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.

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models.

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Activation loop 3 and the 170 loop interact in the active conformation of coagulation factor VIIa.

The initiation of blood coagulation involves tissue factor (TF)-induced allosteric activation of factor VIIa (FVIIa), which circulates in a zymogen-like state. In addition, the (most) active conformation of FVIIa presumably relies on a number of intramolecular interactions. We have characterized the role of Gly372(223) in FVIIa, which is the sole residue in activation loop 3 that is capable of forming backbone hydrogen bonds with the unusually long 170 loop and with activation loop 2, by studying the effects of replacement with Ala [G372(223)A]. G372A-FVIIa, both in the free and TF-bound form, exhibited reduced cleavage of factor X (FX) and of peptidyl substrates, and had increased K(m) values compared with wild-type FVIIa. Inhibition of G372A-FVIIa.sTF by p-aminobenzamidine was characterized by a seven-fold higher K(i) than obtained with FVIIa.sTF. Crystallographic and modelling data suggest that the most active conformation of FVIIa depends on the backbone hydrogen bond between Gly372(223) and Arg315(170C) in the 170 loop. Despite the reduced activity and inhibitor susceptibility, native and active site-inhibited G372A-FVIIa bound sTF with the same affinity as the corresponding forms of FVIIa, and burial of the N-terminus of the protease domain increased similarly upon sTF binding to G372A-FVIIa and FVIIa. Thus Gly372(223) in FVIIa appears to play a critical role in maturation of the S1 pocket and adjacent subsites, but does not appear to be of importance for TF binding and the ensuing allostery.