Requirements and limitations for the preparation of targeted mass spectrometry assays enabling therapeutic drug monitoring

The integration of mass spectrometric methods in the field of therapeutic drug monitoring (TDM) represents a promising approach. Through the application of mass spectrometry, selective and accurate measurements of monoclonal antibodies (mAB´s) in the blood are obtainable, enhancing the reliability and accuracy of TDM analysis and minimizing the effects of autoantibodies on test results, thereby preventing patients" over-and underdosing during therapy. Additionally, mass spectrometry offers the capability of multiplexing, allowing simultaneous detection and quantification of multiple mAB"s in a patient´s sample, especially pivotal for patients under therapy using a combination of antibodies. For those reasons, it was our aim to establish a targeted mass-spectrometric assay facilitating reproducible absolute quantification of peptides from the hypervariable and interacting regions of selected antibodies. Therefore, tryptic peptides and their corresponding stable isotopologue were analysed using UHPLC coupled to a TSQ Altis Triple-Quad mass spectrometer. In this study, exemplary for the antibody infliximab, we demonstrate the substantial influence of peptide selection, choice of hydrolase for digestion, and digestion time on digestion efficiency and absolute peptide yield. Additionally, we showed the robustness and reproducibility of the generated calibration curves including key figures such as LOD and LLOQ. Finally, we have developed a semi-automated platform enabling the rapid (2 days of sample preparation and 30 min of measurement time per sample) and robust absolute quantification of Infliximab antibody concentration in patients" blood samples (Hentschel A et al.; Clin Proteomics. 2024 Feb 29;21(1):16. doi: 10.1186/s12014-024-09464-x.). The use of mass spectrometry also facilitates the straightforward expansion of the method to include additional antibody peptides.