title: O-linked glycosylation analysis of recombinant human granulocyte colony-stimulating factor produced in glycoengineered Pichia pastoris by liquid chromatography and mass spectrometry.
abstract: 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 (rhG-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.
authors: GlycoFi, Biologics Discovery, Merck & Co., Inc., 16 Cavendish Court, Lebanon, NH 03766, USA. Electronic address: bing.gong@merck.com.
authors: Bing
authors: B
authors: Gong
authors: GlycoFi, Biologics Discovery, Merck & Co., Inc., 16 Cavendish Court, Lebanon, NH 03766, USA.
authors: Irina
authors: I
authors: Burnina
authors: GlycoFi, Biologics Discovery, Merck & Co., Inc., 16 Cavendish Court, Lebanon, NH 03766, USA.
authors: Heather
authors: H
authors: Lynaugh
authors: GlycoFi, Biologics Discovery, Merck & Co., Inc., 16 Cavendish Court, Lebanon, NH 03766, USA.
authors: Huijuan
authors: H
authors: Li
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D005818:Genetic Engineering; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008358:Mannose / Q000032:analysis*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D008358:Mannose
keywords: HPAEC-PAD; LC–MS; O-linked mannosylation; rhG-CSF