Performance of a recombinant fusion protein linking coagulation factor IX with recombinant albumin (rIX-FP) in the one-stage assay

A. Veldman1, A. Feussner1, U. Kalina1, K. St. Ledger2 (1Marburg, Germany, 2King of Prussia, PA, USA)

Laboratory tests
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) has been developed to have an improved pharmacokinetic profile compared with standard FIX products, allowing less frequent dosing. During the PROLONG-9FP development program for rIX-FP, a reagent-dependent activity determination was observed in the one-stage (OS) clotting assay. The variability of different OS assay reagents for the activity measurement of rIX-FP was assessed in an international, multicenter field study. In addition, samples from pediatric and adult subjects within the clinical trial program were measured in parallel at local and central laboratories.

Methods: Centers participating in the rIX-FP Phase I/II clinical trial as well as five external laboratories were provided with lyophilized rIX-FP reference plasma for reconstitution and dilution to 0.25, 0.5 and 0.9 IU/mL. Centers participating in the rIX-FP Phase II/III trial (n=8) were provided with rIX-FP plasma samples spiked at concentrations of 0.1, 0.25 and 0.625 IU/mL and assayed samples using the local OS assay with the instrumentation and reagents used routinely in their laboratory. These laboratories, where samples were measured locally, were encouraged to send a paired sample to CSLB’s central laboratory for analysis (n=21).

Results: For both the field study and paired sample analysis, plasma samples at all concentrations showed comparable results across different clinical laboratories using a variety of aPTT reagents. However, when Actin FS and CK Prest (a kaolin-based reagent) were used for analysis, rIX-FP activity was underestimated by approximately 50%.

Conclusion: Following infusion of rIX-FP, plasma FIX activity can be monitored using the OS assay to confirm adequate FIX levels have been achieved and maintained. Results obtained from both the field study and paired sample analysis demonstrated that although the majority of commercially available reagents for the one-stage clotting assay will provide accurate measurement of rIX-FP activity, use of a kaolin-based or Actin FS aPTT reagent is likely to result in an underestimation of FIX activity of up to 50%.