WO2013114165A1

WO2013114165A1 - Process of obtaining glycoprotein composition with increased afucosylation content

Info

Publication number: WO2013114165A1
Application number: PCT/IB2012/057091
Authority: WO
Inventors: Satakarni MAKKAPATI; Vaibhav S NIKAM; Satyam Subrahmanyam
Original assignee: Dr Reddy's Laboratories Limited
Priority date: 2012-01-30
Filing date: 2012-12-08
Publication date: 2013-08-08

Abstract

The invention describes a cell culture process for obtaining a glycoprotein with the enhanced afucosylation content. The method involves culturing cells in a medium supplemented with galactose, at a specific osmolality, and harvesting on about 12th day or at about 50% viability to obtain glycoform composition with enhanced afucosylation.

Description

PROCESS OF OBTAINING GLYCOPROTEIN COMPOSITION WITH INCREASED AFUCOSYLATION CONTENT

RELATED APPLICATION

This application is related to and takes priority from Indian Provisional Application 332/CHE/2012 filed 30 Jan 2012 and is herein incorporated in its entirety.

INTRODUCTION

The invention describes a method for production of glycoprotein with increased afucosylation content. More specifically, the invention describes a cell culture process wherein cells are cultured in a medium supplemented with galactose, at a specific osmolality, and harvested on about 12^th day or at about 50% viability to obtain glycoform composition with enhanced afucosylation.

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 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. 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 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 consequence of the competitive action of diverse enzymes involved during glycosylation and are key to understanding glycoprotein heterogeneity (Marino K. et al., Nature Chemical Biology, 2010, Vol. 6, No. 10, pages 713 to723).

The process of N-linked glycosylation begins co-translationally in the endoplasmic reticulum (ER) 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, and that is then transferred to the appropriate position (Asn297) on the polypeptide chain (Schwarz F. and Aebi M., Current Opinion in Structural Biology, 2011, Vol.21, Issue 5, pages 576 to 582 and Burda P., and Aebi M.,Biochemica et Biophysica acta (BBA) General Subjects, 1999, Volume 1426, Issue 2, pages 239 to 257).

The glycan complex formed in the ER lumen is further modified by action of enzymes in the Golgi apparatus. If the saccharide is relatively inaccessible, it is likely to remain in the original high-mannose form. If it is accessible, then several mannose residues may be cleaved and the saccharide further modified, resulting in the complex type N-glycans structure. In the c/^'s-Golgi, mannosidase-1 may act to cleave/hydrolyzes a high mannose glycan, while further on,fucosyltransferase FUT- 8 fucosylates the glycan in the medial-Go\g\ (Harue Imai-Nishiya et al, BMC Biotechnology, 2007, 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. 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. There is a significant variability observed in terminal galactosylation that is dependent on the medium. It has been shown that feeding cultures with galactose up to a concentration 36mM results in high levels of galactosylation (Davies, J.; Jiang, L.; Pan, L.Z.; LaBarre, M. J.; Anderson, D.; Reff, M. Biotechnol.Bioeng. 2001, Vol.74, Issue 4pages 288 to 294). However in a separate study it was shown that galactose feeding does not affect the sialic acid content. 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 G₀F and G-| F glycoforms, resulting in its faster clearance from the body and thereby reducing its efficacy (Pacis E., Yu, M., Autsen, J., Bayer, R., Li F., Bitechnol Bioeng, 2011, Vol. 108, IssueW, pages 2348 to 2358).

The studies by Kaufman et al and Yoon et a/ show a reduction in protein sialylation upon decrease in temperature {Kaufman, H., Mazur X., Fussenegger, M., Bailey, J.E., Biotechnology and Bioengineering., 1999, Vol. 63, Issue 5, pages 573 to 582;Trummer, E., Fauland, K., et.ai, Biotechnol Bioeng., 2006, Vol. 94, Issue 6, pages 1045 to 1052 andYoon S.K., Song, J. Y., Lee, G.M., Biotechnol Bioeng., 2003, Vol. 82, Issue 3, pages 289 to 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, Etchverry T, Ryll T. Cytotechnology, 1997, Vol.23, pages 47 to 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, ΒΙΟ/technology, Vol.1 Ί , pages 720 to 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., Biotechnol Bioeng. 2003, Vol.83, Issue 3, pages 321 to 334).

