CC BY 4.0 · TH Open 2019; 03(02): e123-e131
DOI: 10.1055/s-0039-1688413
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Molecular Aggregation of Marketed Recombinant FVIII Products: Biochemical Evidence and Functional Effects

Raimondo De Cristofaro
1   Haemorrhagic and Thrombotic Diseases Service, Area of Hematology, Fondazione Policlinico Universitario “A. Gemelli,” IRCCS, Rome, Italy
2   Institute di Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy
,
Monica Sacco
2   Institute di Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy
,
Stefano Lancellotti
1   Haemorrhagic and Thrombotic Diseases Service, Area of Hematology, Fondazione Policlinico Universitario “A. Gemelli,” IRCCS, Rome, Italy
,
Federico Berruti
2   Institute di Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy
,
Isabella Garagiola
3   Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Luigi Villa Foundation, Milan, Italy
,
Carla Valsecchi
2   Institute di Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy
,
Maria Basso
1   Haemorrhagic and Thrombotic Diseases Service, Area of Hematology, Fondazione Policlinico Universitario “A. Gemelli,” IRCCS, Rome, Italy
,
Enrico Di Stasio
4   Institute of Biochemistry and Clinical Biochemistry, Università degli Studi di Milano, Milan, Italy
,
Flora Peyvandi
3   Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Luigi Villa Foundation, Milan, Italy
5   Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy
› Institutsangaben
Funding This work was supported by an unrestricted research grant from Biotest AG (Dreieich, Germany), and by Italo Monzino Foundation.
Weitere Informationen

Publikationsverlauf

23. Februar 2019

08. März 2019

Publikationsdatum:
08. Mai 2019 (online)

Abstract

Background Recombinant (rec-) coagulation factor VIII concentrates available for hemophilia A (HA) treatment differ in cell line production and structure, which could affect their pharmacodynamics and immunogenicity. Clinical trials showed that previously untreated patients with severe HA present higher rates of inhibitor development if treated with rec-FVIII products and that differences do exist as to inhibitor's formation among different rec-FVIII products. This finding could arise from several causes, such as absence of von Willebrand factor, different glycosylation profiles, or processes of molecular aggregation of the recombinant FVIII molecules.

Objectives/Methods In this study, using size exclusion high-performance liquid chromatography (SE-HPLC), dynamic light scattering (DLS) spectroscopy, and functional biochemical assays, we investigated the purity grade, FX activating ability, and aggregation status of three recombinant marketed products (Advate [Baxalta], Refacto AF [Pfizer], and Kogenate [Bayer]).

Results The overall analysis of the results obtained with SE-HPLC and DLS spectroscopy showed that the three recombinant FVIII concentrates contain low but significant amounts of molecular aggregates. This phenomenon was less evident for the Advate product. Molecular aggregation negatively affects the in vitro pharmacodynamics of the concentrates with higher aggregates' content.

Conclusions This study shows that the three pharmaceutical formulations of recombinant FVIII contain variable amounts of molecular aggregates after their reconstitution at therapeutic concentrations. This phenomenon negatively affects the in vitro potency of the products with higher aggregates' content and might be invoked as a contributing cause of their increased risk to induce the formation of FVIII inhibitors.

Authors' Contributions

Monica Sacco, Stefano Lancellotti, and Federico Berruti performed the biochemical experiments and critically read the manuscript. Enrico Di Stasio performed some DLS experiments and analyzed the results of these assays. Isabella Garagiola and Carla Valsecchi analyzed the VWF content of the pd-FVIII products and critically read the manuscript. Flora Peyvandi critically read the manuscript and provided experimental suggestions. Raimondo De Cristofaro designed the study, performed some biochemical experiments, analyzed the data, and wrote the manuscript.


