title: Investigation of the correlation between charge and glycosylation of IgG1 variants by liquid chromatography-mass spectrometry.
abstract: 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.
authors.affiliation: Livzon MabPharm, Zhuhai, Guangdong 519020, China.
authors.forename: Jia-Ming
authors.initials: JM
authors.lastname: Yang
authors.affiliation: Livzon MabPharm, Zhuhai, Guangdong 519020, China.
authors.forename: Junwen
authors.initials: J
authors.lastname: Ai
authors.affiliation: Livzon MabPharm, Zhuhai, Guangdong 519020, China.
authors.forename: Yuemei
authors.initials: Y
authors.lastname: Bao
authors.affiliation: Livzon MabPharm, Zhuhai, Guangdong 519020, China.
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: Livzon MabPharm, Zhuhai, Guangdong 519020, China.
authors.forename: Yumin
authors.initials: Y
authors.lastname: Qin
authors.affiliation: Livzon MabPharm, Zhuhai, Guangdong 519020, China.
authors.forename: Yi-Wu
authors.initials: YW
authors.lastname: Xie
authors.affiliation: Livzon MabPharm, Zhuhai, Guangdong 519020, China.
authors.forename: Desheng
authors.initials: D
authors.lastname: Tao
authors.affiliation: Livzon MabPharm, Zhuhai, Guangdong 519020, China.
authors.forename: Daotian
authors.initials: D
authors.lastname: Fu
authors.affiliation: Livzon MabPharm, Zhuhai, Guangdong 519020, China. Electronic address: pengyucai@livzon.com.cn.
authors.forename: Yucai
authors.initials: Y
authors.lastname: Peng
mesh_terms: D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000032:analysis; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011110:Polymorphism, Genetic; D017434:Protein Structure, Tertiary; D011761:Pyrrolidonecarboxylic Acid / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D008239:Lysine; D011761:Pyrrolidonecarboxylic Acid
keywords: Charge heterogeneity; Glycosylation; Monoclonal antibody; UPLC–MS