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目的建立用于全人源抗人肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)单克隆抗体鉴别的毛细管等电聚焦(capillary isoelectric focusing,c IEF)方法,并进行验证。方法通过优化c IEF过程中各关键实验参数,如阴极稳定剂浓度、阳极稳定剂浓度、两性电解质浓度、3-10与8-10.5两性电解质比例、聚焦时间、聚焦电压,建立c IEF方法,并对方法的专属性、重复性、中间精密度进行验证。结果确定c IEF方法的实验参数为:阴极稳定剂浓度30 mmol/L,阳极稳定剂浓度3.2 mmol/L,3-10两性电解质在样品溶液中体积比为6.0%,聚焦电压为25 k V,聚焦时间为10 min。保护剂在样品出峰时间无紫外吸收峰,阳性对照阿达木单抗与样品出峰时间及峰形一致,阴性对照利妥昔单抗生物类似药、贝伐珠单抗、曲妥珠单抗生物类似药、西妥昔单抗与样品出峰时间及峰形均不一致,且对样品无干扰;样品制备重复性主峰等电点的相对标准差(relative standard deviation,RSD)为0.114%,中间精密度RSD为0.837%。结论建立的c IEF方法 p H梯度和精密度良好,专属性较强,适用于制品的批放行鉴别试验。
Objective To establish and verify the capillary isoelectric focusing (c IEF) method for the identification of all-human monoclonal antibodies against human tumor necrosis factor-α (TNF-α). Methods The c IEF method was established by optimizing the key experimental parameters in c IEF, such as concentration of cathode stabilizer, concentration of anode stabilizer, concentration of ampholytes, ratio of amphiphilic electrolytes 3-10 and 8-10.5, focusing time and focusing voltage The method of specificity, repeatability, intermediate precision verification. Results The experimental parameters of the cEF method were determined as follows: cathode stabilizer concentration of 30 mmol / L, anode stabilizer concentration of 3.2 mmol / L, volume ratio of 3-10 ampholytes in the sample solution of 6.0%, focusing voltage of 25 kV, Focus time is 10 min. Protective agent in the sample peak time without UV absorption peak, positive control adalimumab sample peak time and peak shape, the negative control rituximab bio-analogs, bevacizumab, trastuzumab The bio-analogs, cetuximab and the sample peak time and peak shape are inconsistent, and the sample did not interfere with the sample preparation of the repetitive peak isoelectric point of the relative standard deviation (RSD) was 0.114%, the middle The RSD of precision is 0.837%. Conclusion The established c IEF method has good pH gradient and precision, and has high specificity. It is suitable for batch identification of products.