PB0187 - Impact of One-Stage Clotting Assay Reagents on Factor XIa Levels and Clotting Time with High Activity Factor IX (R338L) Variant

Background: Fidanacogene elaparvovec, a recombinant Adeno-associated virus vector encoding the human Factor IX-R338L high activity variant, is in development for Hemophilia B (HB). Emerging data suggest variation across different one-stage (OS) clotting assays and chromogenic substrate (CS) assays when measuring FIX-R338L activity.

Aims: To gain further insight into the contributions of the OS assay components on the FIX-activity ranges in OS FIX assays.

Methods: Factor XIa, IXa, and Xa levels were determined by CS assays at intervals pre-or post-calcification, mimicking the OS contact and amplification phase, in ellagic acid (Actin FSL; Actin FS) and silica (SynthASil) OS reactions. For like-versus-like, recombinant FIX-WT and FIX-R338L proteins, produced from HEK-293 cells, were spiked at equimolar concentrations into HB plasma for a pair-wise comparison of the absolute clotting times.

Results: Differences in FXIa, FIXa, FXa generation between the ellagic-acid and silica-based OS reagents were observed. In SynthASil, FXIa generation pre-calcification, and FIXa and FXa generation (20 and 40 seconds) post-calcification were lower compared to the ellagic acid reagents. Consistent with the variant’s increased specific activity, FIXa and FXa were ~ 10 and 2-3-fold > FIX-WT, respectively. A range in the absolute clotting times were observed between the three OS reagents with > differences at lower concentrations.

Conclusion(s): Both phospholipid and contact activator composition likely impacted the OS activity ranges observed with FIX-R338L. However, it can be measured using commonly used FIX assays.