FIELD OF THE INVENTION

The invention describes a method for obtaining a glycoprotein with a particular glycoform composition by culturing cells at low pH. Further, the invention describes a cell culture process wherein cells in the growth phase are maintained at a particular pH to attain optimum growth, after which, temperature and pH are reduced simultaneously such that high yield of glycoprotein with a particular glycoform composition is obtained.

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 acids 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 complex type that contain more than two N-acetylglucosamines plus any number of other types of sugars. In both N- and O-glycosylation, there is usually a range of glycan structures associated with each site (microheterogeneity). Macroheterogeneity results from the fact that not all N-glycan or O-glycan consensus sequences (Asn-X-Ser/Thr for N-glycan and serine or threonine for O-glycan present in the glycoproteins) are actually glycosylated. This may be a consequence of the competitive action of diverse enzymes during biosynthesis and are key to understanding glycoprotein heterogeneity. (Marino, K., (2010) Nature Chemical Biology 6, 713-723)

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 appropriate point on the polypeptide chain as it is translocated into the

ER lumen (Schwarz, F. andAebi M., (2011) Current Opinion in Structural Biology, 21:576-582 & Burda, P. & Aebi M., (1999) Biochimica et Biophysica Acta (BBA)General Subjects Volume 1426, Issue 2, Pages 239-257). The glycan complex so formed in the ER lumen is modified by action of enzymes in the Golgi apparatus. If the saccharide is relatively inaccessible, it will most likely stay in the original high-mannose 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-glycans structure. In the m-Golgi, mannosidase-1 may act to generate a high mannose glycan, while further on, fucosyltransferase FUT-8 fucosylates the glycan in the medial-Go\gi(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 machinery enzymes to the glycan structure, the order of action of each enzyme and the stage at which the protein is released from the glycosylation machinery.

External factors affecting glycan structure and composition

In addition to the "in vivo" factors listed above, "external factors" may also affect the glycan structure and composition of a protein. These include the conditions in which the cell line expressing the protein is cultured, such as the medium composition, the composition and timing of the feed, osmolality, pH, temperature etc. Pacis et al has shown that higher osmolality may result in increase in the number of Man5 residues on recombinant antibodies, with a simultaneous reduction in GoF and GiF glycoforms, resulting in its faster clearance from the body and thereby reducing its efficacy (Pacis E., Yu, M., Autsen, J., Bayer, R., Li F., (2011) Bitechnol Bioeng 108 (10) 2348-2358).

Studies by Kaufman et al and Yoon et al show a reduction in protein sialylation upon decrease in temperature {Kaufman, H., Mazur X., Fussenegger, M., Bailey, J.E., (1999) Biotechnol Bioeng. 63, 573-578; Trummer, E., Fauland, K., et.al. (2006) Biotechnol Bioeng. 94 1045-1052); Yoon S.K., Song, J. Y, Lee, G.M., (2003) Biotechnol Bioeng. 82: 289-298). Further, reducing temperature can increase overall protein production by prolonging cell viability, which should, in principle, improve glycosylation. (Moore A, Mercer J, Dutina G, Donahue CJ, Bauer KD, Mather JP, Etcheverry T, Ryll T. (1997), Cytotechnology. 23:47-54).

Likewise Borys et al has shown that a deviation from optimum pH results in decrease in the expression rate as well as the extent of glycosylation of proteins (Borys M.C., Linzer, D.I.H., Papoutsakis (1993), BlO/technology 11 720-724). The culture pH of a hybridoma cell line has been shown to affect the resulting galactosylation and sialylation of the monoclonal antibody (Muthing J, Kemminer SE, Conradt HS, Sagi D, Nimtz M, Karst U, Peter- Katalinic J. (2003). Biotechnol Bioeng 83:321-334).

The structure and composition of the glycan moieties of a glycoprotein can have a profound effect on the safety and efficacy of therapeutic proteins, 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, R. G., Kopp, K. and Schlueter, M. (2007), 96:17-22. doi: 10.1111/j. 1651-2227.2007.00199.x). Removal of terminal galactose residues from the chimeric mouse-human IgGl antibody (alemtuzumab) was shown to reduce complement dependent cytotoxicity (CDC), without effecting FcyR-mediated functions (Boyd, P. N., Lines, A. C. & Patel, A. K.(1995), Mol. Immunol. 32, 1311-1318). Similarly, the (GIF-GIF) glycoform of rituximab triggered a CDC response twice as large as that triggered by the (GOF-GOF) glycoform (http://www. nature. com/nrd/journal/v8/n3/full/nrd2804. html-accessed on 23/12/2011). Hence given the role of the glycan structure and composition in the activity and efficacy of a glycoprotein on the one hand, and the array of factors that affect the glycan composition on the other, methods that control the glycan composition of glycoproteins would be beneficial.

The present invention describes a process that combines reduction of pH with temperature downshift to attain an antibody composition comprising a particular glycoform distribution.

