期刊
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
卷 945, 期 -, 页码 135-140出版社
ELSEVIER
DOI: 10.1016/j.jchromb.2013.11.031
关键词
O-linked mannosylation; rhG-CSF; LC-MS; HPAEC-PAD
资金
- Strain Development, Purification, Analytical, High Through-put Screening and Fermentation groups at GlycoFi
Glycosylation is a major biochemical attribute of therapeutic proteins and detailed analyses including 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 (rhC-CSF) derived from glycoengineered Pichia pastoris with regard to its nature, structure, occupancy, and location. Peptide 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 that Thr134, 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. (C) 2013 Elsevier B.V. All rights reserved.
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