Bioanalysis of an antibody drug conjugate (ADC) PYX-201 in human plasma using a hybrid immunoaffinity LC–MS/MS approach

•First LC-MS/MS assay validation on the investigational ADC, PYX–201 was reported.•A hybrid immunoaffinity LC–MS/MS assay was validated for PYX-201 in human plasma.•2018 U.S. FDA guidance and 2022 U.S. FDA ICH M10 guidance for BMV were followed.•PYX-201 was proven stable after various stabilities were tested.•This bioanalytical LC-MS/MS assay successfully supported a clinical trial. PYX-201 is an anti-extra domain B splice variant of fibronectin (EDB + FN) antibody drug conjugate (ADC) composed of a fully human IgG1 antibody, a cleavable mcValCitPABC linker, and four Auristatin 0101 (Aur0101, PF-06380101) payload molecules. To better understand the pharmacokinetic (PK) profile of PYX-201 after it is administered to cancer patients, the development of a reliable bioanalytical assay to accurately and precisely quantitate PYX-201 in human plasma is required. In this manuscript, we present a hybrid immunoaffinity LC-MS/MS assay used to successfully analyze PYX-201 in human plasma. PYX-201 was enriched by MABSelect beads coated with protein A in human plasma samples. The bound proteins were subjected to “on-bead” proteolysis with papain to release the payload Aur0101. The stable isotope labelled internal standard (SIL-IS) Aur0101-d8 was added and the released Aur0101 was quantified as a surrogate for the total ADC concentration. The separation was performed on a UPLC C18 column coupled with tandem mass spectrometry. The LC-MS/MS assay was validated over the range 0.0250 to 25.0 µg/mL with excellent accuracy and precision. The overall accuracy (%RE) was between −3.8% and −0.1% and the inter-assay precision (%CV) was