治疗性单克隆抗体电荷异质性分析方法比较

目的：对4种常用的单抗电荷异质性分析方法进行比对研究。方法：以重组抗TNFα全人源单克隆抗体为例，分别利用离子交换色谱（IEC）、毛细管区带电泳（CZE）、毛细管等电聚焦电泳（CIEF）和成像毛细管等电聚焦电泳（iCIEF）4种方法对该抗体的电荷异质性进行分析；利用CIEF和iCIEF 2种方法对抗体主峰等电点（pI）进行分析。结果：4种方法能够对重组抗TNFα全人源单克隆抗体的电荷异构体进行有效的分离和定量，其中IEC和CZE 2种方法检测的电荷异构体峰面积百分比（酸性峰、主峰和碱性峰）较为一致，4种方法检测的碱性峰比例较一致，CIEF和iCIEF检测的主峰与酸性峰比例较其他方法偏高。CIEF和iCIEF检测抗体主峰等电点具有一定差异，其中，聚焦时间长短和pI Marker的选择是影响单抗pI值检测的主要因素。结论：通过抗体电荷异质性分析方法的比较研究可见，在单抗电荷异构体比例和主峰pI测定上4种方法具有一定的差异。对单抗的质控应结合产品特性和表征研究，合理选择电荷异质性分析方法。

Objective: To analyze charge heterogeneity of monoclonal antibody by four widely used analytical methods.Methods: Ion exchange chromatography(IEC),capillary zone ectrophoresis(CZE),capillary isoelectric focusing(CIEF)and imaged capillary isoelectric focusing(iCIEF)were used to analyze the charge heterogeneity of the recombinant anti-TNFα fully human monoclonal antibody. The electronic point(pI)value of the main peak was also analyzed by CIEF and iCIEF.Results: The four methods in this study can efficiently isolate and quantitate charge isomers of recombinant humanized anti-TNFα mAb. The main peak and acidic/basic peak area percentage analyzed by IEC and CZE were similar. The basic peak percentage analyzed by four methods was consistent. The main peak and acidic peak area percentage analyzed by CIEF and iCIEF were higher than the two other methods, and pI value of main peak was different between CIEF and iCIEF. The focusing time and pI marker affect the pI value calculation in this study.Conclusion: By comparing the methods of antibody charge heterogeneity analysis, we can see that there are some differences among the four analytical methods in the determination of antibody charge isomer ratio and pI peak. The appropriate method should be chosen for quality control based on the specificity and characterization of mAb.