 
  • References

  • 1 Lai JD, Swystun LL, Cartier D. , et al. N-linked glycosylation modulates the immunogenicity of recombinant human factor VIII in hemophilia A mice. Haematologica 2018; 103 (11) 1925-1936
  • 2 European Medicines Agency. Guideline on immunogenicity assessment of therapeutic proteins. Committee for Medicinal Products for Human Use (CHMP), May 18, 2017 (EMEA/CHMP/BMWP/14327/2006 Rev1). Available at: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-immunogenicity-assessment-therapeutic-proteins-revision-1_en.pdf . Accessed April 8, 2019
  • 3 Brooker M. Registry of Clotting Factor Concentrates. 9th ed. World Federation of Hemophilia Registry; Montreal, QC, Canada: 2012
  • 4 Chtourou S, Porte P, Nogré M. , et al. A solvent/detergent-treated and 15-nm filtered factor VIII: a new safety standard for plasma-derived coagulation factor concentrates. Vox Sang 2007; 92 (04) 327-337
  • 5 Peyvandi F, Mannucci PM, Garagiola I. , et al. A randomized trial of factor VIII and neutralizing antibodies in hemophilia A. N Engl J Med 2016; 374 (21) 2054-2064
  • 6 Collins PW, Palmer BP, Chalmers EA. , et al; UK Haemophilia Centre Doctors' Organization. Factor VIII brand and the incidence of factor VIII inhibitors in previously untreated UK children with severe hemophilia A, 2000-2011. Blood 2014; 124 (23) 3389-3397
  • 7 Calvez T, Chambost H, Claeyssens-Donadel S. , et al; FranceCoag Network. Recombinant factor VIII products and inhibitor development in previously untreated boys with severe hemophilia A. Blood 2014; 124 (23) 3398-3408
  • 8 Butenas S, Parhami-Seren B, Gissel MT, Gomperts ED, Fass DN, Mann KG. Potency and mass of factor VIII in FVIII products. Haemophilia 2009; 15 (01) 63-72
  • 9 Gangadharan B, Ing M, Delignat S. , et al. The C1 and C2 domains of blood coagulation factor VIII mediate its endocytosis by dendritic cells. Haematologica 2017; 102 (02) 271-281
  • 10 Schmitz KS. Introduction to Dynamic Light Scattering by Macromolecules. 1st ed. San Diego, CA: Academic Press; 1990
  • 11 Di Stasio E, Romitelli F, Lancellotti S, Arcovito A, Giardina B, De Cristofaro R. Kinetic study of von Willebrand factor self-aggregation induced by ristocetin. Biophys Chem 2009; 144 (03) 101-107
  • 12 Pecora R. Dynamic Light Scattering: Applications of Photon Correlation Spectroscopy. New York, NY: Plenum Press; 1985
  • 13 Lorber B, Fischer F, Bailly M, Roy H, Kern D. Protein analysis by dynamic light scattering: methods and techniques for students. Biochem Mol Biol Educ 2012; 40 (06) 372-382
  • 14 de Cristofaro R, Rocca B, Bizzi B, Landolfi R. The linkage between binding of the C-terminal domain of hirudin and amidase activity in human alpha-thrombin. Biochem J 1993; 289 (Pt 2): 475-480
  • 15 Fay PJ. Activation of factor VIII and mechanisms of cofactor action. Blood Rev 2004; 18 (01) 1-15
  • 16 Healey JF, Parker ET, Lollar P. Identification of aggregates in therapeutic formulations of recombinant full-length factor VIII products by sedimentation velocity analytical ultracentrifugation. J Thromb Haemost 2018; 16 (02) 303-315
  • 17 Oldenburg J, Pavlova A. Genetic risk factors for inhibitors to factors VIII and IX. Haemophilia 2006; 12 (Suppl. 06) 15-22
  • 18 Peng A, Gaitonde P, Kosloski MP, Miclea RD, Varma P, Balu-Iyer SV. Effect of route of administration of human recombinant factor VIII on its immunogenicity in Hemophilia A mice. J Pharm Sci 2009; 98 (12) 4480-4484
  • 19 Lövgren KM, Søndergaard H, Skov S, Wiinberg B. Non-genetic risk factors in haemophilia A inhibitor management - the danger theory and the use of animal models. Haemophilia 2016; 22 (05) 657-666
  • 20 Álvarez T, Soto I, Astermark J. Non-genetic risk factors and their influence on the management of patients in the clinic. Eur J Haematol 2015; 94 (Suppl. 77) 2-6
  • 21 Peyvandi F, Garagiola I. Product type and other environmental risk factors for inhibitor development in severe hemophilia A. Res Pract Thromb Haemost 2018; 2 (02) 220-227
  • 22 Bachmann MF, Rohrer UH, Kündig TM, Bürki K, Hengartner H, Zinkernagel RM. The influence of antigen organization on B cell responsiveness. Science 1993; 262 (5138): 1448-1451
  • 23 Moussa EM, Panchal JP, Moorthy BS. , et al. Immunogenicity of therapeutic protein aggregates. J Pharm Sci 2016; 105 (02) 417-430
  • 24 Goudemand J, Rothschild C, Demiguel V. , et al; FVIII-LFB and Recombinant FVIII study groups. Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A. Blood 2006; 107 (01) 46-51
  • 25 Gouw SC, van den Berg HM, Fischer K. , et al; PedNet and Research of Determinants of INhibitor development (RODIN) Study Group. Intensity of factor VIII treatment and inhibitor development in children with severe hemophilia A: the RODIN study. Blood 2013; 121 (20) 4046-4055
  • 26 Calvez T, Chambost H, d'Oiron R. , et al; for FranceCoag Collaborators. Analyses of the FranceCoag cohort support differences in immunogenicity among one plasma-derived and two recombinant factor VIII brands in boys with severe hemophilia A. Haematologica 2018; 103 (01) 179-189