Anouk Donners

84 Chapter 5 ABSTRACT Introduction Emicizumab is a new treatment option for patients with haemophilia A. Emicizumab was approved with a body-weight-based dosage regimen, without laboratory monitoring requirements. Guidelines, however, recommend measuring emicizumab concentrations when suspecting the presence of anti-drug antibodies. Furthermore, drug monitoring can be useful in clinical decision-making, in adherence checking, and for research purposes. Therefore we developed a liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method for quantifying emicizumab. We performed a validation study on this LC-MS/ MS method quantifying emicizumab in the plasma of patients with haemophilia A. Methods Sample preparation for LC-MS/MS analysis included ammonium sulphate protein precipitation and trypsin digestion. A signature peptide of emicizumab and a matching stable isotope-labeled internal standard were used to quantify emicizumab by LC-MS/ MS analysis. Validation was performed in accordance with the ‘Guideline on bioanalytical method validation’ of the European Medicines Agency (EMA). The LC-MS/MS method was cross-validated against a modified and calibrated (r2 Diagnostics) one-stage clotting assay (OSA). Results and Conclusion The LC-MS/MS method demonstrated linearity over a wide range of emicizumab concentrations, far exceeding the concentrations observed in patients with haemophilia A. Precision and accuracy were excellent and all other validation parameters were also within the acceptance EMA criteria. Cross-validation showed that the LC-MS/MS method and the OSA-based method can be used interchangeably for drug monitoring of emicizumab, without the application of a correction factor.