Potency estimates for recombinant factor IX in the one-stage clotting assay are influenced by more than just the choice of activated partial thromboplastin time reagent.

The activated partial thromboplastin time (APTT) one-stage clotting (OSC) assay is used to potency label factor IX (FIX) therapeutics and to monitor the treatment of deficient patients. Studies have highlighted differences in potency estimates for FIX therapeutics depending on what activator type the APTT reagent contains. During the study to replace the 4th International Standard (IS) for FIX Concentrate, it was noted that for the recombinant FIX (rFIX) candidates, a potency difference of 20% was obtained by some between laboratories using the reagent DAPTTIN. This study was designed to investigate this discrepancy. Two plasma-derived (pdFIX) and 2 rFIX materials were assayed against the 4th IS for FIX concentrate (07/182) using the OSC assay. Three different APTT reagents were used (DAPTTIN, SynthAFax and SynthASil), plus 4 different activation times. The pdFIX samples were not affected by activation time or APTT reagent, with expected potencies recovered in all assays and at all activation times. For rFIX, expected potencies were recovered using SynthASil, but SynthAFax generated potencies around 20% lower. This was consistent across the activation times. For DAPTTIN, potencies for rFIX dropped progressively and by up to 30% as activation time increased from 120 to 300 seconds. This study demonstrates that activation time as well as choice of APTT reagent can result in discrepancies in the potency estimation of rFIX. These factors should be taken into account when assaying rFIX.