FIXing postinfusion monitoring: Assay experiences with N9-GP (nonacog beta pegol; Refixia^® ; Rebinyn^® )

N9-GP (nonacog beta pegol; Refixia^® ; Rebinyn^® , Novo Nordisk A/S, Bagsvaerd, Denmark) is a glycoPEGylated extended half-life recombinant factor IX (rFIX) that exhibits efficacy and potency comparable to unmodified FIX molecules in non-clinical models. Phase 3 clinical trials have confirmed the efficacy and tolerability of N9-GP for the prevention and on-demand treatment of bleeding episodes in patients with haemophilia B. Recent studies have shown that PEGylation affects clotting times in activated partial thromboplastin time (aPTT)-based one-stage activity assays due to interaction between the FIX molecule and certain aPTT reagents. In recognition of the challenges surrounding FIX activity assessment, the identification of consistent, reproducible and accurate assays to measure FIX activity has been a priority for Novo Nordisk, running in parallel to the clinical development program for N9-GP. N9-GP activity can be reliably measured using chromogenic substrate assays and specific aPTT reagents. The conjugation of the PEG moiety to the FIX molecule may affect one-stage aPTT-based clotting assays in a reagent-dependent manner. Many aPTT reagents that use silica as the contact activator dramatically overestimate N9-GP activity due to premature activation. On the other hand, the contact activator in some other aPTT reagents negatively affects the enzymatic activity of FXIa, causing the underestimation of N9-GP activity. While N9-GP activity cannot be measured consistently with all available aPTT reagents, accurate N9-GP measurements can be achieved with certain aPTT reagents. Here, we review the studies that led to these findings and summarize the current options for accurate measurement of N9-GP in patient samples.