Anouk Donners

161 General Discussion Potential future applications of LC-MS/MS monitoring of FVIII concentrations in haemophilia A Background and rationale The FVIII activity plays an important role in managing haemophilia A. This thesis investigated but did not determine how the FVIII concentration relates to the FVIII activity. Nevertheless, this work is a first step towards explaining the relationship. This step included developing and validating an LC-MS/MS method to quantify the FVIII concentrations in human plasma (Chapters 2 and 3). The LC-MS/MS method was then used in a proof-of-principle study (Chapter 4) that described the initial comparison between the FVIII concentration (using LC-MS/MS analysis) and the FVIII activity (using a clotting assay). This comparison revealed significant variability, which was partially explained by the neutralising FVIII inhibitors and the modified exogenous FVIII product. Most of this variability could not be explained, although it may have been introduced by plasma samples of PwHA, which can contain exogenous, endogenous, inactive FVIII molecules or FVIII peptide fragments. The LC-MS/MS method cannot discriminate between endogenous or exogenous FVIII molecules, nor between active or inactive FVIII fragments. These unknown proportions in the heterogeneous samples added to the uncertainties in the comparison between the FVIII activity and the FVIII concentration, potentially overestimating the functional FVIII concentration. One could further investigate both the person and product variability of FVIII – first separately, then combined – to explain fully the observed variability and to determine the necessary corrections. A follow-up study could obtain pharmacokinetic (PK) profiles by measuring the FVIII activity using different clotting assays, and the FVIII concentration via an LC-MS/ MS analysis. Such a study would preferably be performed for people with severe, mild and moderate haemophilia A and people with normal FVIII activity. This study should also include various modified FVIII products to compare activation rates. These data could then be modelled using PK simulations to explain the total variability and the concentration–biomarker relationship of FVIII. The FVIII concentration’s role must also be thoroughly investigated and established for diagnostic and therapeutic monitoring before implementing the FVIII concentrations with LC-MS/MS analysis in a clinic. The LC-MS/MS technique has become an indispensable tool in proteomics. Analysis using LC-MS/MS is the standard method for determining amino acid sequences and peptides, characterising post-translational modifications, determining absolute and relative protein quantities, and identifying and quantifying multiple proteins in one measurement run (i.e., multiplexing) [16-18]. This technique has been employed to measure small molecules and therapeutic proteins in many laboratories worldwide and will probably become one of the leading techniques in the clinic [19]. The possibilities of this analysis seem endless when adopting mass spectrometry. Four potential applications of LC-MS/MS analysis in haemophilia A are now outlined. 9