WO2013114164A1

WO2013114164A1 - Method for obtaining glycoprotein composition with increased afucosylation content

Info

Publication number: WO2013114164A1
Application number: PCT/IB2012/057090
Authority: WO
Inventors: Asma AHMED; Indraneel DASARI; Mohit NARESH
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 method for obtaining a glycoprotein composition with increased percentage of afucosylated glycoforms.

Description

METHOD FOR OBTAINING GLYCOPROTEIN COMPOSITION WITH INCREASED AFUCOS YLATION CONTENT

RELATED APPLICATION

This application is related to and takes priority from the Indian Provisional Application 335/CHE/2012 filed 30 Jan 2012 and is incorporated herein in its entirety.

INTRODUCTION

Protein glycosylation is one of the most important post-translation

modifications associated with the eukaryotic proteins. The two major types of glycosylations 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 are of further 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) may actually be glycosylated. This may be a consequence of the competitive action of diverse enzymes during biosynthesis and are key to understanding glycoprotein heterogeneity (Marino K. et ai, 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, that is then transferred to the appropriate point 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 so formed in the ER lumen is modified by action of enzymes in the Golgi apparatus. If the saccharide is relatively inaccessible, it is likely to stay in the original high-mannose form. If it is accessible, then many of the mannose residues may be cleaved off and the saccharide further modified, resulting in the complex type N-glycans structure. In the c/^'s-Golgi, mannosidase-1 may cleave/hydrolyze 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 which is found to be 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 4 pages 288 to 294). However in a separate study it was shown that galactose feeding does not affect the sialic acid content.

Pacisef a/ 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 (Pads 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.al, Biotechnol. Bioeng., 2006, Vol. 94, Issue 6, pages 1045 to 1052 and Yoon 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.11, 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 antibodies (Muthing J, Kemminer SE, Conradt HS, Sagi D, Nimtz M, Karst U, Peter- Katalinic J., BiotechnolBioeng. 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 861 and Krapp, S. et al., J. Mol. Biol., 2003, Vol. 325, pages 979 to 989). Further, the binding to the Fcylll 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 and Shields RL, The Journal of Biological Chemistry, 2002, Vol. 277(30), pages 26733 to 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 in quality 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 key to high ADCC activity have been generated using specific inhibitors for e.g. KifunesinefSfren, 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 (edM.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 does not describe 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 an increased percentage of afucosylated glycans. In particular the invention describes a cell culture process wherein the cells are subjected to simultaneous temperature downshift and pH upshift to obtain an antibody composition having increased percentage of afucosylated glycoform.

SUMMARY

The invention describes a process for obtaining a glycoform composition comprising increased percentage of afucosylated glycoforms. In particular the method describes culturing cells at a temperature and pH for a period of time followed by subjecting cells to a second temperature and pH to obtain a glycoform composition having increased percentage of afucosylated glycans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of effect of pH upshift and temperature downshift on the viable cell count during cell culture process, as described in examples 1 to 4. FIG. 2 is an illustration of effect of pH upshift and temperature downshift on afucosylation content of antibodies obtained by cell culture process, as described in examples 1 to 4. DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "glycoprotein" refers to protein or polypeptide having at least one glycan moiety wherein glycan refers to a monosaccharide or polysaccharide moeity. Thus, any polypeptide attached to a saccharide moiety is termed as glycoprotein. 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 "complex glycovariant" as used herein consists of glycan moieties comprising any number of sugars.

"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 (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 "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

Table I : Representative table of various glycans

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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 is defined as the change in pH during the cell culture process. As used herein, cells are first cultured at a pH for a certain period of time after which pH of the cell culture medium is increased, and cells are cultured at this increased 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 or cell viability is defined as number of live cells in the total cell population. The present invention discloses a cell culture process wherein cells are maintained at a particular pH and temperature to attain optimum growth, followed by decrease in temperature to a lower value, while pH is increased to a higher value to obtain a glycoform composition comprising increased percentage of afucosylated glycoforms. In one embodiment, the present invention provides a process for obtaining a glycoprotein composition comprising increased percentage of total afucosylated glycans 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 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 is towards lower value while shift in pH is towards higher values.

In an embodiment the glycoprotein comprises about 10.0% to about 22.0 % total afucosylated glycans.

In another embodiment the glycoprotein comprises about 10.0 %total afucosylated glycans.

In yet another embodiment the glycoprotein comprises about 13.0 % total afucosylated glycans.

In yet another embodiment the glycoprotein comprises about 15.0 % total afucosylated glycans. In yet another embodiment the glycoprotein comprises about 22.0 % total afucosylated glycans.

Various methods described in the art such as Wuhreret. al., Ruhaak L.R., and Geoffrey et. al. can be used for assessing glycovariants present in a glycoprotein composition (Wuhrer M. et ai, Journal of Chromatography B, 2005, Vol.825, Issue 2, pages 124-13;, 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 another embodiment, the application provides method for production of glycoproteins with a particular glycoform composition by first culturing cells at temperatures about 35°C -37°C and pH about 7.05, followed by lowering of temperature by about 2-7°C and increasing pH to about 7.2.

