ABSTRACT
Background: It is essential to measure the activity of factor VIII (FVIII) throughout the life cycle of a coagulation FVIII concentrate. Such measurement in nonclinical pharmacokinetic studies is potentially biased by the presence of endogenous nonhuman FVIII, and certain manufacturing process-related additives can also impact the assay performance. Finally, the presence of FVIII activity-mimicking antibodies poses challenges when measuring FVIII in samples. Therefore, we developed an antibody-based chromogenic FVIII assay, which facilitates the selective and sensitive activity measurement of human FVIII in the presence of animal plasma and interfering agents. Methods: Plate-bound monoclonal anti-FVIII antibody specifically captured human FVIII, which was then measured with a chromogenic activity assay. A human reference plasma preparation was used to construct the calibration curve. Spike recovery was carried out in a citrated cynomolgus monkey plasma-solvent/detergent mixture and in the presence of the bispecific antibody emicizumab. Results: The calibration curve ranged from 3.03 to 97.0 mIU FVIII/ml and was obtained repeatedly with good accuracy. B domain-deleted and full-length FVIII did not differ in their responses. Recovery of spiked human FVIII in citrated cynomolgus monkey plasma was 102.7%, while neither native monkey plasma nor the other animal specimen tested showed any activity. Solvent/detergent solution and the bispecific antibody emicizumab had no influence on the assay. Conclusion: Combining antibody-mediated specific capture of human FVIII and a chromogenic activity assay resulted in a selective and sensitive measurement of human FVIII with no interference by endogenous, nonhuman FVIII, manufacturing process additives, or an FVIII activity-mimicking antibody.
