Modification of Factor VIII in Therapeutic Concentrates after Virus Inactivation by Solvent-Detergent and Pasteurisation

Summary

The addition of a pasteurisation step to a solvent/detergent (SD) treated FVIII concentrate has recently resulted in enhanced inhibitor incidence in patients in Germany and Belgium. We have investigated the effect of virus inactivation procedures on FVIII function by preparing experimental concentrates from the same starting cryoprecipitate with the following procedures: none (N); dry heat (DH); pasteurisation (P); solvent/detergent (SD); solvent detergent + dry heat (SDDH); solvent detergent + pasteurisation (SDP). In addition, several clinical SD concentrates with and without pasteurisation were studied.

There were no significant differences in fibrinogen and vWF content and in the ratio of one-stage/chromogenic FVIII activity among any of the samples studied. In thrombin proteolysis and FXa generation experiments, there were no differences in results on samples N, DH, P, and SDDH from those on sample SD. However sample SDP gave markedly different results from sample SD in the following respects: slower thrombin proteolysis (t½2 = 12.0 min vs 1.9 min); more rapid FXa generation (rate 2.5 times that of SD); enhanced phospholipid binding (K_D = 3.89 × 10^–11M vs 5.53 × 10^–10M). Similar differences between SDP and SD were seen in the clinical samples.

The observed changes in the FVIII activity occurred in combination with SD and pasteurisation, but not with either treatment alone. These results suggest that SDP treatment may enhance exposure of the phospholipid binding site in the C2 domain of FVIII, and since inhibitors to the SDP product are predominantly against C2, these findings could be relevant to the enhanced immunogenicity of the SDP product.

