Selective Measurement of Human Factor VIII Activity in the Citrated Plasma of Laboratory Animals

Objectives: Nonclinical pharmacokinetic analyses monitoring the activity of human coagulation factors are often hampered by the presence of endogenous coagulation factors. Correction for the endogenous activity, measured before administration of human coagulation factor, is usually carried out to overcome this limitation. This approach, however, can introduce considerable bias, observed especially at terminal phases, where the difference between endogenous and administered coagulation factor reaches a minimum. Therefore, we developed a chromogenic activity assay, specific for human factor VIII (FVIII).

Methods: Screening of commercially available anti-FVIII antibodies resulted in the identification of the murine IgG1 (clone GMA-8024, Green Mountain Antibodies), which is described to bind to the A2 domain of human FVIII. Plate-adsorbed antibody facilitated the specific capture of human FVIII. Selectively bound human VIII was then measured with a conventional chromogenic activity assay. B domain-deleted (BDD) human FVIII was used as assay standard, diluted in buffer, and also measured after being spiked to 10% citrated plasma samples of laboratory animals.

Results: The six-point calibration curve ranged from 2.9 to 94 mU BDD FVIII/mL with the back-fitted data demonstrating 100 ± 20% agreement to nominal values. Citrated plasma samples (cynomolgus and rhesus monkeys, sheep, goat, rabbit and rat) and serum samples (mouse, swine dog, hamster and guinea pig) showed no response when measured in the dilution 1/10. Also purified recombinant porcine FVIII did not elicit any response. In contrast, BDD-FVIII spiked to these plasma/serum samples at 100 mU/mL was recovered at rates ranging from 83.3% to 116.9.

Conclusions: The combination of antibody-mediated capture of human FVIII and chromogenic activity assay resulted in the selective and sensitive measurement of human FVIII with no interference by endogenous FVIII.