Characterization of N-Linked Glycosylation on Recombinant Glycoproteins Produced in Pichia pastoris Using ESI-MS and MALDI-TOF

The production of recombinant therapeutic glycoproteins is an active area of research and drug development. Typically, improvements in therapeutic glycoprotein efficacy have focused on engineering additional N-glycosylation sites into the primary amino acid sequence or attempting to control a particular glycoform profile on a protein through process improvements. Recently, a number of alternative expression systems have appeared that are challenging the dominance of mammalian cell culture. Our laboratory has focused on the re-engineering of the secretory pathway in the yeast Pichia pastoris to perform glycosylation reactions that mimic processing of N -glycans in humans. We have demonstrated that human antibodies with specific human N -glycan structures can be produced in glycoengineered lines of Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. In this chapter we provide detailed protocols for the analysis of glycosylation on intact glycoproteins by MALDITOF and site specific N -glycan occupancy on digested glycoprotein using ESI-MS.