Performance Evaluation of Different FIX Activity Assays for Determining Nonacog Beta Pegol (N9-GP, Refixia®) Activity Using Reagent-/Platform-Specific Conversion Factors

Abstract

Background

Nonacog beta pegol (N9-GP) is a glycoPEGylated FIX replacement product with extended half-life for treatment of haemophilia B patients. Monitoring of N9-GP with clotting-based one-stage FIX assays is complicated by high variations, mainly due to reagent-specific interference with polyethylene glycol.

Methods

In 11 distinct specialized coagulation laboratories in Austria, N9-GP spiked samples were measured in replicates in two distinct surveys, 3 years apart, using five different one-stage assay reagents and one chromogenic FIX assay. Regression analysis was used to investigate if back-calculation of N9-GP levels is feasible.

Results

We could demonstrate a linear relationship between the spiked N9-GP concentration and measured FIX activity levels for all examined assays, suggesting that N9-GP activity may be back-calculated using reagent-/platform-specific conversion factors. Within-laboratory variation after 3 years was acceptable in most, but not all, laboratories.

Conclusion

We demonstrate that back-calculation of N9-GP activity levels may be possible when using one-stage FIX assays. However, we recommend that every laboratory ascertain its own conversion factor. When measuring real patient samples, we encourage simultaneous measurement of N9-GP spiked control material with known concentrations to ensure the validity of the current back-calculation.

Keywords

coagulation factor IX - haemophilia B - nonacog beta pegol - Refixia - aPTT

Author Contributions

The potential linear relationship between the measured values, the interim reports prepared for the study participants, and the basis of the evaluation were developed and documented by AH. Under supervision of AH all authors contributed to the conception and design of the study. All authors except GL coordinated sample measurements in their respective laboratories. GL performed statistical analyses. CI wrote the original draft, and all other authors commented on the manuscript. All authors agree to be accountable for all aspects of the work. All authors read and approved the final manuscript.

Ethical Approval

Not applicable.

Consent to Participate

Not applicable.

Availability of Data and Materials

Data available on request from the authors.

