Versatile intact LC-MS method for evaluating the drug-antibody ratio and drug load distribution of antibody-drug conjugates in human plasma.

The average drug-antibody ratio (DAR) and drug load distribution (DLD) of an antibody-drug conjugate (ADC) can be altered by biotransformation after administration. In addition, drug loading affects the clearance and exposure of the ADC. Evaluating alterations in the average DAR and DLD of an ADC in vivo would provide valuable information to better understand of the pharmacokinetic (PK) profile of the ADC. Although the quantitation of antibodies/ADCs using LC-MS is often coupled with affinity capture methods, here, we aimed to develop a versatile intact LC-MS method for evaluating the average DAR and DLD of ADCs in human plasma. The development of the affinity purification process and method validation were performed using healthy human pooled plasma spiked with the model ADCs, commercially available trastuzumab emtansine (T-DM1) and brentuximab vedotin (B-MMAE), and the recombinant proteins HER2 and CD30 were used to capture T-DM1 and B-MMAE, respectively. As unique points of this study, initially, a two-step gradient was established for the sensitive detection of a small amount of ADC. The ADC elution conditions after affinity capture were also optimized considering its application for LC-MS analysis. Furthermore, a validation study of the intact LC-MS approach for analyzing the average DAR and DLD of ADCs in human plasma sample was proposed for the first time. Using the validation study, our analytical method was validated by verifying its performance characteristics, including sensitivity, intermediate precision, accuracy, carryover and autosampler stability. In addition, the feasibility of applying our method was demonstrated by a collaborative study with six laboratories. Finally, our method was shown to be versatile for evaluating the average DAR and DLD of ADCs in human plasma.