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 was compared. 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.
BU Ling-li, ZHANG Rui-ning, HAN Guo-hua, LIU Zhen-dong, WANG Qing-min
. Research and validation of new carrier matrix of iCIEF method for charge heterogeneity analysis of recombinant bispecific antibody[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(11)
: 1927
-1935
.
DOI: 10.16155/j.0254-1793.2022.11.08
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