SUMMARY OF THE INVENTION

A method for producing a glycoprotein having particular glycoform composition is described. The invention describes a process that combines reduction of pH with temperature downshift to attain an antibody composition comprising a particular glycoform distribution.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an illustration of effect of temperature and pH shift on antibody titer as described in Examples 1-4

Figure 2 is an illustration of effect of temperature and pH shift on cell viability as described in Examples 1-4

Figure 3 is an illustration of effect of temperature and pH shift on major glycoforms as described in Examples 1-4

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "glycan" refers to a monosaccharide or polysaccharide moiety.

The term "glycoprotein" refers to protein or polypeptide having at least one glycan moiety. Thus, any polypeptide attached to a saccharide moiety is termed as glycoprotein.

The term "glycoform" or "glycovariant" have been used interchangeably herein, and refers to various oligosaccharide entities or moieties linked in their entirety to the Asparagine 297 (as per Kabat numbering) of the human Fc region of the glycoprotein in question, co translationally or post translationally within a host cell. The glycan moieties 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, GO, G1A, GIB, G2).

GO as used herein refers to protein glycan not containing galactose at the terminal end of the glycan chain.

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 and may change during the cell culture process for e.g. by addition of feed, salts, additives or metabolites.

The term "temperature shift" as used herein refers to any change in temperature during the cell culture process. For the purpose of this invention, the initial temperature of the cell culture process is higher than the final temperature i.e. cells are subjected to a temperature downshift wherein cells are first cultured at a higher temperature for certain time period after which temperature is reduced, and cells are cultured at this lower temperature for a fixed period of time.

The term "pH shift" as used herein refers to a change in pH during the cell culture process. As used herein, cells are first cultured at a higher pH for a certain period of time after which pH of the cell culture medium is reduced, and then cells are cultured at this reduced pH for a certain period of time.

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.

Detailed description of the embodiments

The present invention provides a method for obtaining a glycoprotein with a particular glycoform composition. In particular, the invention provides a cell culture process wherein cells are maintained at a particular pH to attain optimum growth, after which, temperature and pH are reduced simultaneously such that high yield of glycoprotein with a particular glycoform composition is obtained.

In one embodiment the present invention provides, a process for obtaining a glycoprotein composition comprising about 3.2 to about 8.5% high mannose glycans, about 2.2 to 5.3% afucosylated glycans and about 44.7 to 61.3 % of G0F glycan comprising, culturing cells expressing said glycoprotein

a) at a first temperature and a first pH, for a first period of time, followed by

b) subjecting the cell to a second temperature and second pH, for second period of time wherein the temperature and pH shift are carried out simultaneously.

The shift in temperature and pH may be accompanied by addition of nutrient feed, and further wherein the shift in temperature and pH is towards lower values.

In another embodiment, the application provides method for production of glycoproteins with a particular glycoform composition by first culturing cells at temperatures about 35-37°C and pH about 7.05-7.2, followed by lowering of temperature by about 2-7°C and pH about 6.8 accompanied by addition of feed.

In yet another embodiment, the application provides method for expression of protein with particular glycoform composition by growing cells at about 37°C and pH about 7.05 - 7.2,

followed by subjecting cells to about 35°C and pH about 6.8, accompanied by addition of feed.

In another embodiment the invention provides a process for obtaining a glycoprotein composition comprising about 4.18 % to about 4.28 % high mannose glycans, about 2.52 % to about 2.70 % afucosylated glycans and about 51.32 % to about 56.50 % of G0F glycan.

In yet another embodiment the invention provides a process for obtaining a glycoprotein composition comprising about 8.23 % high mannose glycans, about 4.34 % to about 4.67 % afucosylated glycans and about 44.70 % to about 44.74 % of GoF glycan.

In a further embodiment the invention provides a process for obtaining a glycoprotein composition comprising about 3.2 % to about 3.8 % high mannose glycans, about 2.2 % to about 2.7 % afucosylated glycans and about 58.3 % to about 61.1 % of GoF glycan.

In yet another embodiment the invention provides a process for obtaining a glycoprotein composition comprising about 3.9 % to about 4.3 % high mannose glycans, about 2.2 % to about 2.4 % afucosylated glycans and about 60.5 % to about 61.2 % of GoF glycans.

The cell culture media that are useful in the application include but are not limited to, the commercially available products PF CHO (HyClone®), PowerCHO® 2 (Lonza), Zap-CHO (Invitria), CD CHO, CDOptiCHO™ and CHO-S-SFMII (Invitrogen), ProCHO™ (Lonza), CDM4CHO™ (Hyclone), DMEM (Invitrogen), DMEM/F12 (Invitrogen), Ham's F10 (Sigma), Minimal Essential Media (Sigma), and RPMI -1640 (Sigma).

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 are in second manner (e.g. bolus or continuous mode). Further, the feed may be composed of nutrients or other medium components that have been depleted or metabolized by the cells.

