Overestimation of N‐glycoPEGylated factor IX activity in a one‐stage factor IX clotting assay owing to silica‐mediated premature conversion to activated factor IX

Abstract

*Rossix AB, M€ olndal, Sweden; and †Haemophilia Biology, Novo Nordisk A/S, M aløv, DenmarkTo cite this article: Ros en P, Ros en S, Ezban M, Persson E. Overestimation of N-glycoPEGylated factor IX activity in a one-stage factor IX clotting assay owing to silica-mediated premature conversion to activated factor IX. J Thromb Haemost 2016; 14: 1420-7. Essentials• Nonacog beta pegol (N9-GP) activity is overestimated in clot method using silica-based reagents.• Mimicking contact activation phase with silica reveals N9-GP activation before recalcification.• Localization of N9-GP to silica facilitates activation by factor XIa and plasma kallikrein.• Silica-based reagents to be used with caution when monitoring N9-GP therapy using clot method.Summary. Background: Clinical laboratories routinely quantify factor IX (FIX) activity by measurement of the activated partial thromboplastin time (APTT) in a onestage (OS) clotting assay. This assay can be performed with any of a plethora of differently composed APTT reagents, giving variable recovery when applied to nonacog beta pegol (N9-GP), an N-glycoPEGylated recombinant FIX. Objective: To identify the cause of observed overestimations of N9-GP activity in an OS FIX clotting assay when most APTT reagents containing silica are used as the contact activator, and to elucidate the underlying mechanism. Methods: Experiments mimicking the contact activation and clotting phases of the OS assay, combined with the use of plasmas with various deficiencies, were employed to shed light on the unique behavior of N9-GP. Confirmatory activations of N9-GP with purified enzymes and physical adsorption to silica particles were studied, and the influence of free polyethylene glycol (PEG) on these processes was investigated. Results: N9-GP, but not native FIX, added to FIX-deficient plasma was prematurely converted to activated FIX (FIXa) during the contact activation phase of the clotting assay. Activated FXI (FXIa) and plasma kallikrein (PK) were responsible for the activation of N9-GP, an event that appeared to require the presence of a silica-containing APTT reagent. PEG-dependent adsorption of N9-GP to silica particles could be demonstrated. Conclusions: The PEG moiety mediates colocalization of N9-GP with its activators FXIa and PK on silica surfaces, thereby facilitating premature conversion of N9-GP to FIXa during the contact activation phase, and leading to overestimation of the FIX activity in the OS clotting assay.

Performance of a recombinant fusion protein linking coagulation factor IX with recombinant albumin in one‐stage clotting assays

“…These constituents may also explain, to some extent, the discrepancies observed between assays. Overall, the findings presented in this study reinforce the suggestion that certain APTT reagents utilized in OSC assays lead to discrepancies in the assessments of FIX:C in samples containing FIX‐DPL and various rFIX products .…”

“…However, a small number of specific APTT reagents, e.g. SynthAFax, DG Synth, and STA‐Cephascreen, which show acceptable recovery, have been identified for monitoring N9‐GP , as have suitable reagent instrument systems .…”

New Tools to Study Contact Activation

“…The demonstrated stability of FXIa, FXIIa, and kallikrein in MES–BSA buffer pH 5.7 may be conveniently utilized in, e.g., studies on activation kinetics where subsampling and extensive dilutions in MES–BSA buffer followed by chromogenic FXIa determination provides a sensitive and robust tool for such studies (19). …”

“…These products were originally developed for detection of trace amounts of FXIa as contaminant in pharmaceutical products such as intravenous immunoglobulins. However, the chromogenic FXIa method has also been applied for investigation of FXI activation during contact activation on analysis of a one-stage method for potency assignment of the extended half-life rFIX product N9-GP (19). Here, subsampling and dilution into a low pH buffer was used to quench further activation.…”

Recombinant FIX Fc fusion protein activity assessment with the one‐stage clotting assay: A multicenter, assessor‐blinded, prospective study in Japan (J‐Field Study)

“…Furthermore, there is greater variability for some of the newer extended half‐life FIX products compared with the conventional rFIX products. For example, a PEGylated FIX product is incompatible with all commonly used silica‐based aPTT reagents for the one‐stage clotting assay, resulting in pronounced overestimation of plasma FIX levels . Such assay variability is of particular concern when accurate clinical monitoring is required (eg, during surgical procedures).…”