title: Characterization of N-linked glycosylation on recombinant glycoproteins produced in Pichia pastoris using ESI-MS and MALDI-TOF.
abstract: 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.
authors.affiliation: GlycoFi Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Gong
authors.forename: Michael
authors.initials: M
authors.lastname: Cukan
authors.forename: Richard
authors.initials: R
authors.lastname: Fisher
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Tillman
authors.initials: T
authors.lastname: Gerngross
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins