Simultaneous LC-MS/MS Quantification Method for Cassette-Dosing Pharmacokinetics Study of Monoclonal Antibodies Using...

We developed a novel cassette-dosing pharmacokinetic (PK) evaluation method that combines antibody engineering with multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS). The antibody was engineered by introducing alanine mutations or insertions into the GPSVFPLAPSSK sequence on the CH1 domain of IgG1, thereby enabling clear differentiation and simultaneous measurement of multiple antibodies recognizing the same antigen. Twelve antibodies, one unmodified and eleven modified, were selected for this study and evaluated for assay performance and PK. The method was validated with intra-day and inter-day assay data, which demonstrated accuracy ranging from 70.3% to 128.4% and precision (%CV) less than or equal to 28.7% for all antibodies. The mouse PK profiles of modified antibodies after intravenous administration were comparable to that of the unmodified antibody, with no statistical differences in key PK parameters (t_1/2, C₀, AUC_last, and V_ss). These results indicate that the modifications had no substantial impact on PK profiles. Furthermore, cassette-dosing PK results were also consistent with those of single-dosing PK studies for six antibodies, indicating that there were no drug-drug interactions among antibodies. This method enables the efficient selection of lead/clinical candidates and the prediction of human PK for IgG1-type antibodies, which are the most widely used subtype in antibody therapeutics. Consequently, this novel approach complies with the 3Rs principle (Reduction, Refinement, and Replacement) in animal research and can substantially reduce costs in antibody drug development.