Quantification of total and conjugated antibody of a novel ADC in rat by two affinity-capture LC-MS/MS methods using...

GL-2401 is a novel antibody-drug conjugate (ADC) made by site-specific enzymatic conjugation of monomethyl auristatin E (MMAE) to trastuzumab at the N297 glycosylation site. We aimed to develop two reliable bioanalytical methods for quantifying both total antibody (TAb) and conjugated antibody of GL-2401 in rat serum for exploration of its pharmacokinetic (PK) profiles. In this study, we developed two affinity-capture LC-MS/MS methods, of which one for the conjugated antibody and the other for the TAb in rat serum. In both methods, the target analytes in rat serum were firstly enriched using a related biotinylated capture reagent immobilized on streptavidin magnetic beads, followed by washes and on-bead trypsin digestion. Thereafter, the characteristic peptide IYPTNGYTR, derived from the complementarity determining region 2 (CDR2) of trastuzumab, served as the single surrogate analyte for quantification of both conjugated antibody and TAb. The methods were subsequently validated, showing good accuracy and precision for quantifying the conjugated antibody and the TAb within the ranges of 0.50-100.00 μg/mL and 0.50-40.00 μg/mL, respectively. Finally, the validated methods were applied to a rat PK study. GL-2401 demonstrated higher serum and linker stability compared to conventionally conjugated ADCs, providing a basis for further application of GL-2401 in other animal species.