplementation.
2. A cell culture process comprising supplementation of culture medium with arginine for increasing the percentage of G0F glycans and/or decreasing the percentage of galactosylated glycans without affecting the high mannose or afucosylation content in the glycoprotein composition as compared to cell culture 10 process wherein there is no arginine supplementation.
3. A cell culture process according to claims 1 or 2, wherein the concentration of arginine supplemented is about 8mM.
4. A cell culture process according to claims 1 or 2, wherein the glycoprotein composition comprises about 66% of G0F glycans and / or about 25% 15 galactosylated glycans.
5. A cell culture process according to claims 1 or 2, wherein increase in percentage of G0F glycans is about 6% and/ or decrease in percentage of galactosylated glycan is about 18%.
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6. A cell culture process according to claim claims 1 or 2, wherein the process comprises temperature shift and/or pH shift steps.
7. A cell culture process according to claim claims 1 or 2, wherein the wherein the difference in the first and the second temperature is about 2°C to about 7°C and/ or difference in any of the pH shifts is about 0.1 to about 0.5 units of pH. 5
8. A cell culture process according to claim claims 1 or 2, wherein the glycoprotein composition is an anti-VEGF antibody composition.
9. A cell culture process according to claim claims 1 or 2, wherein the cell are CHO cells.
10. A cell culture process according to claims 1 or 2, wherein the process leads to 10 increased titers.