靶向BCMA/CD3的双特异性抗体药物的质量控制研究

Study on quality control of anti⁃BCMA/CD3 bispecific monoclonal antibody drugs

目的 研究并建立靶向B细胞成熟抗原(BCMA)和白细胞分化抗原3(CD3)的双特异性抗体(双抗)药物的关键质量属性(CQA)质量控制方法。方法 从特异性鉴别、纯度、电荷异质性、产品相关杂质、翻译后修饰和生物学活性几个方面对靶向BCMA/CD3的双抗药物开展质量控制研究。采用基于赖氨酰肽链内切酶(Lys-C)酶切和反相高效液相色谱(RP-HPLC)技术的肽图法对靶向BCMA/CD3的双抗药物进行特异性鉴别确认;采用分子排阻色谱法(SEC)和还原/非还原十二烷基硫酸钠毛细管电泳法(CE-SDS)进行纯度控制;采用离子交换色谱法(IEC)测定电荷异质性和产品相关杂质;采用反相超高效液相色谱(RPUHPLC)和在线质谱联用的多属性监测方法(MAM)监测靶向BCMA/CD3双抗中的翻译后修饰;采用T细胞激活和报告基因法测定该双抗的生物学活性。结果 肽图法检测可有效识别双抗中的关键肽段,起到特异性鉴别确认的作用;SEC和CE-SDS法可有效区分双抗中的主成分、聚体和片段,对有效成分的纯度控制发挥关键作用;IEC法可有效区分酸碱变异体、目标双抗及亲代残留同源二聚体,有利于产品相关杂质的控制;MAM法可检测到天冬氨酸异构化和天冬酰胺脱酰胺化等翻译后修饰的存在,可作为CQA控制的有力补充;T细胞激活结合报告基因法可有效评估该双抗的生物学活性。结论 针对靶向BCMA/CD3的双抗药物的理化和生物学特性,研究并建立了针对其CQA的质量控制方法,从质量可控的角度确保该类双抗产品的临床安全性和有效性。

Objective To establish a critical quality attribute(CQA)quality control(QC)method for bispecific antibody(bsAb)targeting B cell maturation antigen(BCMA)and cluster of differentiation 3(CD3).Method QC of anti-BCMA/CD3 bsAb were inves-tigated in terms of identification,purity,charge heterogeneity,product related impurities,post-translational modifications,and biological activity.The peptide map method using Lys-C enzyme digestion and reverse phase high performance liquid chromatography(RP-HPLC)was used to specifically identify the bsAb.Purity control was performed using size exclusion chromatography(SEC)and reduced/non-reduced sodium dodecyl sulfate capillary electrophoresis(CE-SDS).Ion exchange chromatography(IEC)was used to measure charge heterogeneity and product related impurities.Multi-attribute method(MAM)composing of reverse phase ultra high performance liquid chromatography(RP-UHPLC)and online mass spectrometry was used to monitor post-translational modifications(PTMs).The biological activity was determined using T cell activation and reporter gene assay(RGA).Results The key peptide segments were effectively identified by the peptide map method which benefits for specific identification.The main components,aggregates and frag-ments were effectively distinguished by both SEC and CE-SDS methods to control the purity of the active ingredients.The acid-base variants,target bsAb,and parental residual homodimers were also effectively distinguished by IEC method,which is beneficial for controlling product related impurities.The PTMs,such as aspartic acid isomerization and asparagine deamidation,were detected by MAM method to serve as a powerful supplement to CQA control.The biological activity was evaluated by T cell activation with RGA method.Conclusion Based on the physicochemical and biological characteristics of anti-BCMA/CD3 bsAb,a QC method for their CQAs has been studied and established to ensure the clinical safety and effectiveness of bsAb products from a quality controllable per-spective.