Summary
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 MALDI-TOF and site specific N-glycan occupancy on digested glycoprotein using ESI-MS.
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Gong, B., Cukan, M., Fisher, R., Li, H., Stadheim, T.A., Gerngross, T. (2009). Characterization of N-Linked Glycosylation on Recombinant Glycoproteins Produced in Pichia pastoris Using ESI-MS and MALDI-TOF. In: Packer, N.H., Karlsson, N.G. (eds) Glycomics. Methods in Molecular Biology™, vol 534. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-022-5_16
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DOI: https://doi.org/10.1007/978-1-59745-022-5_16
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