Method for increasing n-glycosylation site occupancy on therapeutic glycoproteins produced in pichia pastoris

Abstract

Described is a method for increasing the N-glycosylation site occupancy of a therapeutic glycoprotein produced in recombinant host cells modified as described herein and genetically engineered to express the glycoprotein compared to the N-glycosylation site occupancy of the therapeutic glycoprotein produced in a recombinant host cell not modified as described herein. In particular, the method provides recombinant host cells that overexpress a heterologous single-subunit oligosaccharyltransferase, which in particular embodiments is capable of functionally suppressing the lethal phenotype of a mutation of at least one essential protein of the yeast oligosaccharyltransferase (OTase) complex, for example, the Leishmania major STT3D protein, in the presence of expression of the host cell genes encoding the endogenous OTase complex. The method is useful for both producing therapeutic glycoproteins with increased N-glycosylation site occupancy in lower eukaryote cells such as yeast and filamentous fungi and in higher eukaryote cells such as plant and insect cells and mammalian cells.

1 - 22 . (canceled) 
     
     
         23 . A glycoprotein composition comprising:
 a plurality of antibodies wherein at least 70% of the antibody molecules in the composition have both N-glycosylation sites occupied and about 50 to 70 mole % of the N-glycans are G0, 15-25 mole % of the N-glycans are G1, and about 5 to 15 mole % of the N-glycans comprise a Man 5 GlcNAc 2  core structure and a pharmaceutically acceptable carrier.   
     
     
         24 . The composition of  claim 23 , wherein the antibodies comprise an antibody selected from the group consisting of anti-Her2 antibody, anti-RSV (respiratory syncytial virus) antibody, anti-TNFα antibody, anti-VEGF antibody, anti-CD3 receptor antibody, anti-CD41 7E3 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-CD33 antibody, anti-IgE antibody, anti-CD11a antibody, anti-EGF receptor antibody, and anti-CD20 antibody. 
     
     
         25 - 26 . (canceled)