Rapid quantification of a cleavable antibody-conjugated drug by liquid chromatography/tandem mass spectrometry with microwave-assisted enzymatic cleavage

[Display omitted] •Generic immunocapture for human or humanized ADCs.•96-well based, high-throughput sample preparation.•Microwave-assisted, enzymatic cleavage of antibody-conjugated drug.•The reagents are commercially available.•This method could be easily adapted for all human or humanized ADCs with the same linker but different payloads. Antibody–drug conjugates (ADCs) play an increasingly important role for targeted cancer treatment. One class of ADCs has attracted particular interest in drug development. These ADCs employ a cleavable chemistry linkage for drugs and utilize the reduced interchain disulfide cysteine residues for conjugation. In this work, a novel bioanalytical method for the quantification of a cleavable antibody-conjugated drug in plasma was developed, qualified, and implemented. This novel method significantly improves throughput by combining a microwave-assisted, enzymatic cleavage of conjugated drugs from ADCs with a 96-well based sample preparation procedure to immunocapture ADCs in plasma. The released drug is subsequently quantified using a LC/MS/MS method. Our results represent a high-throughput, generic, and sensitive quantification method for antibody-conjugated microtubule inhibitors (such as MMAE) for preclinical PK/PD studies. The linear range of the standard curve for antibody conjugated drug (MMAE) was from 2.01 to 2010ng/mL with an excellent linearity (r2>0.997). The intra-run precision was below 8.14% and accuracy was from −7.71% to −1.08%. No matrix effect or carryover was observed for this method. This method was successfully used to measure the level of conjugated drug in a preclinical PK/PD study in mice.