Determination of drug-to-antibody ratio of antibody–drug conjugate in biological samples using microflow-liquid chromatography/high-resolution mass spectrometry: Supplementary materials

Background: Antibody–drug conjugates (ADCs) are a promising modality for cancer treatment; however,
considering their complicated nature, analytical complexity in understanding their pharmacokinetics and
pharmacodynamics in the body presents a significant challenge. Results: Vorsetuzumabmaleimidocaproyl
valine-citrulline p-aminobenzyloxycarbonyl monomethyl auristatin E was used to develop pretreatment
and analytical workflows suitable for ADCs. Monomethyl auristatin E release and drug-to-antibody ratio
retention were consistent in mouse plasma but inconsistent in monkey and human plasma. Further,
metabolites were species-specific. Microflow-liquid chromatography/high-resolution mass spectrometry
(LC–HRMS) resulted in a 4–7-fold improvement in detection sensitivity compared with conventional flow
LC–HRMS. Conclusion: Microflow-LC–HRMS can be a useful tool in understanding the complex properties
of ADCs in the body from a drug metabolism and pharmacokinetics point of view.