Monitoring of Emicizumab (ACE910): comparison between clotting and chromogenic assay

Emicizumab is a humanized monoclonal antibody which binds to the coagulation factors (F) IX/IXa and X. By connecting FIXa and FX, Emicizumab mimics the cofactor function of FVIIIa, which is deficient in patients with congenital or acquired hemophilia A. Monitoring of Emicizumab has been done in clinical trials using methods that are not commercially available. So far, Emicizumab plasma levels have not been associated with clinical outcomes and monitoring is not routinely recommended. However, the ability to monitor the drug may be useful in difficult cases, lack of efficacy, intoxication or unexpected adverse events. Emicizumab cannot be monitored with standard APPT-based one-stage factor VIII clot assay (OS), because it does not need to be activated like natural FVIII, resulting in a serious overestimation of its effect. However, a modified OS has been proposed using a high sample dilution (1:80) and Emicizumab as a calibrator (r2 Diagnostics, South Bend, IN, USA, distributed by Haemochrom Diagnostica GmbH). Chromogenic substrate assays (CS) using bovine FIX and FX are not able to detect Emicizumab activity because of lack of cross-species reactivity. However, a commercially available CS using human FIX and FX is available (Biophen FVIII assay, distributed by CoaChrom Diagnostica GmbH). Here we compared Emicizumab detection by these assays using spiked plasma and clinical samples from inhibitor patients. We found the modified OS assay able to quantify Emicizumab plasma concentrations between 10 and 100 μg/ml. The choice of APTT reagent and dilution buffer resulted in variable precision in particular for higher Emicizumab concentrations. The best combination of APTT reagent and buffer was found with a repeatability coefficient of variation (CVr ) of 8.47% and 3.1% for high and low Emicizumab concentrations, respectively. Measurements on different days using the same lots of reagents yielded a reproducibility coefficient of variation (CVR ) of 10.64% and 13.26% for high and low Emicizumab concentrations, respectively. The Biophen CS assay was able to quantify Emicizumab plasma concentrations between 10 and 150 μg/ml. The Emicizumab calibration curve was more dynamic for higher drug concentrations compared to the modified OS assay. We found a CVr of 3.88% and 5.59% for high and low Emicizumab concentrations, respectively. Both assays are not specific for Emicizumab. Human FVIII or porcine FVIII added to Emicizumab samples yielded false high results with both assays. In summary, Emicizumab can be monitored in with sufficient precision using a modified OS or CS assay. Choice of APTT and buffer reagents may affect the precision of OS assay. Human or porcine FVIII will disturb quantification of Emicizumab with these assays.