Mammalian cell culture process for producing glycoproteins

Details Mammalian cell culture process for producing glycoproteins Mammalian cell culture process for producing glycoproteins

1999-05-26

2003-03-04

Saunders, David (Department: 1644)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S070100, C435S200000, C435S358000

Reexamination Certificate

active

06528286

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to processes for producing glycoproteins in eukaryotic cell culture. The invention provides cell culture processes which preserve oligosaccharide structures of nascent glycoproteins and greatly facilitate the recovery of glycoproteins containing oligosaccharides terminating in one or more sialic acid residues from the cell culture.
BACKGROUND
Many eukaryotic cell surface- and secreted proteins are post-translationally processed to incorporate N-linked and O-linked carbohydrate (Kornfeld and Kornfeld (1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785-838). Protein glycosylation is thought to subserve a variety of functions including augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion (Fieldler and Simons (1995) Cell 81:309-312; Helenius (1994) Mol. Biol. of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473; Caton et al., (1982) Cell 37:417-427; Alexander and Elder (1984) Science 226:1328-1330; Flack et al. (1994) J. Biol. Chem. 269:14015-14020). In higher organisms, the nature and extent of glycosylation can markedly affect the circulating half-life and bio-availability of secreted proteins by mechanisms involving receptor mediated uptake and clearance (Ashwell and Morrell (1974) Adv. Enzymol. 41:99-128; Ashwell and Harford (1982) Ann. Rev. Biochem. 51:531-54). Receptor systems have been identified that are thought to play a major role in the clearance of serum proteins through recognition of various sugar components of the oligosaccharide on the glycoproteins (Stockert (1995) Physiol. Rev. 75:591-609; Kery et al., (1992) Arch. Biochem. Biophys. 298:49-55). Since the terminal sialic acid component affects absorption, serum half life, and clearance from the serum as well as the physical, chemical and immunogenic properties of the glycoprotein (Parekh, R. B., suira; Varki, A., (1993) Glycobiology 3:97-100; Paulson, J. (1989), TIBS, 14:272-276; Goochee, et al., (1991) Biotechnology 9:1347-1355; Kobata, A, (1992) Eur. J. Biochem. 209:483-501) production strategies which preserve the terminal sialic acid component can advantageously lengthen protein bioavailability and serum half-life.
Much attention has been paid to factors which affect glycosylation during recombinant protein production such as growth mode (adherent or suspension), fetal bovine serum in media formulation, culture density, oxygenation, pH, ammonium concentration, purification schemes and the like (Werner, R. and Noe, W. (1993), Drug Res. 43:1134-1249; Hayter et al., (1992) Biotech. and Bioeng. 39:327-335; Borys et al., (1994) Biotech and Bioeng. 43:505-514; Borys et al., (1993) Bio/Technology 11:720-724; Hearing et al., (1989) J. Cell Biol. 108:339-353; Goochee et al., in Frontiers in Bioprocessing II, Todd et al., eds (1992) American Chemical Society pp.199-240; U.S. Pat. No. 5,096,816; Chotigeat, W., (1994) Cytotech. 15:217-221; Gawlitzek et al., (1998) Biotech. Bioeng. 57:518-528). Several groups have investigated the process parameters that surround the production of recombinant proteins and especially the effect of media composition in various production strategies (Park et al., (1992) Biotech. Bioeng. 40:686-696; Cox and McClure, (1983) In Vitro, 19:1-6; Mizutani et al., (1992) Biochem. Biophys. Res. Comm. 187:664-669; Le Gros et al. , (1985) Lymph. Res. 4(3):221-227). For example U.S. Pat. No. 5,705,364 discloses methods of altering the sialic acid content of a glycoprotein by controlling factors that affect cell productivity, such as the addition of an alkanoic acid to the culture medium, controlling osmolality of the culture medium, and controlling growth temperature.
