Recombinant factorIX: discrepancies between one‐stage clotting and chromogenic assays

Abstract

New and modified recombinant factor IX (rFIX) products are in development and accurate potency estimation is important to ensure the consistency of production and efficacy of these therapeutics. Collaborative study data obtained during the replacement of the 3rd International Standard (IS) for FIX concentrate suggested that there was a discrepancy between potency estimates for rFIX using clotting and chromogenic methods, when the rFIX candidate was measured against the plasma-derived FIX (pdFIX) IS. This study explores potential chromogenic and one-stage clotting method discrepancies in more detail. Five batches each of rFIX and pdFIX were assayed against the 4th IS FIX concentrate (a pdFIX) by activated partial thromboplastin time (APTT) one-stage clotting assay and specific functional chromogenic assay. The potency of rFIX by chromogenic assay was consistently around 70% of the one-stage clotting potency (average 78 and 108 IU mL(-1) respectively). These differences were not observed with pdFIX, which had similar potencies (average 96 IU mL(-1) ) by each assay method. In addition, different APTT reagents yielded different potency estimates for rFIX when assayed against the pdFIX IS, with a variation of up to 23%. In all cases, the differences were largely resolved when a rFIX reference was used as the standard. This study highlights some of the challenges associated with assay of rFIX products in the laboratory and that careful consideration needs to be given to the choice of reference material used. This is especially important with the imminent arrival of new and modified rFIX products.

Evaluation of chromogenic factor IX assays by automated protocols

“…Recoveries of two rFIX products (Benefix ® and Rixubis ® ) and the EHL product (Alprolix ® ) demonstrated that CSAs generally give closer and more consistent agreement to target levels compared to any of the three OSA protocols employed, where two of three APTT reagents tended to over‐recovery for Benefix ® and Rixubis ® (Figure ). This pattern of similar recovery of pdFIX and discrepant recovery of rFIX products using plasma calibration standards between OSA and CSA by Hyphen test has been previously reported . Consistent with the present study, in a collaborative study assessing postinfusion FIX assays, median CSA results were around 25% lower than median OSA results ( P < .01) with Benefix ® .…”

“…Barrowcliffe reported that a chromogenic assay under‐estimated potency of recombinant FIX (rFIX) relative to OSA. More recent studies have shown FIX estimates for rFIX to be significantly lower by CSA than for OSA, but differences were not observed when testing plasma derived FIX (pdFIX) products . Potency discrepancies for both recombinant and extended half‐life (EHL) FIX products have been described between the OSA and CSA and for different OSAs related to APTT reagents…”

Recombinant to modified factor VIII and factor IX – chromogenic and one‐stage assays issues

“…Differences between chromogenic and one‐stage FIX assays in the presence of standard half‐life recombinant FIX have been reported. Chromogenic FIX assay results were consistently around 70% of one‐stage results when two rFIX concentrates were assayed and in postinfusion samples containing one of these products (Benefix) . Furthermore, one‐stage assay results in samples containing full length recombinant FIX may be influenced by the assay reagents used in the case of Benefix and Rixubis, for which the product information states that FIX potency results can be affected by the type of APTT reagent used in the assay and that differences of up to 40% have been observed.…”

Role of chromogenic assays in haemophilia A and B diagnosis

“…It is known that using different FIX assays disparate results may be obtained in the same sample. Differences in postinfusion samples of recombinant ( r ) FIX, with CSA 30% lower than OSA FIX, have been described . The discrepancy for some extended half‐life (EHL) FIX concentrates has also been observed .…”