Anouk Donners

181 Summary internal standard selection, chromatographic separation, sample purification, digestion, and method validation. Measuring and monitoring FVIII using LC-MS/MS Then, the LC-MS/MS technique was used to quantify FVIII in human plasma. The development and validation of this method were described in Chapter 3. Samples were prepared by first triggering the coagulation cascade, which freed FVIII from Von Willebrand factor, and then a selective immunoaffinity step was performed to selectively purify the active FVIII with camelid nanobodies. After heat denaturation and trypsin digestion, a FVIII specific peptide was used as a surrogate for quantification. The validation results were all well within the acceptance criteria of the current European Medicines Agency (EMA) guideline on bioanalytical method validation. This validated method allowed us to further investigate the relationship between the FVIII plasma concentration and the FVIII activity in PwHA in Chapter 4. A proof of principle, crosssectional study was conducted on remnant plasma samples (n = 87) from PwHA from our clinic. The FVIII concentrations using LC-MS/MS analysis and the FVIII activities using a clotting assay were compared. An overall mean relative difference of -1% with an SD of 64% was demonstrated. Despite a good overall correlation between the two methods, several relative differences were large. Large differences between concentration and activity were correlated with the presence of anti-FVIII antibodies and use of exogenous FVIII products (e.g., plasma-derived and modified FVIII products). The clinical impact thereof is yet unclear. Therefore, more research is needed to determine the value of FVIII plasma concentration in comparison with FVIII activity. Measuring and monitoring emicizumab using LC-MS/MS Similarly, the LC-MS/MS technique was used to quantify emicizumab’s concentration. A method validation study was performed on a generic sample preparation method with LC-MS/MS analysis in Chapter 5. The method demonstrated linearity over a wide range of emicizumab concentrations, far exceeding the concentrations observed in PwHA. Precision and accuracy were excellent and all other validation parameters were also well within the acceptance criteria of the current EMA guideline on bioanalytical method validation. A cross validation against a common clotting-based method demonstrated that both methods can be used interchangeable for drug monitoring of emicizumab, without the application of a correction factor. Emicizumab has been approved with body-weight-based dosing without the necessity for laboratory monitoring. This assumes a clear dose–concentration–bleed relationship, with acceptable variability due to factors other than body weight. To investigate this assumption, a systematic review on the pharmacokinetics (PK) and associated efficacy of emicizumab in humans has been reported in Chapter 6. Data from 15 clinical studies 10