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