Method for increasing N-glycosylation site occupancy on therapeutic glycoproteins produced in Pichia pastoris

摘要

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.