title: Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry.
abstract: CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC-MS) is a versatile technique for characterizing N- and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N- and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.
authors.affiliation: a International Joint Cancer Institute; Second Military Medical University ; Shanghai , China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhu
authors.forename: Qingcheng
authors.initials: Q
authors.lastname: Guo
authors.forename: Huaizu
authors.initials: H
authors.lastname: Guo
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: Yingxin
authors.initials: Y
authors.lastname: Zheng
authors.forename: Peiming
authors.initials: P
authors.lastname: Gu
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.forename: Sheng
authors.initials: S
authors.lastname: Hou
authors.forename: Yajun
authors.initials: Y
authors.lastname: Guo
mesh_terms: D000069594:Abatacept; D000595:Amino Acid Sequence; D059451:Biosimilar Pharmaceuticals / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D018796:Immunoconjugates / Q000032:analysis* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis
chemical_list: D059451:Biosimilar Pharmaceuticals; D006020:Glycopeptides; D018796:Immunoconjugates; D011134:Polysaccharides; D000069594:Abatacept
keywords: 2-AB, 2-aminobenzamide; CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; CTLA4-Ig fusion protein; DTT, dithiothreitol; EMA, European Medicines Agency; ESI, electrospray ionization; FA, formic acid; FDA, Food and Drug Administration; GFP, [Giu1]-Fibrinopeptide B; IAM, Iodoacetamide; LC, Liquid chromatography; MS, mass spectrometry; PNGase F, peptide N-glycosidase; PTMs, post-translational modifications; Q-Tof quadrupole-time of flight; RA, Rheumatoid arthritis; TIC, Total Ion Chromatography; Tof, Time of flight; UPLC, Ultra-performance liquid chromatography; characterization; glycan; glycosylation modification; intact protein; mass spectrometry; peptide mapping; similarity