目的:探索和优化单克隆抗体电荷异构体分析的酶切预处理和高效液相色谱检测方法。方法:采用和优化基于Thermo ProPacTM WCX-10色谱柱的离子交换色谱检测方法,以含10 mmol·L-1磷酸盐和1 mol·L-1氯化钠缓冲液(pH 6.3)为流动相进行梯度洗脱,流速0.5 mL·min-1,检测信号为波长280 nm吸收度,采用免疫球蛋白G降解酶(IdeS)切前后的电荷异构体检测,并对方法的专属性、线性、重复性、精密度等指标进行考察。结果:基于阳离子交换色谱(CEX)方法和酶切处理,以几种商品化抗体为例,展示该方法的应用,对完整的IgGs与IdeS酶切产生的F(ab’)2和Fc区域进行比较分析提供更详细和更特异的电荷异构体信息。通过方法学验证,空白溶剂无干扰峰出现,进样量在10~80 μg范围内,进样量与峰面积的线性关系良好(R2=1.000);6次进样测得的峰面积RSD为0.52%;日间精密度3.3%;耐用性良好;在24 h内样本基本稳定,测得的峰面积RSD为3.5%;3批样品检测结果的峰面积RSD为2.5%。结论:该方法可用于全流程的抗体药物研发,包括早期阶段的分子筛选,中期产业化的工艺开发,直至产品上市质量标准和稳定性研究。
Objective: To explore and optimize enzyme digestion pretreatment and high-performance liquid chromatography for charge isomer analysis of monoclonal antibody. Methods: An ion-exchange chromatography method based on Thermo ProPacTM WCX-10 column was adopted and optimized. The phosphate buffer solution (pH 6.3) containing 10 mmol·L-1 phosphate and 1 mol·L-1 sodium chloride was used as mobile phase for gradient elution. The flow rate was 0.5 mL·min-1,and the detection wavelength for protein adsorbance was 280 nm. Charge isomers were detected before and after IdeS cutting, and the specificity, linearity, repeatability and precision of the method were investigated. Results: The method was applied to several commercial antibody products as examples of demonstrating the method application based on cation exchange chromatography (CEX) and enzyme digestion. Results of comparative analyses of the intact IgG versus F(ab’)2 and Fc sub-domains generated by IdeS digestion provide more specific information about the location of the charge differences. There was no interference peak in the blank solvent;the linear relationship between the sample volume and the absorption peak was good within the range of 10-80 μg, with R2 being 1.0;the RSD of the peak area for six injections was 0.52%;The daytime precision was 3.3%;the assay has good durability;the sample was stable within 24 h, and the RSD of the peak was 3.5%;the RSD of the peak area for the three batches of samples was 2.5%. Conclusion: The method could be applied to the whole process of antibody drug development, including molecular screening in the early stage, industrial process development in the middle stage, and product quality standards and stability studies.
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