紫外可见光谱法测定抗体偶联药物药物-抗体偶联比的方法建立和验证

Development and validation of the determination of drug-antibody ratio for an antibody-drug conjugate by UV spectroscopy

目的 建立基于紫外可见光谱检测抗体偶联药物(ADC)药物-抗体偶联比(DAR)的检测方法并进行验证。方法 使用盐酸胍作为变性剂,修正基于氨基酸组成推算的抗体在280 nm处消光系数。利用比尔-朗伯定律测定抗体和小分子药物在252nm处的消光系数及小分子药物在280nm处的消光系数。确定基于紫外可见光谱检测ADC的DAR公式参数和检测方法。根据《中华人民共和国药典》(2020)指导原则,对方法进行验证。结果 抗体在252nm和280nm处的摩尔消光系数分别为91258和228913L/(mol·cm);tubulysin衍生物小分子药物在252和280nm处的摩尔消光系数分别为12798和3186 L/(mol·cm)。DAR=(A280-ADC×91258–A252-ADC×228913)/(A252-ADC×3186–A280-ADC×12798)。方法专属性符合要求。模拟DAR值为1.881~15.045的ADC药物进行准确性验证,回收率为79.38%~99.13%。重复性相对标准偏差(RSD%)为1.93%,中间精密度RSD%为2.36%。以252 nm处吸光值与280 nm处吸光值的比值为纵坐标,以模拟DAR值为横坐标进行线性回归的方程为y=0.0408x+0.414,相关系数r2为0.9954。根据DAR测定公式推算该方法的检测范围为0.60~82.56。不同检测波长[(280±1)或(252±1)nm]、不同放置时间(0~60 min)、不同批次的比色皿条件下,DAR值检测结果的RSD%均<5.0%。结论 基于紫外可见光谱法进行抗体偶联药物DAR测定的方法简单、可操作性强,专属性、准确度、精密度、耐用性满足检测需求,该方法可以用于抗体偶联药物的质量分析和质量控制。

Objective To develop and validate the determination of drug-antibody ratio(DAR)for an antibody-drug conjugate(ADC)by ultraviolet/visible(UV)spectroscopy.Methods Guanidine hydrochloride was used as a denaturant to modify the inferred extinction coefficient of an antibody at 280 nm based on amino acid numbers.The extinction coefficients of the antibody and small molecule drugs at 252 nm and small molecule drugs at 280 nm were determined according to the Beer-Lambert law.The parameters of a formula for determination of DAR of an ADC by UV spectroscopy were defined.The method was validated according to the Pharmacopoeia of the People's Republic of China(ChP,2020).Results The molar extinction coefficients of the IgG1 antibody at 252 nm and 280 nm were 91258 L/(mol·cm)and 228913 L/(mol·cm),respectively.The molar extinction coefficients of tubulysin small molecule drug at 252 nm and 280 nm were 12798 L/(mol·cm)and 3186 L/(mol·cm),respectively.The DAR=(A280-ADC×91258–A252-ADC×228913)/(A252-ADC×3186–A280-ADC×12798).The specificity of the method met the requirements.The accuracy was validated by different analogs of the payloads and different DAR values(1.881-15.045)of the ADC drugs,and the recovery was 79.38%-99.13%.The relative standard deviation(RSD%)of repeatability of the same analysts was 1.93%and the intermediate precision RSD%among different analysts was 2.36%.Linear regression equation with the ratio of the absorbance value at 252 nm to that at 280 nm as the ordinate and the simulated DAR value as the abscissa was y=0.0408x+0.414,and the correlation coefficient r2 was 0.9954.Detection range of the method was 0.60-82.56 according to the defined DAR formula.Under the conditions of different detection wavelengths(280±1)nm or(252±1)nm,different solution storage times(0-60 min),and different batches of cuvettes,the RSD%of measured DAR numbers was all less than 5.0%.Conclusion The method for the determination of DAR of an ADC by UV spectroscopy is simple and robust.The specificity,accuracy,precision and robustness of the method met the requirements of accurate determination and it can be applied for the quality characterization and quality control of ADCs.