帕博利珠单抗电荷异构体阳离子交换高效液相色谱检测方法的建立及验证

Development and verification of cation exchange high performance liquid chromatography for detection of charge variants of pembrolizumab

目的建立帕博利珠单抗电荷异构体阳离子交换高效液相色谱(cation exchange high performance liquid chromatography,CEX-HPLC)检测方法,并进行验证。方法采用MabPac SCX-10色谱柱将帕博利珠单抗与交换柱基质结合,通过逐渐增加流动相盐浓度洗脱带不同电荷的异构体,并验证该方法的专属性、精密性、线性范围、准确性和耐用性。采用建立的方法对3批帕博利珠单抗成品的电荷异构体进行检测。结果帕博利珠单抗最后1个酸性异构体峰和第1个碱性异构体峰与主峰的分离度分别为1.28和1.42。流动相A和制剂缓冲液在样品出峰处无明显干扰峰;精密性验证RSD均<2.0%;标准品总峰面积、主峰面积、酸性异构体峰面积和碱性异构体峰面积均与其理论浓度呈良好的线性关系,R^(2)均为1.00;3个浓度标准品的总峰面积和主峰面积的回收率为96.81%~106.07%;流动相pH在6.30±0.10范围内,标准品总峰面积和主峰面积百分比RSD分别为1.5%和1.9%;色谱柱温度在(35±4)℃范围内,标准品总峰面积和主峰面积百分比RSD分别为0.4%和0.3%。3批帕博利珠单抗成品主峰保留时间RSD为0。结论建立的CEX-HPLC法能够有效分离帕博利珠单抗的酸性异构体、主峰及碱性异构体,具有良好的专属性、精密性及准确性,可用于帕博利珠单抗的后续研发、扩大生产的工艺检定及稳定性研究。

Objective To develop and verify a cation exchange high performance liquid chromatography(CEX-HPLC)method for the detection of charge variants of pembrolizumab.Methods Pembrolizumab was bound to the exchange column matrix by using MabPac SCX-10 column,and the variants with different charges were eluted by gradually increasing the salt concentration of the mobile phase.The specificity,precision,linear range,accuracy and durability of the method were verified,and the charge variants of three batches of pembrolizumab finished products were detected by using the developed method.Results The resolution of the last acidic isomer peak and the first basic isomer peak of pembrolizumab from the main peak were 1.28 and 1.42,respectively.The mobile phase A and preparation buffer had no obvious interference peaks at the peak of the sample;The RSD values of the precision verification were all less than 2.0%;The total peak area,main peak area,acidic isomer peak area and basic isomer peak area of the standard all exhibited good linear relationship with the theoretical dilution concentration with each R^(2) of 1.00;The recovery rates of the total peak area and main peak area of the standard at three concentrations were between 96.81%and 106.07%;When pH value of the mobile phase was within the range of 6.30±0.10,the RSD values of the total peak area and main peak area percentage of the standard were1.5%and 1.9%,and when the column temperature was within the range of(35±4)℃,the RSD values of the total peak area and main peak area percentage of the standard were 0.4%and 0.3%,respectively.The RSD value of the main peak retention time of the three batches of finished products was 0.Conclusion The developed CEX-HPLC method can effectively separate the acidic isomers,main peaks and basic isomers of pembrolizumab with good specificity,precision and accuracy,which can be used for the follow-up research and development of pembrolizumab,the process verification of expanding production and the stability research.