Anouk Donners

180 Chapter 10 Introduction In Chapter 1 a general introduction with background and rationale for the thesis was provided. Herein is explained that haemophilia A is a bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII). The endogenous FVIII activity is used to classify severe (<1%), moderate (1 to 5%) or mild (>5 to 40%) haemophilia A. People with haemophilia A (PwHA) experience recurrent bleeding, predominantly into major joints, such as ankles, knees and elbows, leading to painful and disabling arthropathy. Currently, effective protection against bleeding is provided by replacement therapy using FVIII products or by emicizumab. Because FVIII products and emicizumab are costly, they can pressure healthcare budgets and limit patient access. Measuring and monitoring are key in haemophilia A care. The FVIII activity is measured for diagnosing, but also to determine the dose of FVIII products. Monitoring emicizumab is not routinely performed, although guidelines recommend to measure its concentration in suspicion of anti-drug antibodies (ADA). The liquid chromatography (LC) coupled with tandem-mass spectrometry (MS/MS) analysis is a beneficial technique for quantifying therapeutic protein concentrations in human plasma. Prior to this thesis, the potential value of concentration measurements with LC-MS/MS analysis for monitoring PwHA in clinical practice was unclear. The knowledge gaps regarding this process concerned the relationship between the FVIII concentration (measured with LC-MS/MS) versus the FVIII activity (measured with clotting assays) and a rationale for emicizumab’s concentration in routine daily practice. In this thesis, the objective was to optimise drug monitoring of FVIII and emicizumab in PwHA by: - developing and validating LC-MS/MS methods for their quantification in plasma; - investigating the concentration–biomarker relationship of FVIII and the dose– concentration–response relationship of emicizumab; and - proposing and evaluating a cost-efficient dosing strategy for emicizumab. General LC-MS/MS method development In recent years, LC-MS/MS methods have enabled absolute quantification of therapeutic proteins. These methods have additional benefits, such as a large linear dynamic range, a high specificity and the option of multiplexing. In Chapter 2 we discussed the strategies for the quantification of therapeutic proteins in several biological matrices using LC-MS analysis based on top-down and middle-down quantitative proteomics. Then, we presented the widely used bottom-up method in a six-step workflow as a tutorial for quantitative LC-MS/MS method development. Considerations for signature peptide selection were provided and critical method parameters were discussed for