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 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 × 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 20.0 µg/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.