Recombinant <scp>FVIII</scp> Concentrates Show Distinct Stability Profiles During Heat Treatment Prior to Inhibitor Testing

Patients with hemophilia A (HA) receiving factor VIII (FVIII) replacement therapy are at risk of developing anti-FVIII neutralizing antibodies (nAbs), a major complication in severe HA. Accurate detection and quantification of nAbs are essential for patient monitoring and treatment management. Laboratory assessment of FVIII inhibitors in congenital HA is primarily performed using the Bethesda or Nijmegen assay (NA), with both the International Society on Thrombosis and Haemostasis (ISTH) and the World Federation of Hemophilia (WFH) recommending the Nijmegen assay as the method of choice [1]. Briefly, the principle of the factor VIII (FVIII) inhibitor assay is based on the inactivation of FVIII from a known source by inhibitors present in patient plasma during a 2-h incubation. The inhibitor titer is determined by measuring residual FVIII activity in the assay mixture relative to a control [2]. A critical feature of the Nijmegen assay is a pre-analytical heat treatment at 56°C for 30 min, which inactivates endogenous FVIII and thereby enhances assay specificity and sensitivity. Patient samples may contain exogenous FVIII due to recent coagulation factor concentrate (CFC) infusions for prophylaxis or treatment of bleeding episodes. The presence of exogenous FVIII can interfere with inhibitor detection, potentially causing underestimation or false-negative results [3]. Heat treatment overcomes this interference by denaturing FVIII and disrupting immune complexes. Accordingly, the World Federation of Hemophilia (WFH) recommends that samples with FVIII activity greater than 5 IU/dL be heated to 56°C for 30 min prior to testing. However, the thermal stability of newer recombinant FVIII products, particularly extended half-life (EHL) concentrates, has not been fully characterized. Structural modifications in these molecules may render the standard incubation insufficient to completely inactivate all FVIII, raising concerns about assay accuracy. With the introduction of efanesoctocog alfa, a novel recombinant FVIII with an ultra-extended half-life (~37–41 h), new analytical challenges have emerged. Its molecular design incorporates an IgG1 Fc domain, two XTEN polypeptides, and a modified von Willebrand factor (VWF) D′D3 domain, enabling VWF-independent activity. This architecture allows once-weekly dosing that maintains FVIII levels above 40 IU/dL for most of the dosing interval, potentially impacting the detection and quantification of inhibitors in standard assays [4]. However, the unique structure of efanesoctocog alfa complicates laboratory assessment. For example, while chromogenic substrate assay (CSA) can be used to monitor recombinant FVIII, it substantially overestimates FVIII activity in samples containing efanesoctocog alfa [5]. This limitation has recently been addressed through the development of a molecule-specific chromogenic calibrator, which enables accurate measurement of efanesoctocog alfa activity, even in the context of combination therapy with emicizumab [6]. octocog alfa (Kogenate, Bayer)—a full-length recombinant FVIII. efmoroctocog alfa (Elocta, Sobi)—an Fc-fusion extended half-life FVIII. efanesoctocog alfa (Altuvoct, Sobi). Products were prepared using FVIII inhibitor–free pooled plasma derived from patients with inherited severe hemophilia A (Cryopep, Montpellier, France) and spiked with each FVIII concentrate to achieve a target activity of 100 IU/dL. Samples were incubated at 56°C for 5, 10, 15, 30, 60, and 90 min in a dry water bath equipped with a calibrated temperature probe. Following centrifugation (10 min at 14 000 rpm), FVIII activity (FVIII:C) was measured at each time point using a one-stage clotting assay performed on an ACL TOP 750 analyzer with SynthasIL reagent and HemosIL FVIII-deficient plasma (Werfen). Although the reagent underestimates efanesoctocog activity, this limitation is inconsequential, as only the relative degradation from baseline (T0) was assessed. Statistical analyses were performed using paired t-tests and Kruskal–Wallis tests in GraphPad Prism (GraphPad Software, San Diego, CA), with a significance threshold of p = 0.05. In our study, FVIII:C decreased to less than 1UI/dL for octocog alfa and efmoroctocog alfa, indicating that inactivation was essentially complete after 30 min of incubation at 56°C. By contrast, efanesoctocog alfa retained measurable activity at 30 min, with a relative mean inactivation of 95.7% (range 90%–99%), although complete inactivation was observed by 60 min (Figure 1). The superior thermal stability of efanesoctocog alfa compared with the other two products was evident from the earliest time points: at 5 and 10 min of incubation, degradation percentages were significantly lower for efanesoctocog alfa, whereas no significant difference was observed between octocog alfa and efmoroctocog alfa (Figure 1). Nonetheless, complete inactivation of all recombinant FVIII concentrates was achieved between 30 and 60 min of heating at 56°C (Figure 1). These results indicate that efanesoctocog alfa is more resistant to thermal degradation than previously used recombinant FVIII products. Very few data are currently available regarding the thermal stability of efanesoctocog alfa. Only two recent studies using different assay platforms and reagents have addressed this question, and both confirmed that standard heat inactivation may be insufficient to achieve complete FVIII degradation [7, 8]. Although their findings align with ours in showing increased heat resistance, the optimal incubation time varied between studies, from 60 to 120 min at 56°C. This likely reflects differences in reagents. As also highlighted by Pipe et al. [5] in a global comparative field study, efanesoctocog alfa activity measured by one-stage assays (OSA) is highly dependent on the APTT reagent and FVIII concentration, with CK Prest markedly overestimating activity, while Actin FSL and SynthASil perform better at low concentrations (≈5 IU/dL). This reagent-dependent effect should therefore be carefully considered when defining standardized heat-inactivation protocols for efanesoctocog alfa. Given these findings, caution is warranted when screening for and titrating anti-FVIII inhibitors in patients treated with efanesoctocog alfa. Residual FVIII activity in plasma after standard heat treatment may interfere with inhibitor assays, potentially leading to false-negative results or underestimation of inhibitor titers. Although increasing the temperature to 58°C for 30 min could enhance efanesoctocog alfa inactivation, this approach is not recommended, as it may reduce anti-FVIII antibody levels [9]. To reduce the risk of false-negative results, particularly in the detection of low-titer FVIII inhibitors, alternative methodological strategies may be considered. These include dosing FVIII activity after the incubation step to ensure complete degradation of FVIII from inhibitory complexes, thereby minimizing assay interference. In addition, extending the incubation time to 60 min or longer represents a feasible adjustment, as this modification has been shown not to compromise anti-FVIII antibody stability [10, 11]. Collectively, these approaches may improve assay sensitivity without adversely affecting antibody integrity. In conclusion, efanesoctocog alfa appears more heat-resistant than earlier recombinant FVIII products. The optimal preanalytical inactivation conditions remain to be validated across assay platforms and reagents, and larger studies with fresh plasma are warranted to establish standardized recommendations for laboratory practice. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. The authors have nothing to report. The authors have nothing to report. The authors have nothing to report. The authors declare no conflicts of interest. All data analyzed during this study are included in this published article. No additional data are available.