The 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. 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 feed may include, but are not limited to:

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 POWER CHO® 2 (Lonza, Catalog no: 12-771Q) at 37 °C and pH 7.05-7.2. The cells were cultured for 3 hrs after which profile feeding of Feed I was done till 72 hrs. At 72 hrs, pH was reduced to 6.8 and temperature to 35 °C followed by addition of Feed II. The culture was finally harvested after 180 - 288 hrs or at greater than 50% viability and the resulting antibody yield (IA) was determined.

In an alternate, after 72 hours, temperature of the cell culture was shifted to 35 °C and feed II was added (pH shift to 6.8 was not performed). The culture was finally harvested after 180 -288 hrs or at greater than 50% viability and the resulting antibody yield (IB) was determined.

The antibody yield IA and IB and cell viability has been disclosed in Table III. The glycan profile is shown in Table IV respectively.

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 grown in a PF CHO (HyClone®, Catalog no: SH30335 & SH30334) at 37°C and 7.05-7.2 pH. The cells were cultured for 3 hrs after which profile feeding of Feed I was done till 72 hrs. At 72 hrs, pH of the cell culture was reduced to 6.8 and the temperature was lowered to 35° C, subsequently

Feed II was added. The culture was finally harvested after 180 - 288 hrs or at greater than 50% viability and the resulting antibody yield (IIA) determined.

In an alternate, after 72 hours, temperature of the cell culture was shifted to 35 °C and feed II was added. The culture was finally harvested after 160 - 300 hrs or at greater than 50% viability and the resulting antibody yield (IIB) was determined.

The antibody yield at (IIA) and (IIB) and cell viability has been disclosed in Table III. The glycan profile is shown in Table IV respectively.

Example HI

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 grown in POWER CHO® 2 (Lonza, Catalog no: 12-771Q) at 37°C. During the growth phase, feeding of nutrients was done at 24 hours and 48 hours with Feed I. On attainment of optimum cell growth (Ivcc of 3.0 to 10.0 million cell-days/ml), the pH of the cell culture medium was reduced to pH 6.8, temperature was lowered to 35 °C and subsequently Feed II was added. The culture was finally harvested after 180-288 hrs or at greater than 50% viability and the resulting antibody yield determined. The antibody yield (III) and the glycan profile are disclosed in Table III and IV respectively.

Example IV

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 grown in POWER CH02 (Lonza, Catalog no: 12-771Q) at 37 °C. During the growth phase, feeding of nutrients was done at 24 hours and 48 hours with Feed I. On attainment of optimum cell growth (Ivcc of 3.0 to 10.0 million cell-days/ml), the pH of the cell culture medium was reduced to pH 6.8, temperature was lowered to 35 °C and subsequently Feed III was added. The culture was finally harvested after 180-288 hrs or at greater than 50% viability and the resulting antibody yield determined. The antibody yield (IV) and the glycan profile are disclosed in Table III and IV respectively.

CLAIMS

We claim:

1) A process for obtaining a glycoprotein composition comprising about 3.2% to about 8.5% high mannose glycans, about 2.2% to about 5.3% afucosylated glycans and about 44.7% to about 61.2% of GOF glycan comprising, culturing cells expressing said glycoprotein

a) at a first temperature and a first pH, for a first period of time, followed by

b) subjecting cells to a second temperature and second pH, for a second period of time wherein the temperature and pH shift are carried out simultaneously.

2) A process according to claim 1, wherein the said glycoprotein composition comprises about 4.2%o to about 4.3% high mannose glycans, about 2.5% to about 2.7% afucosylated glycans and about 51.3% to about 56.5% of GOF glycan.

3) A process according to claim 1, wherein the said glycoprotein composition comprises about 8.2%) high mannose glycans, about 4.3%) to about 4.7% afucosylated glycans and about 44.7% of GOF glycan.

4) A process according to claim 1, wherein the said glycoprotein composition comprises about 3.2% to about 3.8% high mannose glycans, about 2.2% to about 2.7% afucosylated glycans and about 58.3% to about 61.1% of GOF glycan.

5) A process according to claim 1, wherein the said glycoprotein composition comprises about 3.9%) to about 4.3% high mannose glycans, about 2.2% to about 2.4% afucosylated glycans and about 60.5% to about 61.2% of GOF glycans.

6) A process according to claim 1, wherein the cells are cultured in step a) at a temperature of about 35°C to about 37°C and a pH of about 7.05 to about 7.2.

7) A process according to claim 1, wherein the cells are cultured in step a) at a temperature of about 37°C.

8) A process according to claim 1, wherein the temperature in step b) is reduced in the range of about 2°C to 7°C.

9) A process according to claim 1, wherein cells are cultured in step b) at a temperature of
about 35°C.

10) A process according to claim 1, wherein cells are cultured in step b) at a pH of about 6.8.