Underestimation of N-glycoPEGylated factor IX one-stage clotting activity owing to contact activator-impaired activation

Background: In clinical practice, factor IX (FIX) activity is routinely quantified by measurement of the activated partial thromboplastin time (APTT) in a one-stage (OS) FIX clotting assay. APTT reagents provide a contact activator and phospholipid surfaces required for triggering and sustaining the plasma clotting process. The large diversity in reagent components is reflected in the variable recovery of nonacog beta pegol (N9-GP; N-glycoPEGylated recombinant FIX) activity when assayed against a FIX standard. This variation warrants mechanistic studies and is plausibly attributable to the nature and amount of contact activator. Objective: To identify the cause of the N9-GP activity underestimation observed with a heterogeneous group of APTT reagents. Methods: Experiments mimicking the clotting phase (omitting the contact activation phase) of the OS assay, complemented by measurements of activated factor XI (FXIa) activity, were performed to characterize and explain the influence of APTT reagents/contact activators on the conversion of N9-GP and regular FIX (N9) to activated FIX (FIXa). Results: In the presence of an intact underestimating APTT reagent or the isolated contact activator, clotting phase activation of N9-GP proceeded at a reduced rate compared with that of N9. APTT reagent and contact activator negatively affected the activity of FXIa, conceivably as a consequence of FXIa adsorption. Thus, activation of FIX apparently poses a greater steric challenge after polyethylene glycol (PEG) conjugation. Conclusions: Some OS clotting assay contact activators reduce FXIa-mediated activation of N9-GP to a larger degree than that of N9, causing underestimation of N9-GP activity of potential clinical significance.