Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers

Background: Andexanet alfa (AnXa) is a modified human FXa protein approved in the US and EU for reversal of direct factor Xa (FXa) inhibitors (apixaban, rivaroxaban) due to life-threatening or uncontrolled bleeding. AnXa reverses the anticoagulant effect of FXa inhibitors by binding and sequestering FXa inhibitor. Commercially available anti-FXa assays measure FXa inhibitors using drug specific calibrators and controls. However, there are limitations when these assays are used for measuring AnXa-containing patient samples. One of the limitations is the large sample dilution in the assay setup, which causes dissociation of the inhibitor from the AnXa-inhibitor complex (due to the reversible binding equilibrium of the AnXa-inhibitor), resulting in an erroneous elevation of the anti-FXa activity following AnXa administration. We have developed modified assay parameters for the drug-specific anti-FXa activity assays, apixaban and rivaroxaban, on the ACL TOP Family instruments, which minimizes the effect of sample dilution suitable for analyzing AnXa-containing samples. Methods: Pooled normal human plasma was spiked with different concentrations of apixaban (230, 460, 920 ng/mL, 0.5 - 2.0 μM) or rivaroxaban (218, 436, 872 ng/mL, 0.5 - 2.0 μM) and AnXa at different AnXa:inhibitor molar ratios (0, 0.5,1.0, 2.0). The reversal activity of AnXa for apixaban and rivaroxaban was measured by a modified anti-FXa assay in a 96-well format previously used in the AnXa preclinical and clinical studies. The same set of contrived samples was assessed using the apixaban and rivaroxaban assays on the ACL TOP 700 with HemosIL Anti-Xa with HemosIL Apixaban or HemosIL Rivaroxaban Calibrators and Controls according to the manufacturer's recommendations. The ACL TOP 700 assay parameters were modified with an optimized plasma to reagent volume ratio to reduce the overall sample dilution. The calibration curves for both assays were optimized to report in the 0-100 ng/mL range. Results: When assessed by modified anti-FXa assays in the 96-well format (with an overall 2-fold sample dilution), AnXa dose-dependently reversed the anti-FXa activity of both apixaban and rivaroxaban spiked in plasma. % Reversal increased as a function of the molar ratios (AnXa:inhibitor) with ~50% (1:2), > 95% (1:1), and >99% reversal (2:1) respectively. Conversely, the Anti-FXa setup with the HemosIL Anti-Xa reagents and calibrators showed