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.

Activity of transgene-produced B-domain–deleted factor VIII in human plasma following AAV5 gene therapy

“…28,[46][47][48][49][50] For EHL-FIX products, OS/CS assay discrepancies can be pronounced. 13,[51][52][53][54] This complexity has led to guiding recommendations for potency assignment of recombinant FVIII/FIX products. 55 A probable root cause underlying many of these assay discrepancies is a deviation from the like-vs-like principle: that is, defined molecular differences between nonnative FVIII/FIX analytes and native FVIII/FIX calibrators, leading to divergent behavior in the assays.…”

Normal activated partial thromboplastin time in Chinese patients with mild hemophilia B

“…Our study was consistent with Park et al, both patients showed normal APTT, using reagents from Diagnostica Stago. A recent study showed discrepancies on the determination of FIX:C of rFIX fused with albumin (rFIX-FP) with one-stage and chromogenic methods [14]. Recent studies found that different APTT reagents showed highly variable sensitivity to single factor deficiency.…”

An Update on Laboratory Diagnostics in Haemophilia A and B

“…[66][67][68] Interestingly, it has been recently proposed that the latter is due to impaired activation of rFIX-FP during Pathromtin SL (and colloidal silica)-based OSC measurements. 69 On the basis of the data published to date, an OSCA for the measurement of Idelvion appears to be preferable; however, the above-described aPTT reagents should not be applied (►Table 1).…”