Investigation of the correlation between charge and glycosylation of IgG1 variants by liquid chromatography–mass spectrometry

• Published in: Analytical biochemistry Vol. 448; pp. 82 - 91
• Main Authors: Yang, Jia-Ming, Ai, Junwen, Bao, Yuemei, Yuan, Zhijun, Qin, Yumin, Xie, Yi-Wu, Tao, Desheng, Fu, Daotian, Peng, Yucai
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2014
• Subjects: Animals; Carbohydrate Sequence; Charge heterogeneity; CHO Cells; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cricetinae; Cricetulus; Glycosylation; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - metabolism; Immunoglobulin G - analysis; Immunoglobulin G - genetics; Immunoglobulin G - metabolism; Lysine - analysis; Molecular Sequence Data; Monoclonal antibody; Peptide Mapping; Polymorphism, Genetic; Protein Structure, Tertiary; Pyrrolidonecarboxylic Acid - analysis; Recombinant Proteins - analysis; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Tandem Mass Spectrometry; UPLC–MS; Glycosylation; Monoclonal antibody; Charge heterogeneity; UPLC–MS
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2013.11.020

A recombinant IgG1 monoclonal antibody (mAb) showed multiple charge variants in a cation exchange chromatography profile. To better understand the correlation between charge heterogeneity and glycosylation, a rapid reversed phase ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method with integrated mass analysis has been developed for simultaneous determination of N-terminal pyroglutamate, C-terminal lysine truncation, and Fc glycosylation. The results show that various degrees and/or types of N-terminal pyroglutamate formation and C-terminal lysine (Lys) cleavage account for the majority of charge heterogeneity; and the charge variants showed Fc glycosylation patterns in relation to their terminal modifications. The amount of G1F decreased in the basic variants, whereas Man5 and G0F-GN increased. The complement-dependent cytotoxicity (CDC) activity of purified charge variants also suggested the potential impact of the charge differences on the glycosylation profile.