Recombinant FIX Fc fusion protein activity assessment with the one‐stage clotting assay: A multicenter, assessor‐blinded, prospective study in Japan (J‐Field Study)

Abstract

Introduction The one‐stage clotting assay is used to measure factor IX (FIX) activity in patients’ plasma samples and in FIX products for hemophilia treatment. However, the diversity of reagents and instruments has resulted in significant FIX assay variability. Methods The accuracy of the one‐stage clotting assay to measure recombinant FIX Fc fusion protein (rFIXFc) activity was evaluated by major Japanese hemophilia treatment centers and commercial laboratories that measure factor IX activity for a majority of hemophilia B patients in Japan. Plasma‐derived FIX (pdFIX) and recombinant FIX (rFIX) products were used as comparators. FIX‐deficient plasma was spiked with four levels of FIX products based on label potency and measured under blinded conditions by routine one‐stage clotting assay procedures in 19 participating laboratories. Interlaboratory coefficient of variation and spike recovery were calculated. Results Interlaboratory coefficient of variation of rFIXFc was not significantly different from that of rFIX, but appeared larger than that of pdFIX. Mean spike recovery for rFIXFc was generally comparable to rFIX and pdFIX. However, larger discrepancies between pdFIX and rFIX were observed in three of nine laboratories using ellagic acid‐based activated partial thromboplastin time reagents. Conclusion Recombinant FIX Fc fusion protein activity was found to be similar to that of rFIX or pdFIX by the one‐stage clotting assay. However, minimizing interlaboratory variability is vital for optimizing future patient care.

Real‐world assay variability between laboratories in monitoring of recombinant factor IX Fc fusion protein activity in plasma samples

“…This tendency towards over-recovery at lower FIX levels is not a unique characteristic of rFIXFc, since similar findings have been reported in previous field studies An approximately two-fold overestimation at low levels of FIX (0.03 IU/mL) was also observed in a recent field study that measured FIX activity in samples spiked with a plasma-derived FIX product, demonstrating that the overestimation is not due to an intrinsic property of recombinant FIX products. 29 The approximately 30% higher rFIXFc recovery at 0.05 IU/mL vs 0.80 IU/mL by most aPTT reagents is therefore likely caused by the assay calibration method.…”

An Update on Laboratory Diagnostics in Haemophilia A and B

“…73 Furthermore, another field study also demonstrated interlaboratory differences during analysis of Alprolix, which, however, were not different from that of pFIX and rFIX processed in parallel. 74 In summary, one may conclude that, due to over-or underestimation of Alprolix, by SynthAFax or CK Prest, respectively, these two aPTT reagents should not be used in OSCAs applied for the determination of Alprolix activity. Furthermore, local evaluation of (other) ellagic acid-based aPTT reagents should be performed (►Table 1).…”

“… 73 Furthermore, another field study also demonstrated interlaboratory differences during analysis of Alprolix, which, however, were not different from that of pFIX and rFIX processed in parallel. 74 …”