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US 2013/0295608 A1Published application
Yeast strain for the production of proteins with modified o-glycosylation
AssigneeMerck Sharp & Dohme Corp.
InventorsPiotr Bobrowicz, William J. Cook, Stephen Hamilton, Juergen Nett, Terrance A. Stadheim, Stefan Wildt
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Abstract
Lower eukaryotic host cells have been recombinantly engineered to produce glycoprotein having human-like O-glycosylation. The glycoproteins are useful for the production of glycoprotein compositions with advantages for the production of human therapeutics.
Claims20
A method of producing a recombinant glycoprotein having O-Man-GlcNAc;
- O-Man-GlcNAc-Gal;
- or O-Man-GlcNAc-Gal-Sia as the predominant O-glycan thereof in a lower eukaryotic host cell which comprises:
- (a) providing lower eukaryotic host cells which
- (i) do not express functional beta-mannosyltransferase enzymes Bmt 1, 2, 3 and 4, and phospho-mannose transferase enzymes Mnn4a, Mnn4b and Pno1;
- and
- (ii) do express functional protein O-linked mannose β-1,2-N-linked acetylglucosaminyltransferase 1 (“POMGnT1”), UDP-GlcNAc transporter and α-1,2-mannosidase enzymes;
- (b) transfecting or transforming the lower eukaryotic host cells with nucleic acid encoding the glycoprotein of interest;
- and
- (c) culturing the host cells so that the recombinant glycoprotein is produced.
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A lower eukaryotic host cell capable of producing a recombinant glycoprotein having O-Man-GlcNAc;
- O-Man-GlcNAc-Gal;
- or O-Man-GlcNAc-Gal-Sia as the predominant O-glycan which:
- (a) does not express functional beta-mannosyltransferase enzymes Bmt 1, 2, 3 and 4, and phospho-mannose transferase enzymes Mnn4a, Mnn4b and PNO1;
- and
- (b) which is transformed or transfected with exogenous nucleic acid encoding POMGnT1, UDP-GlcNAc transporter, α-1,2-mannosidase, and the glycoprotein of interest;
- wherein the predominant O-glycan is present at a level greater than 50% and O-Man2 is present at less than 30%.
3 more elements
Dependencies are inferred from the claim text.