Publication History

Received: 22 January 2025

Accepted: 05 May 2025

Article published online:
29 August 2025

• References

• 1 Bolton-Maggs PH, Pasi KJ. Haemophilias A and B. Lancet 2003; 361 (9371) 1801-1809
• 2 Iorio A, Stonebraker JS, Chambost H. et al; Data and Demographics Committee of the World Federation of Hemophilia. Establishing the prevalence and prevalence at birth of hemophilia in males: a meta-analytic approach using national registries. Ann Intern Med 2019; 171 (08) 540-546
• 3 Blanchette VS, Key NS, Ljung LR, Manco-Johnson MJ, van den Berg HM, Srivastava A. Subcommittee on Factor VIII, Factor IX and Rare Coagulation Disorders of the Scientific and Standardization Committee of the International Society on Thrombosis and Hemostasis. Definitions in hemophilia: communication from the SSC of the ISTH. J Thromb Haemost 2014; 12 (11) 1935-1939
• 4 Santagostino E, Mancuso ME, Tripodi A. et al. Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile. J Thromb Haemost 2010; 8 (04) 737-743
• 5 Kulkarni R, Presley RJ, Lusher JM. et al. Complications of haemophilia in babies (first two years of life): a report from the Centers for Disease Control and Prevention Universal Data Collection System. Haemophilia 2017; 23 (02) 207-214
• 6 Richards M, Lavigne Lissalde G, Combescure C. et al; European Haemophilia Treatment and Standardization Board. Neonatal bleeding in haemophilia: a European cohort study. Br J Haematol 2012; 156 (03) 374-382
• 7 Aviña-Zubieta JA, Galindo-Rodriguez G, Lavalle C. Rheumatic manifestations of hematologic disorders. Curr Opin Rheumatol 1998; 10 (01) 86-90
• 8 Syed YY. Nonacog beta pegol: a review in haemophilia B. Drugs 2017; 77 (18) 2003-2012
• 9 Srivastava A, Santagostino E, Dougall A. et al; WFH Guidelines for the Management of Hemophilia panelists and co-authors. WFH Guidelines for the Management of Hemophilia, 3rd edition. Haemophilia 2020; 26 (Suppl. 06) 1-158
• 10 Bowyer AE, Hillarp A, Ezban M, Persson P, Kitchen S. Measuring factor IX activity of nonacog beta pegol with commercially available one-stage clotting and chromogenic assay kits: a two-center study. J Thromb Haemost 2016; 14 (07) 1428-1435
• 11 Powell JS, Pasi KJ, Ragni MV. et al; B-LONG Investigators. Phase 3 study of recombinant factor IX Fc fusion protein in hemophilia B. N Engl J Med 2013; 369 (24) 2313-2323
• 12 Santagostino E, Martinowitz U, Lissitchkov T. et al; PROLONG-9FP Investigators Study Group. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial. Blood 2016; 127 (14) 1761-1769
• 13 Collins PW, Young G, Knobe K. et al; paradigm 2 Investigators. Recombinant long-acting glycoPEGylated factor IX in hemophilia B: a multinational randomized phase 3 trial. Blood 2014; 124 (26) 3880-3886
• 14 Negrier C, Knobe K, Tiede A, Giangrande P, Møss J. Enhanced pharmacokinetic properties of a glycoPEGylated recombinant factor IX: a first human dose trial in patients with hemophilia B. Blood 2011; 118 (10) 2695-2701
• 15 Østergaard H, Bjelke JR, Hansen L. et al. Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide. Blood 2011; 118 (08) 2333-2341
• 16 Negrier C, Young G, Abdul Karim F. et al; paradigm™ 2 and 4 Investigators. Recombinant long-acting glycoPEGylated factor IX (nonacog beta pegol) in haemophilia B: assessment of target joints in multinational phase 3 clinical trials. Haemophilia 2016; 22 (04) 507-513
• 17 Young G, Collins PW, Colberg T. et al. Nonacog beta pegol (N9-GP) in haemophilia B: a multinational phase III safety and efficacy extension trial (paradigm™4). Thromb Res 2016; 141: 69-76
• 18 Carcao M, Zak M, Abdul Karim F. et al. Nonacog beta pegol in previously treated children with hemophilia B: results from an international open-label phase 3 trial. J Thromb Haemost 2016; 14 (08) 1521-1529
• 19 Escobar MA, Tehranchi R, Karim FA. et al. Low-factor consumption for major surgery in haemophilia B with long-acting recombinant glycoPEGylated factor IX. Haemophilia 2017; 23 (01) 67-76
• 20 Pipe SW, Leebeek FWG, Recht M. et al. Gene therapy with etranacogene dezaparvovec for hemophilia B. N Engl J Med 2023; 388 (08) 706-718
• 21 Ezban M, Hermit MB, Persson E. FIXing postinfusion monitoring: assay experiences with N9-GP (nonacog beta pegol; Refixia^®; Rebinyn^® ). Haemophilia 2019; 25 (01) 154-161
• 22 Müller J, Miesbach W, Prüller F, Siegemund T, Scholz U, Sachs UJ. Standing Commission Labor (STAEKOLA) of the Society of Thrombosis and Haemostasis Research (GTH). An update on laboratory diagnostics in haemophilia A and B. Hamostaseologie 2022; 42 (04) 248-260
• 23 Kihlberg K, Strandberg K, Rosén S, Ljung R, Astermark J. Discrepancies between the one-stage clotting assay and the chromogenic assay in haemophilia B. Haemophilia 2017; 23 (04) 620-627
• 24 Sørensen MH, Andersen S, Ezban M. Factor IX-deficient plasma spiked with N9-GP behaves similarly to N9-GP post-administration clinical samples in N9-GP ELISA and FIX activity assays. Haemophilia 2015; 21 (06) 832-836
• 25 Rosén P, Rosén 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 (07) 1420-1427
• 26 Persson E, La Cour Christoffersen C. Underestimation of N-glycoPEGylated factor IX one-stage clotting activity owing to contact activator-impaired activation. Res Pract Thromb Haemost 2017; 1 (02) 259-263
• 27 Augustsson C, Norström E, Andersson NG, Zetterberg E, Astermark J, Strandberg K. Monitoring standard and extended half-life products in hemophilia: assay discrepancies for factor VIII and IX in pre- and postinfusion samples. Res Pract Thromb Haemost 2020; 4 (07) 1114-1120
• 28 Peyvandi F, Kenet G, Pekrul I, Pruthi RK, Ramge P, Spannagl M. Laboratory testing in hemophilia: impact of factor and non-factor replacement therapy on coagulation assays. J Thromb Haemost 2020; 18 (06) 1242-1255
• 29 Ovanesov MV, Williams SC, Nübling CM. et al. Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products. Biologicals 2020; 67: 88-93
• 30 Gray E, Kitchen S, Bowyer A. et al. Laboratory measurement of factor replacement therapies in the treatment of congenital haemophilia: a United Kingdom Haemophilia Centre Doctors' Organisation guideline. Haemophilia 2020; 26 (01) 6-16
• 31 Duboscq C, Sueldo E, Rosa C. et al. High variability in factor IX one-stage assay in samples spiked with nonacog beta pegol among different pairs of reagent/detection system. Int J Lab Hematol 2024; 46 (01) 128-134
• 32 Tiefenbacher S, Bohra R, Amiral J. et al. Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol. J Thromb Haemost 2017; 15 (10) 1901-1912
• 33 Holm PKSM, Hermit BM. et al. The activity of GlycoPEGylated recombinant FIX (N9-GP) can be measured in two-stage chromogenic and one-stage clotting assays. J Thromb Haemost 2013; 11 (Suppl. 02) 828
• 34 External quality Control of diagnostic Assays and Tests (ECAT) foundation. Available aPTT assays. Accessed November 16, 2024 at: https://ecat.nl/ecat-education/assays/aptt/
• 35 Hermit BMSM, Ezban M. et al. Pegylation of fix influences the clotting time in aptt assays. use of product specific standard eliminates peg interference. Haemophilia 2013; 19 (Suppl. 02) 45
• 36 European Parliament. Regulation (EU) 2017/746 of the European Parliament and of the Council of 5 April 2017 on in vitro diagnostic medical devices and repealing Directive 98/79/EC and Commission Decision 2010/227/EU. Accessed January 14, 2025 at: https://eur-lex.europa.eu/eli/reg/2017/746/oj