Performance of a recombinant fusion protein linking coagulation factor IX with recombinant albumin in one‐stage clotting assays

ESSENTIALS: Performance of the one‐stage clotting (OSC) assay varies with the clotting activator used. Recombinant FIX‐albumin fusion protein (rIX‐FP) was reliably monitored with most OSC reagents. rIX‐FP shows comparable reagent‐dependent variability to other rFIX products in the OSC assay. Actin(®) FS and kaolin‐based reagents underestimated rIX‐FP activity by around 50% in the OSC assay. SUMMARY: BACKGROUND: Measuring factor IX activity (FIX:C) with one‐stage clotting (OSC) assays, based on the activated partial thromboplastin time (APTT), is the current mainstay of diagnostic techniques for hemophilia B. Assessing the performance of new recombinant FIX (rFIX) products in OSC assays is essential, as APTT reagents from different manufacturers yield different potency estimates for rFIX. OBJECTIVES: To evaluate the extent to which choice of reagent composition influences rFIX potency measurements of recombinant FIX–albumin fusion protein (rIX‐FP, IDELVION) activity in OSC assays. METHODS: rIX‐FP was added to FIX‐deficient plasma, and FIX:C was assessed centrally and locally in a multicenter international field study with a variety of commercial OSC APTT reagents. Paired sample analysis of clinical samples was performed to compare values of FIX:C from local and central laboratories. In‐house bioanalytical investigations with spiked samples were conducted to compare the APTT‐reagent dependent variability of rIX‐FP with unmodified rFIX and rFIX Fc fusion protein (rFIXFc). RESULTS: Central and local assessments of FIX:C from 10 countries and 21 participating centers showed comparable results to those from the central laboratory across the majority of 18 different APTT reagents from both clinical and spiked samples. There was a consistent underestimation of rIX‐FP activity of ≈ 50% with OSC assays using Actin FS or kaolin‐based APTT reagents. In the bioanalytical study, rIX‐FP showed comparable variability in OSC assays to unmodified rFIX and rFIXFc. CONCLUSIONS: rIX‐FP activity can be accurately measured by the use of OSC assays with the majority of commercial reagents. Actin FS or kaolin‐based reagents will probably lead to a 50% underestimation of activity.