Optimization and evaluation of a two-stage chromogenic assay procedure for measurement of emicizumab plasma levels

PLOS ONE RESEARCH ARTICLE Optimization and evaluation of a two-stage chromogenic assay procedure for measurement of emicizumab plasma levels Nasim Shahidi Hamedani ID*, Johannes Oldenburg, Bernd Pötzsch, Jens Müller Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Hamedani NS, Oldenburg J, Pötzsch B, Müller J (2022) Optimization and evaluation of a two-stage chromogenic assay procedure for measurement of emicizumab plasma levels. PLoS ONE 17(7): e0271330. https://doi.org/10.1371/ journal.pone.0271330 Editor: Arijit Biswas, Institute of Experimental Hematology and Transfusion Medicine, University Clinic of Bonn, GERMANY Received: April 13, 2022 Accepted: June 29, 2022 Published: July 14, 2022 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0271330 Copyright: © 2022 Hamedani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The data set required to replicate the study findings reported in the article Abstract Emicizumab mimics the hemostatic activity of activated factor VIII (FVIIIa) within the tenase complex. Despite functional similarities between FVIIIa and emicizumab, conventional laboratory methods designed for monitoring of FVIII activity are inappropriate for the measurement of emicizumab. At present, a modified one stage (FVIII) assay (mOSA) is mainly used for emicizumab monitoring. Two-stage chromogenic FVIII assays based on human factors can be used, although limited performance due to lack of corresponding optimization might be observed. Furthermore, the presence of FVIII or anticoagulants in the patient sample may falsify assay results. To address these issues, we optimized and evaluated a two-stage chromogenic assay (emi-tenase) for measurement of emicizumab in plasma samples. Heat inactivation of samples was established to abolish the influence of endogenous or substituted FVIII. The lower limit of quantification (LLoQ) was found to be 2 μg/ml in a manual assay format and 9.5 μg/ml on an automated coagulation analyzer. Intra- and inter-assay coefficients of variation (CV) did not exceed 20%. Analysis of 17 patient plasma samples with severe haemophilia A under emicizumab treatment showed good correlation of results between the emi-tenase assay and the mOSA (Cohens Kappa coefficient = 0.9). Taken together, the emi-tenase assay allows specific measurement of emicizumab plasma levels over a broad concentration range (10 μg/ml to 100 μg/ml). The assay can be applied on an automated coagulation analyzer, demonstrating its applicability within a routine laboratory setting. Introduction Routine administration of exogenous factor VIII (FVIII) for bleeding prophylaxis in patients with haemophilia A (HA) improves patients’ quality of life by reducing bleeding episodes and bleeding-related complications [1, 2]. However, 25 to 30% of patients with severe HA and receiving FVIII replacement therapy develop alloantibodies to FVIII and therefore show spontaneous and traumatic bleeding episodes [3]. For HA patients developing autoantibodies against FVIII, on-demand treatment or regular prophylaxis with bypassing agents (BPAs) such as administration of activated prothrombin complex concentrate (aPCC), recombinant PLOS ONE | https://doi.org/10.1371/journal.pone.0271330 July 14, 2022 1 / 13 PLOS ONE are openly available in Zenodo.org at https:// zenodo.org/record/6697795#.YrQxiNNCSUk. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interest exist. Emicizumab tenase assay activated FVII (rFVIIa), and plasma-derived Factor VIIa/Factor X complex are available or under development [4–7]. From 2017, the bioengineered antibody named emicizumab (Hemlibra1, F Hoffmann-La Roche, Basel, Switzerland) has been approved for routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with HA with or without inhibitors [8–11]. Emicizumab mimics the procoagulant function of activated (co)factor VIII [FVIIIa] by simultaneous binding to (activated) factors IXa (FIX) and X (FX) [12, 13]. It is therefore able to take over the tasks executed by FVIIIa, albeit at lower turnover rates. The catalytic efficiency (kcat/Km) by which FVIIIa enhances activated factor X (FXa) generation is 10 times higher than that of emicizumab while patients under prophylactic treatment with emicizumab show annualized bleeding rates and hemostatic potentials close to that of patients with moderate hemophilia [14, 15]. In the HAVEN 4 cohort study, a steady state plasma concentration of approximately 40 μg/ml emicizumab was achieved 24 weeks after initiation of an every 4-week treatment scheme [16]. In addition, in a phase 3 study of emicizumab prophylaxis in children aged <12 years with HA with inhibitors, mean steady-state concentrations were maintained at effective levels of 38–50 μg/ml [17]. Plasma levels of emicizumab can be measured directly or indirectly. Direct methods measure emicizumab mostly through immunological methods while indirect methods quantify the functional procoagulant potential of emicizumab [12, 13]. Moreover, immunological methods are in general hampered by long turn-around times while functional testing is less time-consuming and also well established in monitoring of FVIII replacement approaches and monitoring of anticoagulant drugs [18]. Previous studies showed that emicizumab plasma levels can be measured by a modified FVIII one-stage assay (mOSA), a two-stage chromogenic FVIII-assay or can be estimated by subjecting prediluted plasma samples to a standard FVIII one-stage assay (sOSA) [12, 19, 20]. The mOSA is basically a FVIII-activity assay that operates at higher sample predilutions and the use of emicizumab calibrators for the construction of a corresponding standard curve [18, 21]. The mOSA method was shown to have good precision and reproducibility and emicizumab levels determined using the mOSA correlated well with the ELISA method previously used in the HAVEN clinical trials [21, 22]. In a recently published study, pre-diluted plasma samples were subjected to the sOSA and the calculated FVIII activity was used for emicizumab plasma level approximation (ELA), resembling the emicizumab level in μg/ml of the undiluted sample. Although this method demonstrated substantial agreement with the mOSA method, increased variation of ELA was (...truncated)