Anouk Donners

39 Tutorial on LC-MS/MS methods quantifying mAbs Method validation After optimization of assay conditions the method is subjected to validation following FDA or EMA guidelines for bioanalytical method validation [36, 103]. Parameters, such as LLOQ, accuracy, precision, matrix effect, linearity, stability and carry-over should comply with requirements as states in these guidelines. Furthermore, it is strongly recommended to carry out a cross-validation against an established method to determine whether the methods are strongly correlated. In regards to the latter, it should be stressed that measurement of different therapeutic mAb fractions, e.g., free or total, can result in interassay differences. Moreover, inter-assay variation can occur, evenwhen the same fraction is measured. This is exemplified by the comparison of active infliximab quantification in human serum with LC-MS/MS versus ELISA-based assay [72]. Here, the sandwich type ELISA required two free mAb paratopes, one paratope for fixation and the other for detection, while the LC-MS/MS assay only required one free paratope. This meant that the infliximab fraction with only one free paratope could not be quantified with the ELISA assay which may have resulted in an underestimation of free infliximab in serum. CONCLUSION AND DISCUSSION An overview of state-of-the-art LC-MS/MS methods used for quantification of therapeutic mAbs in biomatrices is provided. Current literature on peptide level quantification is summarized in six workflow steps, and benefits and drawbacks in each step of method development have been critically evaluated. We conclude that LC-MS/MS instruments offer fast method development and multiplexing capabilities and will continue to replace ligand-binding assays as these instruments get cheaper, improve in terms of sensitivity and mass accuracy with each generation. Innovations and improvements in materials, such as the immobilization of trypsin and magnetic beads conjugated with various ligands, will aid in speeding analysis times while providing high recoveries and sensitivities. Also, with increasing availability of stable isotopically labeled mAbs, method robustness, precision and accuracy will further improve. Top-down and middle-down quantitative proteomics are expected to become more important as newly developed mAb are mostly fully human or humanized. Improvement to instrument hardware and software are needed to facilitate the growth in this area. As more awareness in the scientific community is growing to the possibilities that these methods have to offer, LC-MS/MS methods have the potential to become the technique of choice for mAb quantification in preclinical and clinical settings. Author’s contribution AD helped the first author with study design, retrieved and checked data, assisted in the comprehensiveness of the literature search, drafted the first versions of the manuscript together with the first author, helped in implementation of significant contribution from co-authors up to the final publication. 2