Selective human factor VIII activity measurement after analytical in-line purification

Area of Science:

• Biochemistry
• Pharmacology
• Analytical Chemistry

Background:

• Measuring factor VIII (FVIII) activity is crucial for coagulation FVIII concentrates.
• Assays can be biased by nonhuman FVIII, manufacturing additives, and FVIII-mimicking antibodies.
• Accurate FVIII measurement is vital for pharmacokinetic studies and quality control.

Purpose of the Study:

• To develop a selective and sensitive assay for human FVIII activity.
• To overcome interference from animal plasma, additives, and antibodies.
• To ensure accurate FVIII measurement in complex biological matrices.

Main Methods:

• Developed an antibody-based chromogenic assay using a plate-bound monoclonal anti-FVIII antibody for specific human FVIII capture.
• Utilized a human reference plasma for calibration curve construction.
• Validated the assay in cynomolgus monkey plasma with solvent/detergent treatment and emicizumab presence.

Main Results:

• The assay demonstrated good accuracy and reproducibility with a calibration curve from 3.03 to 97.0 mIU FVIII/ml.
• Both B domain-deleted and full-length FVIII showed similar responses.
• Spiked human FVIII recovery in monkey plasma was 102.7%, with no activity detected in native monkey plasma or from additives/emicizumab.

Conclusions:

• The developed assay selectively and sensitively measures human FVIII activity.
• It effectively eliminates interference from endogenous nonhuman FVIII, manufacturing additives, and FVIII-mimicking antibodies.
• This antibody-based chromogenic assay provides a reliable method for FVIII activity quantification.