Field Study Compares OSA With CSA for Measuring Efanesoctocog Alfa

Introduction: Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life.

Aim: Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs).

Methods: Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N = 35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed.

Results: Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations.

Conclusion: Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.

Here is an open-access 2026 follow-up report of OSA versus CSA assays of efanesoctocog alfa in patient plasma: Müller J, Büchsel M, Pezeshkpoor B, et al. On factor VIII assay discrepancies in post-infusion samples obtained from patients treated with efanesoctocog alfa. Hamostaseologie. 2026. doi: 10.1055/a-2717-3413. Epub ahead of print. PMID: 41554518.

Abstract

Efanesoctocog alfa (efa) is a recombinant coagulation factor VIII (FVIII) concentrate, engineered for improved extended half-life in hemophilia A treatment. Its design results in discrepancies in FVIII diagnostic tests, as has so far been demonstrated using spiked sample material (efa added to FVIII-deficient plasma). The aim of the present study was to evaluate FVIII assay discrepancies in post-infusion samples obtained from patients treated with efa. This retrospective analysis included 43 male patients on efa prophylaxis (26 on once weekly and 17 on twice weekly treatment scheme) summoned for determination of incremental recoveries. FVIII activity was measured at trough (3.5 or 7 days post-infusion) and peak levels (30 minutes post-infusion) using three diagnostic tests: two one-stage clotting assays (OSAs) based on Actin FSL (AFSL) or Actin FS (AFS), and a chromogenic substrate assay (FVIII CSA). Incremental recoveries as determined by the recommended AFSL-based OSA were found to be comparable between patient groups. At peak levels (64 to 214 IU/dL), comparable overestimation (1.9-fold) of plasma efa activity relative to Actin FSL-based OSA was observed for both, the AFS-based OSCA and the FVIII CSA. In contrast, at trough levels (5 to 64 IU/dL), rate of overestimation relative to the AFSL-based OSCA results was found to be lower for the FVIII CSA (1.3-fold) when compared with the AFS-based OSCA (1.9-fold). Further analysis demonstrated different behavior of spiked and post-infusion samples within the FVIII CSA. Future studies will reveal underlying mechanisms and assess if drug-specific calibration will sufficiently correct for this phenomenon.

A highlight from the article sent by co-author Ali Sadeghi-Khomami, PhD:

“This manuscript highlights the importance of conducting clinical studies following in vitro field studies to ensure data commutability across studies. They found that the degree of overestimation by the FVIII chromogenic substrate assay (CSA) relative to AFSL-based OSA results was more pronounced at peak levels than at trough levels in patient groups. Rather than using a general conversion factor (CF) of /2.5, the authors advocate for product-specific CFs validated at each site for specific lots.

In their center, using the Sysmex CN6000 with clinical samples, they found a /1.9 CF works well for Actin FS (both peak and trough levels), but for the Siemens bovine chromogenic assay, it only works well at peak levels. For trough levels, a /1.3 CF appears more accurate.”