Most of the therapeutic monoclonal antibodies (rMAb) that have been licensed and developed as medical agents are of the human lgG1 isotype. The effectiveness of these antibodies is dependent on sensitization of target cells for subsequent killing by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) mediated through the interaction of the Fc with FcyRs receptors or complement respectively (Jefferis, R. Expert Opinion on Biological Therapy, 2007, Vol.7, Number 9, pages 1401 to 1413;Shields, R. L. et al., The Journal of Biological Chemistry, 2002, Vol.277 (30), pages 26733 to 26740; Shinkawa, T. et al. The Journal of Biological Chemistry, 2003, Vol.278 (5), pages 3466 to 3473;Niwa, R. et al. Journal of Immunological Methods, 2005, Vol.306, Issues 1-2, pages 151 to 160;Ferrara, C. et al., Biotechnol. Bioeng., 2006, Vol. 93, Issue 5, pages 851 to 861andKrapp, S. et ai, J. Mol. Biol., 2003, Vol. 325, pages 979 to 989). Further, the binding to the FcylM receptor is dependent on the fucose content of the Fc glycans wherein a reduction in fucose can increase effector function. Fucose-deficient IgGI s have shown a significant enhancement of ADCC up to 100-fold (Mori K., Cytotechnology, 2007, Vol. 55(2-3), pages 109 to 114& Shields RL, The Journal of Biological Chemistry, 2002, Vol. 277(30), pages 26733to 26740). Hence, non-fucosylated antibodies may be the promising next- generation therapeutic antibodies with improved efficacy and reduced dose based toxicities.

Robust stable production of completely non-fucosylated therapeutic antibodies consistent inquality has been achieved by the generation of a unique host cell line, in which the endogenous a-1 ,6-fucosyltransferase (FUT8) gene is knocked out. Further, antibodies with high oligomannose content having high affinity for FCYRI I IA, which is a key to high ADCC activity have been generated using specific inhibitors for e.g. kifunesine (Shen, A., Ng, D., Joly, J., Snedecor, B., Lu, Y., Meng, G., Nakamura, G., and Krummen, L. Metabolic engineering to control glycosylation,in cell culture and upstream processing (ed M. butler) 2007, Taylor and Francis group). Antibodies with the altered glycoform profiles have been produced using glycosylation enzyme mutants/knockouts or by using glycosylation inhibitors in cell culture medium such as deoxymannojirimycin (J Bischoff J., Liscum L., and Kornfeld R., The Journal of Biological Chemistry, 1986, Vol. 261(10), pages 4766 to 4774). However, the prior art lack in describing a cell culture process or modifications in process parameters to modify afucosylated glycan content in a glycoprotein composition. The present invention describes a process of obtaining an antibody composition comprising a higher percentage of afucosylated glycans. In particular the invention describes a cell culture process wherein the cells are cultured in a medium supplemented with galactose at specific osmolality and harvested on the 12^th day or at 50% viability. SUMMARY

The present invention describes a method for obtaining a glycoprotein composition comprising higher percentage of afucosylated glycans. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of viable cell count as described in Example 1 .

FIG. 2 is an illustration of major glycoforms of glycoproteins as described in Example 1 . DETAILED DESCRIPTION

Definitions

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

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 that 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.

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.

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

"Total afucosylated glycans" described here, consists of glycan moieties wherein fucose is not linked to the non reducing end of N-acetlyglucosamine.

Without limitation, examples of afucosylated glycans include GO, G1 A, G1 B, G2, M3-M9NAG, M3-M9.

The term "osmolality" as used herein is defined as a measure of the osmoles of solute per kilogram of solvent (mOsm/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.

Table I : Representative table of various glycans

The term "temperature shift" as used herein is defined as the 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

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 or cell viability is defined as number of live cells in the total cell population.

The present invention discloses a cell culture method for obtaining a glycoprotein composition comprising increased percentage of afucosylated glycoforms.

In one embodiment, the present invention provides a process for obtaining a glycoprotein composition comprising about 14% to about 18% of total afucosylated glycans comprising, culturing cells expressing said glycoprotein in a cell culture media a) comprising galactose

b) at an osmolality of about 310 mOsm to about 340 mOsm c) at a first temperature for a first period of time

d) at a second temperature for a second period of time and

harvesting cells on about 12^th day or about 50% viability,

In an embodi ment the glycoprotein comprises about 14.0% of total afucosylated glycans.

