Development and validation of a hybrid immunoaffinity LC–MS/MS assay for quantitation of total antibody (TAb) from an antibody drug conjugate (ADC) PYX-201 in human plasma

•A hybrid immunoaffinity LC-MS/MS assay was fully validated to measure PYX–201 tAb concentrations in human K2EDTA plasma.•This assay validation followed 2018 U.S. FDA guidance and 2022 ICH M10 guidance for industry in bioanalytical method validation.•This is the first bioanalytical assay validation on tAb of the investigational ADC, PYX–201 in human plasma.•This bioanalytical LC-MS/MS assay successfully supported first-in-human (FIH) phase I clinical trial. A hybrid immunoaffinity LC-MS/MS assay was developed and validated for the quantitation of total antibody (TAb) from an antibody drug conjugate (ADC) PYX-201 in human plasma. PYX–201 was proteolyzed using trypsin, and a characteristic peptide fragment PYX-201 P1 with ten amino acids IPPTFGQGTK from the complementarity-determining regions (CDRs) was used as a surrogate for the quantitation of the TAb from PYX-201. Stable isotope labelled (SIL) peptide I(13C6, 15N)PPTFG(13C9, 15N)QGTK was used as the internal standard (IS). We performed chromatographic analysis using a Waters Acquity BEH Phenyl column (2.1 mm x 50 mm, 1.7 µm). Quantification of PYX-201 TAb was carried out on a Sciex triple quadrupole mass spectrometer API 6500 using multiple reaction monitoring (MRM) mode with positive electrospray ionization. To validate PYX–201 TAb, a concentration range of 0.0500 µg/mL to 20.0µg/mL was used, yielding a correlation coefficient (r) of ≥ 0.9947. For intra-assay measurements, the percent relative error (%RE) ranged from -23.2% to 1.0%, with a coefficient of variation (%CV) of ≤14.2%. In terms of inter–assay measurements, the %RE was between -10.5% and -5.7%, with a %CV of ≤12.7%. The average recovery of the analyte was determined to be 81.4%, while the average recovery of the internal standard (IS) was 97.2%. Furthermore, PYX–201 TAb demonstrated stability in human plasma and human whole blood under various tested conditions. This assay has been successfully applied to human sample analysis to support a clinical study.