Isolation and characterization of charge variants of infliximab biosimilar HS626

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1162; p. 122485
• Main Authors: Yuan, Jun-Jie, Gao, Dong, Hu, Feng, Shi, Yang, Wu, Zhen-Hua, Hu, Chuan-Qin, Huang, Xiao-Dong, Fang, Wei-Jie, Zhang, Hai-Tao, Wang, Hai-Bin
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2021
• Subjects: Amino Acid Sequence; Animals; Biological function; Biosimilar Pharmaceuticals - analysis; Biosimilar Pharmaceuticals - chemistry; Biosimilar Pharmaceuticals - isolation & purification; Cell Line; Characterization; Charge heterogeneity; Charge variant; CHO Cells; Chromatography, Ion Exchange - methods; Cricetinae; Cricetulus; Glycosylation; Infliximab; Infliximab - analysis; Infliximab - chemistry; Infliximab - isolation & purification; Mice; Monoclonal antibody; Characterization; Biological function; Charge heterogeneity; Monoclonal antibody; Infliximab; Charge variant
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2020.122485

•Charge variants of infliximab biosimilar HS626 were isolated by CEX.•Acidic variants resulted from fragments, deamidation, sialylation, and galactose.•Basic variants resulted from aggregates, fragments, and Met oxidation.•The detected biological functions were not affected by charge heterogeneity. Charge variants are the most commonly observed sources of heterogeneity in the routine manufacturing of monoclonal antibodies. To gain further insight into the structural foundation of charge heterogeneity and its influence on biological functions, an infliximab biosimilar HS626 from a biopharmaceutical facility was isolated by semipreparative cation exchange chromatography (CEX) to obtain fractions of acidic and basic charge variants and determine the main species. It was assessed again by CEX to ensure purities. Through a series of structural and physicochemical characterizations, we concluded that the acidic variants were caused by fragments, Met oxidation, Asn deamidation, higher levels of sialylation and galactosylation of N-linked glycans, and less high mannose. The basic variants resulted mainly from aggregates, fragments, and Met oxidation. Through further analysis of antigen binding affinity, cell death inhibitory activity, ADCC, and CDC, as well as FcRn, FcγRIIIa, and C1q affinity, we demonstrated that the charge heterogeneity did not affect biological functions. This research enhances the understanding of charge variants, which are usually effective components that should not be intentionally reduced unless biological functions are affected.