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

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.

Characterization of thrombin derived from human recombinant prothrombin.

Thrombin (FIIa) is the key enzyme in haemostasis and acts on several substrates involved in clot formation, platelet activation and feed-back regulation of its own formation. During activation of blood coagulation, FIIa is formed by proteolytic cleavage of prothrombin (FII). In the production of recombinant human FII (rhFII), a key question is whether the thrombin formed has the same properties as endogenous thrombin. We have investigated whether FIIa formed from rhFII and plasma-derived human FII (pdhFII) have the same enzymatic and haemostatic properties against a number of substrates and the same haemostatic capacity in plasma, whole blood and on platelets. Pure FIIa was isolated from rhFII and pdhFII cleaved by recombinant ecarin, and analytical methods were developed to compare the activity of FIIa against different substrates. FIIa derived from rhFII and pdhFII were found to have very similar properties in activating FVIII, FXIII, protein C, platelet aggregation and plasma or whole blood coagulation. Further, the same turnover for S-2366 was found with similar KM. However, activation of FV with rhFIIa was approximately 25% more effective than with pdhFIIa and heparin-enhanced inhibition of rhFIIa by antithrombin was significantly more efficient compared with pdhFIIa with 10% higher inhibition both at steady state and at initial rate conditions. Although differences between the two FIIa preparations using ecarin cleavage were observed, FIIa derived from rhFII administered to human would likely be very similar in activity and function as FIIa formed from endogenous FII.