O-linked glycosylation analysis of recombinant human granulocyte colony-stimulating factor produced in glycoengineered Pichia pastoris by liquid chromatography and mass spectrometry

摘要

Glycosylation is a major biochemical attribute of therapeutic proteins and detailed analyses includ- ing the structures and sites of such modifications are often required for product quality control and assurance. Using liquid chromatography and tandem mass spectrometry techniques, we analyzed the O-linked glycosylation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) derived from glycoengineered Pichia pastoris with regard to its nature, structure, occupancy, and location. Pep- tide mappings using protease and chemical cleavages were performed to determine the specific O-linked glycosylation site used by Pichia-derived rhG-CSF. Our results demonstrated thatThr134, the equivalent O-linked glycosylation site found on endogenous human G-CSF, is the only site modified with a single mannose, allowing glycoengineered P. pastoris to be used as a viable production platform for therapeutic rhG-CSF.