Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry.
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.
['Zhu|Lei|L|', 'Guo|Qingcheng|Q|', 'Guo|Huaizu|H|', 'Liu|Tao|T|', 'Zheng|Yingxin|Y|', 'Gu|Peiming|P|', 'Chen|Xi|X|', 'Wang|Hao|H|', 'Hou|Sheng|S|', 'Guo|Yajun|Y|']
D000069594:Abatacept D000595:Amino Acid Sequence D059451:Biosimilar Pharmaceuticals D002853:Chromatography, Liquid D006020:Glycopeptides D006031:Glycosylation D006801:Humans D018796:Immunoconjugates D013058:Mass Spectrometry D008969:Molecular Sequence Data D011134:Polysaccharides
characterization glycosylation modification CTLA4-Ig fusion protein intact protein peptide mapping glycan similarity 2-AB, 2-aminobenzamide CTLA-4, cytotoxic T-lymphocyte-associated antigen 4 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 mass spectrometry