重组双特异性抗体药物电荷异质性分析iCIEF方法新载体基质研究及验证

Research and validation of new carrier matrix of iCIEF method for charge heterogeneity analysis of recombinant bispecific antibody

目的:建立应用成像毛细管等电聚焦电泳(iCIEF)法测定重组双特异性抗体(bsAb)电荷异质性的新载体基质的方法。方法:首先,对2种bsAbs在传统的载体基质甲基纤维素(MC)/羟丙甲基纤维素(HPMC)与新的甘油基质中的电荷异构体分离效果进行对比;然后,对甘油基质方法进行进一步优化,以研究甘油浓度、两性电解质种类及其终浓度、不同pH范围两性电解质的组合方式等对电荷异构体分离效果的影响,获得基于甘油基质最佳的分析条件;最后,应用甘油基质的最优分离条件进行了iCIEF方法验证,包括精密度、准确度、线性范围及稳定性指示性验证等。结果:2种bsAbs在甘油基质中的电荷异构体的分离效果均优于HPMC载体基质,且方法的稳健性较好;方法验证结果显示:对于bsAb 1,酸区、主峰、碱区峰峰面积百分比的RSD均小于2.0%,精密度良好;在蛋白终浓度0.10~0.30 mg·mL^(-1)范围内,主峰平均峰面积与蛋白浓度回归曲线r大于0.99,线性良好;每个浓度点的回收率均在95%~105%范围内,准确度良好。对于bsAb 2,酸区、主峰峰面积百分比RSD均小于2.0%,碱区峰面积百分比小于20.0%,精密度良好。该方法能够指示出产品电荷异构体的含量变化情况,可用于产品的质控。结论:使用甘油作为新的载体基质代替传统的HPMC载体基质进行iCIEF分析,具有分离度较好,稳定性强,准确性高,成本低等优势,解决了结构较复杂的重组双特异性抗体类药物在传统MC/HPMC载体基质中无法实现电荷异构体良好分离的难题,尤其适用于复杂的双特异抗体类药物的产品质量评估及质量控制。

Objective:To develop a method with new carrier matrix for the detection of charge heterogeneity of recombinant bispecific antibody(bsAb)by imaging capillary isoelectric focusing electrophoresis(iCIEF).Methods:Firstly,the separation efficiency of charge heterogeneity of two bispecific antibodies products in the traditional carrier matrix methyl cellulose(MC)/hydroxypropyl methyl cellulose(HPMC)and glycerol matrix wascompared.Subsequently,the method of glycerol matrix was further optimized to study the effects of glycerol concentration,ampholyte brand and its final concentration,and the combination of ampholyte in different pH ranges on the separation effect of the two bsAbs charge isomers,so as to obtain the best analytical conditions based on glycerol matrix.Finally,the iCIEF method was verified by the optimal separation conditions of glycerol matrix,and the validation indexes included precision,accuracy,linearity range and stability indicator test.Results:The separation efficiency of charge isomers of the two bispecific antibodies in glycerol matrix was better than that in HPMC carrier matrix and the method had good robustness.The results of the method validation showed that for bsAb 1,the RSD of the peak area percentages of acid region,the main peak and the base region were all less than 2.0%,and the precision was good.In the final protein concentration range of 0.10-0.30 mg·mL^(-1),the r score of the linear regression cure of the main peak was greater than 0.99,and the linearity was good;the recovery rate of each concentration was in the range of 95%-105%,and the accuracy was good.For bsAb 2,the RSD of peak area percentages of acid region and main peak were less than 2.0%,and the peak area percentage of base region was less than 20.0%,the precision was good.This method could indicate the change of the content of charge variants of the products,and could be used for quality control of products.Conclusion:Using glycerol as a new carrier matrix instead of the traditional carrier matrix for iCIEF analysis has the advantages of good separation efficiency,strong stability,good accuracy and low cost.It solves the problem that the recombinant bispecific antibody drugs with more complex structure can not achieve good separation of charge heterogeneity in traditional MC/HPMC carrier matrix.It is suitable for the quality evaluation and quality control of complex bispecific antibody drugs.