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 MALDI-TOF and site specific N-glycan occupancy on digested glycoprotein using ESI-MS.
Gong
Bing
B
GlycoFi Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA.
Cukan
Michael
M
Fisher
Richard
R
Li
Huijuan
H
Stadheim
Terrance A
TA
Gerngross
Tillman
T
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*
D006023:Glycoproteins; D011994:Recombinant Proteins