College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China; Zhejiang Hisun Bioray Biopharmaceutical Co., Ltd., Taizhou, Zhejiang 318000, China.
Zhejiang Hisun Bioray Biopharmaceutical Co., Ltd., Taizhou, Zhejiang 318000, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jan 1;1162:122485. doi: 10.1016/j.jchromb.2020.122485. Epub 2020 Dec 8.
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.
电荷变异体是单克隆抗体常规生产中最常见的异质性来源。为了更深入地了解电荷异质性的结构基础及其对生物学功能的影响，从一家生物制药厂分离出英夫利昔单抗生物类似药 HS626，采用半制备阳离子交换色谱（CEX）获得酸性和碱性电荷变异体的馏分，并确定主要物质。然后再次通过 CEX 评估以确保纯度。通过一系列结构和物理化学特性分析，我们得出结论，酸性变异体是由片段、Met 氧化、Asn 脱酰胺、N 连接糖基化的唾液酸化和半乳糖基化水平升高以及高甘露糖水平降低引起的。碱性变异体主要由聚集体、片段和 Met 氧化引起。通过进一步分析抗原结合亲和力、细胞死亡抑制活性、ADCC 和 CDC 以及 FcRn、FcγRIIIa 和 C1q 亲和力，我们证明电荷异质性不会影响生物学功能。这项研究增强了对电荷变异体的理解，通常情况下，电荷变异体是有效成分，除非影响生物学功能，否则不应故意降低。
