FIX potency of rFIX-Albumin fusion protein is underestimated by one-stage methods using silica-based APTT reagents

Abstract

Introduction: Higher potency is obtained with chromogenic substrate (CS) methods and one-stage (OS) method with SynthAFax vs silica-based OS methods on analysis of albutrepenonacog alpha (rFIX fused with albumin, rFIX-FP).

Aim: Investigation of the effect of contact activator in search for explanation of discrepancy between methods.

Methods: Chromogenic Rox Factor IX method and OS methods with Pathromtin SL, SynthAFax or new OS method variants using different phospholipid emulsions and addition of either colloidal silica to create APTT reagents or addition of human FXIa together with calcium ions, in the latter case omitting contact activation. The effect of (a) adding different amounts of colloidal silica or (b) mixtures of Pathromtin SL and purified phospholipids immediately before addition of FXIa and calcium chloride was also explored. FIX activation via tissue factor/FVIIa was also made.

Results: FIX potency of rFIX-FP when using APTT reagents with pure phospholipid emulsions with added colloidal silica was similar to OS method with Pathromtin SL. In contrast, close to 80% higher FIX potency for rFIX-FP, and similar to OS method with SynthAFax and to the CS method, was obtained when FXIa replaced contact activation. No discrepancies were obtained for plasma-derived FIX. Gradual decrease of colloidal silica or decreasing proportion of Pathromtin SL added just before addition of FXIa raised rFIX-FP potency to that obtained with SynthAFax and Rox Factor IX. Supportive results were obtained with the tissue factor/FVIIa method.

Conclusion: Colloidal silica and Pathromtin SL impair activation of rFIX-FP, causing underestimation of rFIX-FP potency.

Keywords: assay discrepancies; chromogenic assay; factor IX; haemophilia B; one-stage assay; silicon dioxide.