Development and validation of an efficient and sensitive LC-MS/MS assay for simultaneous determination of the high frequency used antibody drug conjugate payload MMAE and anti-vascular tyrosine kinase inhibitor lenvatinib in human plasma.

Antibody drug conjugates (ADCs)-based combination therapies are increasingly studied in clinics. Monomethyl auristatin E (MMAE) is the most frequently used payload in approved ADCs and lenvatinib is a classic anti-vascular tyrosine kinase inhibitor. This study aimed to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for simultaneous determining these two analytes in human plasma. Protein precipitation by methanol was used in sample preparation with deuterated agents as internal standards. A Kinetex C18 column (3.0 × 50 mm, 2.6 μm) was used with gradient elution (0.5 mL/min, 40 °C). Positive electrospray ionization in multiple reaction monitoring (MRM) mode was used for detection. Each run needs 50 μL sample and 3 min. The developed assay was linear over 0.20-100 ng/mL for MMAE and over 1.00-500 ng/mL for lenvatinib, which covered reported pharmacokinetic (PK) ranges for both agents. This assay was validated according to ICH M10 and was qualified in terms of selectivity, linearity, accuracy (-6.7 % to 4.5 %), precision (1.5-7.4 %), matrix effects, dilution test, carry-over and stability in human plasma. The in vitro studies suggested no significant in-process release for conjugated MMAE from two ADCs with protease-cleavable maleimidocaproylvaline-citrulline-p-aminobenzyloxycarbonyl linker. Clinical samples were efficiently analyzed and values fell in reported PK ranges. Overall, the developed method is robust and efficient and will potentially support clinical PK studies or therapeutic drug monitoring (TDM) for MMAE-containing ADCs and lenvatinib alone or in combinations. This method also serves as a starting point to build cassette assay by including more tyrosine kinase inhibitors.