Anouk Donners

94 Chapter 5 Strengths and limitations This is the first report on clinical use of an LC-MS/MS method quantifying emicizumab in plasma, further building on our previous work measuring FVIII in plasma with LC-MS/MS [36, 37]. The strengths of this LC-MS/MS method over the mcOSA method are the lack of interference, a high-throughput and easy-to-implement design, and the opportunity to multiplex with other therapeutic monoclonal antibodies. In addition, the sampling volume for this LC-MS/MS method is only 0.25 mL (minimal required volume of tube), which is particularly beneficial to the pediatric population. Furthermore, the LC-MS/ MS-based methods have become the standard for measuring drug concentrations in clinical laboratories worldwide [38]; making this method for emicizumab quantification accessible to routine practice. Another form of assay-interference might result from the formation of ADAs against emicizumab. This immune response generally enhances drug clearance and removal from the circulation but might also form neutralized emicizumab−ADA complexes that remain in the circulation [39, 40]. These neutralized complexes could potentially lead to falsely high emicizumab concentrations using the LC-MS/MS method. The occurrence of such complexes remaining in the circulation has rarely been reported for therapeutic monoclonal antibodies and has not been reported for emicizumab as well. Unfortunately, no robust assays for ADA detection or neutralized complexes are commercially available. The presence of ADAs in our study samples is, however, highly unlikely, as it is extremely rare (reported incidence of <0.8% [41, 42]) and the clinical response of our patients was excellent [31]. This validation study was not powered for the development of emicizumab−ADA. Therefore, future studies should further investigate the impact of potential interference by this phenomenon, especially for the mcOSA and the ELISA, and to demonstrate the complementary role of LC-MS/MS. A limitation of the LC-MS/MS method is the sample preparation time of 24 hours, due to the overnight trypsin digestion step, and an analysis run-time of 13 min per sample. Fast drug monitoring of emicizumab is not required according to clinical guidelines, but might be supportive in an acute bleeding setting [15, 34]. Consequently, the work-flow may need to be optimized. In conclusion, the LC-MS/MS method for the quantification of emicizumab in the plasma of patients with haemophilia A was performed successfully in this validation study. The strong correlation between the current reference method and the LC-MS/MS method allows interchangeable use. This LC-MS/MS method can be implemented for drug monitoring of emicizumab. Author’s contribution AD designed the study, assisted in analytical analysis, performed data management and analysis, prepared the first draft of the manuscript, and implemented significant