Development of a Continuous Thrombin Generation-Based Test to Measure Potency in Factor VIII Concentrates

Abstract 1206 The potency assigned to a Factor (F) VIII-containing product should be predictive of its clinical effectiveness in treating Hemophilia A patients. Potency is assigned using assays that are calibrated against reference standards traceable to an international standard for FVIII concentrates. Therefore, FVIII products are labeled in International Units (IU). Most manufacturers are currently using either a one-stage clotting (OC) or 2-stage chromogenic substrate (CS) assays. However, for some FVIII products, discrepancies in potency values derived from these 2 assays can exceed 50 %. This presents a challenge to manufacturers, regulators and clinicians alike. Recently, a continuous thrombin generation test (TGT) has been introduced in clinical research. The TGT is physiologically more relevant; however, its utility in drug characterization is not yet established. In this report, we describe the development of a FVIII-TGT assay that is suitable for the measurement of FVIII activity in FVIII concentrates. We systematically optimized each of the TGT parameters to obtain the strongest response for a FVIII sample diluted in FVIII-deficient plasma to a concentration of 1 IU/mL. The relative response (ratio of levels of thrombin generation with and without FVIII), and the level of thrombin generation above instrument noise (signal-to-noise ratio) were used as criteria for optimization. In order to limit the variability of the assay, the final parameters were chosen such that small variations in the concentrations of reagents would have a minimal impact on assay outcome. Concentrations of FVIII-deficient plasma, tissue factor (TF), and corn trypsin inhibitor (CTI, inhibits contact activation) had strongest effects on the relative response to FVIII. Concentrations of procoagulant lipids, fluorogenic substrate and calcium ions were more important to achieve an optimal signal-to-noise ratio. Inhibition of contact activation mediated by the contact of plasma with plastic surfaces, control of temperature prior to the start of recording, pipetting technique and internal standards were important for assay reproducibility. Use of low levels of intrinsic factors IXa and XIa in conjunction with TF or omission of CTI allowed for the greatest sensitivity and linearity range of the assay. The performance and utility of the optimized FVIII-TGT were evaluated by measuring the potency of 6 plasma-derived and recombinant FVIII concentrates, and comparing the values to those by the CS assay and on the product label. Most of the products showed parallel dose-response curves between 0.01 and 0.3 IU/mL of plasma FVIII activity in the TGT-FVIII assay. One product had an activity of at least 20 % higher and another product 20 % lower than the labeled potency. The CS assay demonstrated a lack of parallelism in dose-responses for some products but identified the 2 products with the higher- and lower- than-labeled activities. In conclusion, the FVIII-TGT assay can be used to assign and compare the potency of FVIII concentrates. Additionally, the FVIII-TGT, being physiologically more relevant, may act as a reference in understanding the discrepancies in potency assignments in different FVIII concentrates. DISCLOSURE OF INTEREST: MJG, SAS, TKL, and MVO are employees of the U.S. FDA. The findings and conclusions in this presentation have not been formally disseminated by the FDA and should not be construed to represent any Agency determination or policy. Disclosures: No relevant conflicts of interest to declare.