Another means of affecting sialic acid content of glycoprotein is by controlling the activity of cellular sialidases (U.S. Pat. No. 5,510,261). Sialidases are cytosolic and membrane-associated enzymes that cleave sialic acid from glycosyl moiety of a glycoprotein. The activity of synaptosomal membrane-associated sialidase of bovine brain was inhibited by copper ion at high concentration following pre-saturation in situ (Yohe, H. C. and Rosenberg, A. (1978) Neurochemical Research (1978) 3:101-113). Cytosolic and membrane-associated sialidase I was shown to be inhibited by copper ion at approximately 1 mM concentration in partially purified extracts from rat liver or rat skeletal muscle (Miyagi, T. et al. (1993) Glycoconjugates Journal 10:45-49).
Culture media and additives frequently contain trace elements and metal ions such as copper for optimal cell growth (for example, “Protease Peptone 2 and 3,” “Primatone RL” and “Primatone HS”, which are commercially available (Sheffield, England; Difco, USA); Japanese Patent No. 93JP-0171420; International Publication No. WO 90/03430; for composition of various media, for example, DMEM and HAM F12 media, see culture media formulations in American Type Culture Collection Catalogue of Cell Lines and Hybridomas, Sixth Edition, 1988, pages 346-349). A serum-free medium containing Zn (5-100 &mgr;M) or Cu (0.1-50 &mgr;M) for growth of vascular endothelial cells was proposed in Japanese Patent No. 247618. Lanier and Volkman reported an increase in yield of baculovirus expression vectors from recombinant baculoviruses generated in insect (lepidopteran) tissue culture in a medium containing 2 mM CuSO
4
(Lanier, L. M. and Volkman, L. M. (1996) In Vitro 32(3) Pt.2:8A). Hultberg et al. found that metals increased the amount of glutathion reduced in HeLa cell culture medium and that copper ion also increased the amount of reduced homocysteine in the medium at copper ion concentrations that did not interfere with cell growth (1-100 &mgr;m). (Hultberg, B. et al. (1997) Toxicology 117:89-97). Divalent metal ions such as Cu
2+
and Zn
2+
in soluble form at a concentration of 70-120 mg/L have been added to serum and the serum added to cell culture medium to promote cell growth (German Patent No. 155 328).
However, eukaryotic cells, especially mammalian cells such as Chinese hamster ovary (CHO) cells have been shown to be sensitive to Cu
2+
concentration in culture media (Camakaris et al., (1995) Human Molecular Genetics 4:2117-2123; Steinebach & Wolterbeek (1994) J. Inorganic Biochemistry, 53:27-48; German Patent 155 328; Sakai, Y. et al. (1994) Cytotechnology 14 (Suppl. 1):7-36). CHO-K1 cells were found to exhibit an LD
50
of 126 &mgr;M Cu
2+
in copper-supplemented culture medium (Camakaris, J. et al. (1995) Human Molecular Genetics 4:2117-2123).
The present invention provides for processes for producing glycoproteins by mammalian cell culture which maintain the sialic acid component of oligosaccharides of glycoproteins produced.
SUMMARY OF THE INVENTION
The present invention provides processes for producing glycoproteins by eukaryotic cell culture which provide a glycoprotein product containing oligosaccharides terminating in one or more sialic residues. Advantageously, the cell culture processes of the present invention allow for the recovery of a glycoprotein product whose oligosaccharides are not compromised by degradative events associated with standard cell culture procedures. The processes of the present invention overcome the problem of desialylation of a glycoprotein's oligosaccharide side chains that are associated with standard glycoprotein production methods. Advantageously the invention provides economic and commercial benefits through the recovery of greater useful quantities of glycoprotein product.
Accordingly, the invention provides processes for producing glycoproteins by eukaryotic and especially mammalian cell culture comprising culturing a host cell expressing a glycoprotein in the presence of copper ion in a cell culture medium in a concentration effective to minimize the loss of sialic acid. The present invention therefore provides various cell culture processes to preserve particular glycofo

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