In another embodiment the glycoprotein comprises about 1 6.0% of total afucosylated glycans. In yet another embodiment the glycoprotein comprises about 18.0% of total afucosylated 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 andGeoffrey, R. G. et. al. Analytical Biochemistry 1996, Vol. 240, pages 210-226).

In one aspect of the invention, the method may additionally be accompanied by culturing cells at first temperature for a first period of time and then subjecting cells to a second temperature for a second period of time.

In yet another embodiment of the invention, the process comprises culturing cells at a first temperature of about 35 °C -37°C for about 72 hours, followed by lowering of temperature by about 2°C -7°C and harvesting cells on 12^th day or at about 50% viability, whichever is early.

In yet another embodiment of the invention, the process comprises culturing cells at about 37°C for about 72 hours, followed by culturing cells at 33 °C and harvesting cells on 12^th day or about 50% viability whichever is earlier.

In one aspect of the invention, the cell culture media comprises galactose. The concentration of galactose for the purpose of the invention is about 6g/L.

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 -1 640 (Sigma).

In one aspect of the invention, the method may additionally be accompanied by the addition of feed. The feed is composed of nutrients or other medium components that have been depleted or metabolized by the cells. The feed may include hormones, growth factors, ions, vitamins, nucleoside, nucleotides, trace elements, amino acids, lipids or glucose. These supplementary components may be added at one time or in series of additions to replenish. Thus feed may 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:

Table II : Representative feeds and feed composition

Specific embodiment of the invention is more fully defined by reference to the following example. This example should not, however, be construed as limiting the scope of the invention.

Example I

An anti-CD20 antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 7381560 which is incorporated herein by reference. rCHO cells expressing antibody at a seeding density of 0.4-0.6 million cells/ml are seeded in PF CHO (HyClone®, Catalog no: SH30335 & SH30334) at an osmolality of 320-340mOsm/Kg at 37°C and pH 7.05, supplemented with 6g/L galactose. The cells are cultured for 3 hrs after which profile feeding of Feed lis done till 72 hrs. At 72 hrs, temperature is lowered to 33°C and simultaneously bolus Feed His added. The culture is finally harvested after 180-288 hrs or at greater than 50% viability and the resulting antibody yield is determined. The disclosed process was used to produce three batches of the anti-CD20 antibody (I -III). The % viability, IVCC and antibody titer are shown in Table II I while % afucosylation is disclosed in Table IV. The viable cell count and the glycan profileare disclosed in Figures 1 -2.

Table III : %Viability of cells, IVCC and antibody titer of cells at harvest

Table IV: % Total Afucosylation of the antibody

Claims

CLAIMS We claim:

1 . A cell culture process for obtaining a glycoprotein composition comprising about 14% to about 18% total afucosylated glycans comprising culturing cells expressing said glycoprotein in a cell culture media

a) comprising galactose

b) seeding cells at an osmolality of about 31 OmOsm/Kg to about 340mOsm/Kg

c) at a first temperature for a first period of time

d) at a second temperature for a second period of time and harvesting cells on about 12^th day or about 50% viability.

2. A process according to claim 1 , wherein the said glycoprotein comprises about 14% total afucosylated glycans.

3. A process according to claim 1 , wherein the said glycoprotein comprises about 16% total afucosylated glycans.

4. A process according to claim 1 , wherein the said glycoprotein comprises about 18% total afucosylated glycans.

5. A process according to claim 1 , wherein cells are cultured in a cell culture media comprising about 6 g/L galactose.

6. A process according to claim 1 , wherein cells are cultured in step c) at a temperature of about 35^QC to about 37^QC.

7. A process according to claim 1 , wherein cells are cultured in step c) at a temperature of about 37^QC.

8. A process according to claim 1 , wherein cells are cultured in step d) at a temperature reduced in a range of 2- 7^QC.

9. A process according to claim 1 , wherein cells are cultured in step d) at a temperature of about 33^QC.

10. A process according to claim 1 , wherein the process further comprises addition of feed.