title: Rapid Fc glycosylation analysis of Fc fusions with IdeS and liquid chromatography mass spectrometry.
abstract: We developed a rapid method to analyze Fc glycosylation of Fc fusion proteins, especially those with mutated Fc hinge regions. Fc fusion proteins were digested with IdeS, an IgG specific protease with exosites for substrate recognition and cleavage. The resultant fragments were directly analyzed through liquid chromatography mass spectrometry. The structures and relative quantities of Fc glycans were deduced from their masses and intensities. The separated substrate recognition and cleavage property of IdeS makes this method applicable to a broad range of Fc fusion proteins having either standard or non-canonical hinge regions.
authors.affiliation: GlycoFi; Biologics Discovery; Merck & Co. Inc.; Lebanon, NH USA.
authors.forename: Heather
authors.initials: H
authors.lastname: Lynaugh
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Bing
authors.initials: B
authors.lastname: Gong
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D010843:Pichia / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D007141:Immunoglobulin Fc Fragments; C442815:Mac-1-like protein, Streptococcus; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases
keywords: Fc fusion protein; Glycosylation analysis; IdeS protease; LC-MS