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.
The activated partial thromboplastin time (APTT) one-stage clotting (OSC) assay is used to potency label factor IX (FIX) therapeutics and to monitor the treatment of deficient patients. Studies have highlighted differences in potency estimates for FIX therapeutics depending on what activator type the APTT reagent contains. During the study to replace the 4th International Standard (IS) for FIX Concentrate, it was noted that for the recombinant FIX (rFIX) candidates, a potency difference of 20% was obtained by some between laboratories using the reagent DAPTTIN. This study was designed to investigate this discrepancy. Two plasma-derived (pdFIX) and 2 rFIX materials were assayed against the 4th IS for FIX concentrate (07/182) using the OSC assay. Three different APTT reagents were used (DAPTTIN, SynthAFax and SynthASil), plus 4 different activation times. The pdFIX samples were not affected by activation time or APTT reagent, with expected potencies recovered in all assays and at all activation times. For rFIX, expected potencies were recovered using SynthASil, but SynthAFax generated potencies around 20% lower. This was consistent across the activation times. For DAPTTIN, potencies for rFIX dropped progressively and by up to 30% as activation time increased from 120 to 300 seconds. This study demonstrates that activation time as well as choice of APTT reagent can result in discrepancies in the potency estimation of rFIX. These factors should be taken into account when assaying rFIX.
The 1st International Standard (IS) for blood coagulation factor XI (FXI), plasma, has been successfully used for potency labeling of FXI therapeutics and for diagnosis of FXI deficiency in patients. With stocks of the 1st IS near depletion, a replacement is required. In addition to the functional activity value, assignment of an antigen value to the 2nd IS would allow harmonization of antigen assay methods and differentiation of patients who have low functional activity but normal antigen FXI levels from patients who have both low functional and antigen FXI levels. The aims of this study were, therefore, to assign FXI functional activity to the 2nd IS for FXI, plasma, and to additionally assign a new analyte, FXI antigen, to the same International Standard. The candidate material was prepared from double-spun, virology negative, normal plasma, which was pooled and filled into siliconized glass ampoules and subsequently freeze-dried. Assignment of the functional activity (FXI:C) value in International Units (IUs) was performed by one-stage clotting assay by 29 laboratories, relative to the 1st IS. The overall geometric mean (GM) was 0.71 IU/amp with extremely low inter-laboratory variability (expressed as geometric coefficient of variation) of 1.8%. The antigen value assignment was performed by 11 laboratories and was calculated relative to normal plasma pools, as is customary with new coagulation factor analytes. The amount of antigen present in 1 ml of normal plasma was taken to be 1 U. The overall GM for the antigen assays was 0.78 IU/amp with an inter-laboratory variation of 10%. The candidate (National Institute for Biological Standards and Control code, 15/180) was established by the World Health Organization (WHO) Expert Committee on Biological Standardization in 2016 as the WHO 2nd IS for blood coagulation FXI, plasma, with a functional activity value (FXI:C) of 0.71 IU/amp and an antigen value (FXI:Ag) of 0.78 IU/amp.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Until recently, the role of factor XII (FXII) in hemostasis was not considered to be important since patients with FXII deficiency do not present with bleeding. The activation of FXII by agents including mast cells and platelet polyphosphates suggests that it may have a role in thrombogenesis. The inhibition of FXII therefore presents an option for antithrombotic therapy, and antibodies and inhibitors are already in development. Assays for FXII will be required to support these technologies, and an international standard (IS) for FXII would be useful for the development of these methods and for the clinical monitoring of patients. The purpose of this study was to develop an IS for FXII, with values for functional activity (FXII:C) and antigen (FXII:Ag). Double-spun normal plasma was pooled, filled into siliconized glass ampoules, and freeze-dried to prepare the candidate material. Data from 20 laboratories using the one-stage clotting assay were used to assign the functional activity value in units (u). The antigen value was calculated using data from eight laboratories that carried out antigen assays. Each laboratory was requested to collect two local normal plasma pools. Units of activity and antigen were calculated relative to these pools, as is usual for new coagulation factor analytes. The amount of activity or antigen in 1 ml of normal plasma from each pool was taken to be 1 unit. A total of 566 donors were used across the pools for the FXII:C study and 216 donors for the FXII:Ag study. The overall geometric mean per ampoule for FXII:C was 0.86 u and for FXII:Ag was 0.80 u. The inter-laboratory variation was 10 and 11%, respectively (expressed as the geometric coefficient of variation). Based on these data, the candidate was deemed suitable for use as an IS for FXII. In 2017, the candidate was established by the World Health Organization (WHO) Expert Committee on Biological Standardization as the WHO first IS for blood coagulation FXII, Plasma (National Institute for Biological Standards and Control code 15/180). The values assigned were 0.86 international units (IU) of functional activity (FXII:C) per ampoule and 0.80 IU/ampoule of antigen (FXII:Ag).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.