双波长分子排阻色谱法测定半胱氨酸偶联一甲基澳瑞他汀E抗体药物偶联物的药物抗体偶联比

目的：建立双波长分子排阻色谱（SEC）测定抗体药物偶联物（ADC）的药物抗体偶联比（DAR）的方法。方法：采用凝胶色谱柱，柱温25℃，以0.1 mol·L^-1磷酸盐-0.1 mol·L^-1氯化钠缓冲液为流动相对100 μg样品进行等度洗脱，流速1.0 mL·min^-1，在248 nm及280 nm双波长下分别对ADC的小分子药物和抗体进行检测；采用紫外分光光度计在248 nm及280 nm处进行吸收度测量。结果：双波长SEC可准确地对ADC的DAR进行测定，测得的DAR为4.17，RSD为0.24%；紫外分光光度（UV）法测得的DAR为3.97，RSD为2.8%。结论：双波长SEC检测可用于ADC样品的DAR测定，同时可对样品的大小异构体进行分析。SEC及UV 2种方法测得的DAR结果相似，都可用于ADC的药物抗体偶联比分析。

Objective:To establish a dual wavelength size exclusion chromatography (SEC)method for drug antibody ratio (DAR)determination in antibody drug conjugate (ADC).Methods:100 μg ADC sample was analyzed by size exclusion chromatography with dual wavelength,using gel column and 0.1 mol·L^-1 phosphate -0.1 mol·L^-1 NaCl solution as the mobile phase.The 248 nm and 280 nm wavelengths were used to detect the low molecule drug and ADC,respectively;Optical density at 248 nm and 280 nm was measured with UV spectrophotometry.Results:The dual wavelength SEC method showed good precision in DAR determination,the DAR value was 4.17 and RSD was 0.24%.The DAR was 3.97 and RSD was 2.8% by UV spectrophotometry analysis.Conclusion:The dual wavelength SEC method is suitable for DAR determination in ADC samples and the size heterogeneity can be detected at the same time.The results of DAR measured by SEC and UV were similar to those of the two methods,and the two methods can both be applied to the analysis of drug antibody ratio of ADC.

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