In yet another embodiment, the application provides methods for expression of protein with particular glycoform composition by growing cells at about 37°C and pH about 7.05, followed by subjecting cells to about 35 °C and pH about 7.2. In yet another embodiment, the application provides methods for expression of protein with particular glycoform composition by growing cells at about 37°C and pH about 7.05, followed by subjecting cells to about 33 °C and pH about 7.2.

In yet another embodiment, the application provides methods for expression of protein with particular glycoform composition by growing cells at about 37°C and pH about 7.05, followed by subjecting cells to about 31 °C and pH about 7.2.

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).

The feeds in the present invention may be added in a continuous, profile or a bolus manner. Also it may be that one or more feeds are in one mode (e.g. profile mode) and others are in second mode (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. It 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 can be a solution of depleted nutrient(s), mixture of nutrient(s) or a mixture of cell culture medium/feed providing such nutrient(s). In one aspect of the invention, specific commercial feeds may be used while in other concentrated basal media may be added to the cell culture medium.

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.

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 0.8-1 .0 million cells/ml are seeded in PF CHO (HyClone^®, Catalog no: SH30335 & SH30334) comprising galactose (6 g/L) at 37°C and pH 7.05. The cells are cultured to attain VCC of -2.0 million cells/ml, at which 75ml/L Cell Boost 2 (HyClone^®) is added and temperature is lowered to 35^QC.An additional feed is added at an IVCC of 10 million cells/mL.The culture is harvested after 180 - 288 hrs or at greater than 50% viability whichever is early. The antibody titer at the time of harvest has been shown in Table 2. The VCC values and afucosylation content are disclosed in Figures (FIG.) 1 and 2.

Example II

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 0.8-1 .0 million cells/ml are seeded in PF CHO (HyClone^®, Catalog no: SH30335 & SH30334) comprising galactose (6g/L) at 37C and pH 7.05. The cells are cultured to attain VCC of -2.0 million cells/ml after which 75ml/L Cell Boost 2 (HyClone^®) is added,temperature is lowered to 35°C and pH of the culture medium is raised to 7.2. The culture is harvested after 180 - 288 hrs or at greater than 50% viability whichever is early. The resulting antibody (II) titre at the time of harvest is disclosed in Table 2. The VCC values and afucosylation content are shown in FIG. 1 and 2.

Example III

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 0.8-1 .0 million cells/ml are seeded in PF CHO (HyClone^®, Catalog no: SH30335 & SH30334) comprising galactose (6 g/L) at 37°C and pH 7.05. The cells are cultured to attain VCC of -2.0 million cells/ml, at which 75ml/L Cell Boost 2 (HyClone^®) is added, temperature is lowered to 33°C and pH of the culture medium is raised to 7.2. The culture is harvested after 180 - 288 hrs or at greater than 50% viability whichever is early. The resulting antibody (III) titre at the time of harvest has been shown in Table 2. The VCC values and afucosylation content are disclosed in FIG. 1 and 2.

Example IV 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 0.8-1 .0 million cells/ml are seeded in PF CHO (HyClone^®, Catalog no: SH30335 & SH30334) comprising galactose (6 g/L) at 37°C and pH 7.05. The cells are cultured to attain VCC of -2.0 million cells/ml, at which 75ml/L Cell Boost 2 (HyClone^®) is added, temperature is lowered to 31 °C and pH of the culture medium is raised to 7.2. The culture is harvested after 180 - 288 hrs or at greater than 50% viability whichever is early. The resulting antibody (IV) titre at the time of harvest has been shown in Table 2. The VCC values and afucosylation content are disclosed in FIG. 1 and 2.

Table 2: Antibody titre at the time of harvest (180-288 hrs)

Claims

We claim

A process for obtaining a glycoprotein composition comprising increased percentage of total afucosylated glycans comprising, culturing cells expressing said glycoprotein at a first temperature and a first pH, for a first period of time, followed by subjecting cells to a second temperature and second pH, for a second period of time wherein the temperature shift and pH shift are carried out simultaneously.

A process according to claim 1 , wherein the said glycoprotein comprises about 10.0% to about 22.0% total afucosylated glycans.

A process according to claim 1 , wherein the said glycoprotein comprises about 10.0% total afucosylated glycans.

A process according to claim 1 , wherein the said glycoprotein comprises about 13.0% total afucosylated glycans.

A process according to claim 1 , wherein the said glycoprotein comprises about 15.0% total afucosylated glycans.

A process according to claim 1 , wherein the said glycoprotein comprises about 22.0% total afucosylated glycans.

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

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

A process according to claim 1 , wherein cell are cultured in step a) at a pH of about 7.05.

A process according to claim 1 , wherein cells are cultured in step b) at a temperature of about 31 °C to about 35 °C. 1 1 ) A process according to claim 1 , wherein cells are cultured in step b) at a temperature of about 35 °C.

12) A process according to claim 1 , wherein cells are cultured in step b) at a temperature of about 33 °C.

13) A process according to claim 1 , wherein cells are cultured in step b) at a temperature of about 31 °C.

14) A process according to claim 1 , wherein cells are cultured in step b) at a pH of about 7.2.

15) A process according to claim 1 , wherein step b) further comprises addition of a feed.