85 LC-MS/MS-method validation quantifying emicizumab INTRODUCTION Haemophilia A is a congenital bleeding disorder resulting from a deficiency or malfunction of coagulation factor VIII (FVIII) [1]. This cofactor is required to bridge activated factor IX (FIXa) and factor X (FX) for adequate hemostasis. The recommended treatment to prevent bleeding for patients with FVIII levels of ≤1 IU/dL is FVIII replacement therapy on a regular basis [2, 3]. Despite its efficacy, prophylaxis is burdensome due to frequent intravenous injections [4, 5]. In addition, a major complication is the formation of antiFVIII antibodies (called inhibitors), which renders treatment with FVIII products less effective [6]. Emicizumab (ACE910, Hemlibra®; by Roche and Chugai) is the first licensed non-factor replacement product. The European Medicines Agency (EMA) and the US Food and Drug Administration approved emicizumab for the prophylaxis of patients with haemophilia A in 2018 [7, 8]. This recombinant, humanized, and bispecific immunoglobulin G (IgG)-4 antibody binds both FIXa and FX, and mimics the function of activated FVIII in coagulation reactions [9]. The advantages of emicizumab compared with FVIII products are subcutaneous instead of intravenous administration, longer dosing intervals, and lack of interference by anti-FVIII antibodies [10]. Emicizumab has been approved with a body-weight-adjusted regimen without the requirement of drug monitoring [11, 12]. Guidelines recommend, however, measuring the emicizumab plasma concentration when suspecting the presence of anti-drug antibodies (ADAs) against emicizumab [13-17]. In addition, drug monitoring of emicizumab can be useful in clinical decision-making, in detecting lack of adherence, and for research purposes [18, 19]. Consequently, efforts have been made to determine emicizumab concentrations in human plasma. An enzyme-linked immunosorbent assay (ELISA) was used for this purpose in the HAVEN premarket approval studies, but is not commercially available [20-23]. Instead, the manufacturer supplies emicizumab-specific calibrators and controls to use in combination with a modified activated partial thromboplastin time (APTT)- based one-stage clotting assay (OSA), commonly used in a clinical setting [18, 24]. The modified, calibrated OSA (mcOSA) has shown agreement with the non-commercial ELISA [25]. However, disadvantages of the mcOSA are interference by FVIII or by ADAs and its availability at specialized hematologic laboratories [19, 26, 27]. A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of emicizumab was developed by our research group [28]. The objective was to perform a validation study on this LC-MS/MS method quantifying emicizumab in the plasma of patients with haemophilia A. 5
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