Robust and selective chromogenic measurement of factor VIII activity with an antibody-based factor VIII chromogenic assay

Objective Chromogenic factor VIII (FVIII) activity measurement is essential through the whole life cycle of a coagulation factor concentrate or a gene therapy-based treatment approach. While the presence of endogenous non-human FVIII potentially biases the nonclinical pharmacokinetic study analysis, also certain manufacturing process-related additives can impact the assay performance. In particular, those used for the solvent/detergent viral inactivation process interfere with the assay. 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. Furthermore, this assay enabled the reliable measurement of FVIII activity even in presence of the solvent/detergent mixture.

Material and Methods Plate-adsorbed murine IgG1 (GMA-8024, Green Mountain Antibodies), binding to the A2 domain of human FVIII, was used. Bound human FVIII was measured with a chromogenic activity assay. A human reference plasma preparation was used to construct the calibration curve. Spike-recovery was carried out in citrated cynomolgus monkey plasma and solvent/detergent mixture. Assay robustness was confirmed by the results of the assay control obtained by three different analysts.

Results The six-point calibration curve ranged from 3.03 to 97.0 mIU/mL with the back-fitted data for a total of 108 curves demonstrating 100 ± 14% agreement to the nominal values. Recovery of spiked human FVIII (about 80 mIU/mL) in citrated cynomolgus monkey plasma was 102.7% and 92.4% for the full length and B domain-deleted preparation, respectively, while native monkey plasma did not show any activity. Relative standard deviations (RSDs) for the mean of the triplicate spikes did not exceed 2.8%. Solvent/detergent solution (1% Triton X-100, 0.3% polysorbate 80 and 0.3% tri-n-butyl phosphate) was shown to have no influence on the assay. Finally, assay robustness was demonstrated by the data obtained for the assay control: 108 tests resulted in an RSD of 10.4% with no statistically significant difference between the results obtained by three analysts.

Conclusion Combining antibody-mediated specific capture of human FVIII and a chromogenic activity assay resulted in the selective and sensitive measurement of human FVIII with no interference by endogenous, non-human FVIII or manufacturing process additives like solvent/detergent solution.

Publication History

Article published online:
18 June 2021