阿达木单抗理化特性的关键质量属性分析

Critical Quality Attributes Analysis of Adalimumab in Physicochemical Properties

阿达木单抗是作用于肿瘤坏死因子α(TNF-α)的全人源单克隆抗体药物,由1 330个氨基酸组成,相对分子质量约1.48×105。本研究通过分子排阻色谱(SEC-HPLC)、还原和非还原毛细管电泳(CE-SDS)测定了7批阿达木单抗注射液的高分子量物质(HMWS)、单体及低分子量物质(LMWS);采用毛细管电泳法和UPLC-Q-TOF MS法分析其N-糖;应用阴离子色谱法和UPLC法测定其唾液酸化水平;采用成像毛细管等电聚焦电泳和阳离子交换色谱法分析了其中的电荷异质体。为进一步研究电荷异质体来源,采用UPLC-Q-TOF MS肽图法分析阿达木单抗发生的翻译后修饰。单抗药物作为大分子生物药物,分子结构复杂,很难用单一方法进行质量分析和控制。本试验利用不同的质量分析方法,从HMWS、LMWS、N-糖基化、唾液酸、电荷异质体5个方面有针对性地检测了阿达木单抗注射液的关键质量属性,可为阿达木单抗及其制剂的质量控制提供参考。

Adalimumab is a human monoclonal antibody against TNF-alpha consisting of 1 330 amino acids and has a molecular weight of approximately 1.48×10^5. In this study, the important physicochemical properties of seven batches of adalimumab injections were investigated by combination of various specific analytical methods, including SEC-HPLC for the quantitation of high molecular weight substances （HMWS）, reduced and non-reduced CE-SDS for the determination of low molecular weight substances （LMWS）, capillary electrophoregram and UPLC-Q-TOF MS for the analysis of N-glycans, anion chromatography and UPLC for the assay of sialic acid, as well as capillary isoelectric focusing with whole column imaging detection and cation exchange chromatography for the determination of charge varients. Furthermore, a UPLC-Q-TOF MS peptide mapping method was applied for the determination of post-translational modifications occurred in adalimumab, to investigate the source of charge heterogeneity. As a bio- macromolecule drug, monoclonal antibodies have complex structures and it is a hard work to complete its quality analysis and control by single method. Here from HMWS, LMWS, N-glycosylation, sialic acid and charge varients, we applied various anlytical methods for the specific determination of five critical quality attributes of adalimumab. It would provide some reference for the quality control of adalimumab and its preparations.