• References

• 1 Krenz W, Escuriola-Ettingshausen C, Martinez-Saguer Güngör T, Korn-huber B. Epidemiology of inhibitors in haemophilia A. Vox Sang 1996; 70 (suppl): 2-8.
• 2 Peerlinck K, Rosendaal FR, Vermylen J. Incidence of inhibitor development in a group of young hemophilia A patients treated exclusively with lyophilized cryoprecipitate. Blood 1993; 81: 3332-5.
• 3 Peerlinck K, Arnout J, Giambattista MD, Gilles JG, Laub R, Jacquemin M, Saint-Remy JMR, Vermylen J. Factor VIII inhibitors in previously treated haemophilia A patients with a double virus-inactivated plasma derived factor VIII concentrate. Thromb Haemost 1997; 77: 80-6.
• 4 Rosendaal FR, Nieuwenhuis HK, van den Berg HM, Heijboer H, MauerBunschoten EP, van der Meer J, Smit C, Strengers PFW, Briët E. The Dutch Haemophilia Study Group. A sudden increase in factor VIII inhibitor development in multitransfused haemophilia A patients in the Netherlands. Blood 1993; 81: 2180-6.
• 5 Peerlinck K, Arnout J, Gilles JG, Saint-Remy JMR, Vermylen J. A higher than expected incidence of factor VIII inhibitors in multitransfused haemophilia A patients tested with an intermediate purity pasteurised factor VIII concentrate. Thromb Haemost 1993; 69: 115-8.
• 6 Saldanha J, Minor P. Detection of human parvovirus B19 DNA in plasma pools and blood products derived from these pools: implications for efficiency and consistency of removal of B19 DNA during manufacture. Br J Haematol 1996; 93: 714-9.
• 7 Vermylen J, Peerlinck K. Review of the hepatitis A epidemics in haemophiliacs in Europe. Vox Sang 1994; 67: 8-11.
• 8 Ruymann FB, Krill CE, Halpin TJ, Churchill WH, Ewenstein B, DeMaria A, MancoJohnson MJ, Hoffman RE. Hepatitis A among persons with haemophilia who received clotting factor concentrate. MMWR 1996; 45: 29-32.
• 9 Schwinn H, Stadler M, Josic Dj, Bal F, Gehringer W, Nur I, Schütz R. A solvent/detergent treated, pasteurised and highly purified factor VIII concentrate. Arzneim Forsch/Drug Res 1994; 44: 188-91.
• 10 Laub R, Giambattista MD, Fondu P, Brackmann HH, Lenk H, Scandella D. Restricted epitope specificity for factor VIII inhibitors which appeared in previously treated haemophiliacs after infusion with OCTAVI SD plus. Thromb Haemost 1997; suppl: 590.
• 11 Barrowcliffe TW. Inhibitor development and activated factor VIII in concentrates. Thromb Haemost 1993; 70: 1065-6.
• 12 Schwinn H, Smith A, Wolter D. Progress in purification of virus-inactivated factor VIII concentrates. Three generations of solvent/detergent treated plasma derivatives. Arzneim Forsch/Drug Res 1989; 39: 1302-5.
• 13 Barrowcliffe TW, Stocks J, Gray E. Preparation of a stable phospholipid reagent for coagulation assays. Haemostasis 1982; 11: 96-101.
• 14 Modi GJ, Blajchman MA, Ofosu FA. The isolation of prothrombin, factor IX and factor X from human FIX concentrates. Thromb Res 1984; 36: 537-47.
• 15 Gray E, Walker D, Heath A, Barrowcliffe TW. Collaborative study on assays of activated FIX (FIXa). On behalf of the factor VIII and factor IX subcommittee of the International Society on Thrombosis and Haemostasis (ISTH). Thromb Haemost 1996; 76: 1114-7.
• 16 Investigation of a prolonged activated partial thromboplastin time. In: Laboratory Haematology: an account of laboratory technique. Chanarin I. ed. Churchill Livingstone; UK: 1989. pp 293-306.
• 17 Kemball-Cook G, Tubbs JE, Dawson NJ, Barrowcliffe TW. The behaviour of different factor VIII concentrates in a chromogenic factor X-activating system. Brit J Haematol 1993; 84: 273-8.
• 18 van Dieijen G, van Rijn JLML, Govers-Riemslag JWP, Hemker HC, Rosing J. Assembly of the intrinsic factor X activating complex: interactions between factor IXa, factor VIIIa and phospholipid. Thromb Haemost 1985; 53: 396-400.
• 19 Mertens K, van Wijngaarden A, Bertina RM. The role of factor VIII in the activation of human blood coagulation factor X by activated factor IX. Thromb Haemost 1985; 54: 654-60.
• 20 Jönsson U, Fagerstam L, Ivarson B, Johnsson B, Karlsson R, Lundh K, Lofas S, Persson B, Roos H, Ronnberg I, Sjolander S, Stenberg E, Stahlberg R, Urbaniczky C, Ostlin H, Malmquist M. Real-time biospecific interaction analysis using surface plasmon resonance and sensor chip technology. Biotechniques 1991; 11: 620-7.
• 21 Karlsson R, Michaelsson A, Mattsson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immun Methods 1991; 145: 229-40.
• 22 MacDonald RC, MacDonald RI, Menco BPM, Takeshita K, Subbarao NK, Hu LR. Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta 1991; 1061: 297-303.
• 23 BIAtechnology NOTE 106, BIAcore AB. 1995 pp 1-4.
• 24 BIAcore AB. Evaluating kinetic data. In: BIAevaluation 2.1 Software Handbook. BIAcore AB 1995; pp 4.1-4.24.
• 25 Raut S, Barrowcliffe TW. Interaction of factor VIII with phospholipid (PC:PS 3:1): a kinetic evaluation using surface plasmon resonance (SPR) technology. (In preparation).
• 26 Biggs R, Austen DEG, Denson KWE, Borrett R, Rizza CR. The mode of action of antibodies which destroy factor VIII. II. Antibodies which give complex concentration graphs. Brit J Haematol 1972; 23: 137-55.
• 27 Scandella D, Gilbert GE, Shima M, Nakai H, Eagleson C, Felch M, Prescott R, Rajalakshmi KJ, Hoyer LW, Saenko E. Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site. Blood 1995; 86: 1811-9.
• 28 Prescott R, Nakai H, Saenko EL, Scharrer I, Nilsson IM, Humphries JE, Hurst D, Bray G, Scandella D. The inhibitor antibody response is more complex in hemophilia A patients than in most nonhemophiliacs with factor VIII autoantibodies. Recombinate and Kogenate Study Groups. Blood 1997; 89: 3663-71.
• 29 Hoyer LW, Scandella D. Factor VIII inhibitors: structure and function in autoantibody and hemophilia A patients. Semin Hematol 1994; 31: 1-5.
• 30 Scandella D, Mattingly M, Prescott R. A recombinant factor VIII A2 domain polypeptide quantitatively neutralizes human inhibitor antibodies that bind to A2. Blood 1993; 82: 1767-75.
• 31 Scandella D, Mattingly G, de Graaf S, Fulcher CA. Localisation of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralisation. Blood 1989; 74: 1618-26.
• 32 Rosendaal FR. Factor VIII inhibitors on a SD-treated and pasteurized concentrate associated with specific batches and batch characteristics. Thromb Haemost 1997; suppl: 590.
• 33 Kemball-Cook G, Edwards SJ, Sewerin K, Andersson LO, Barrowcliffe TW. Factor VIII procoagulant protein interacts with phospholipid vesicles via its 80 kDa light chain. Thromb Haemost 1988; 60: 442-6.
• 34 Foster PA, Fulcher CA, Houghten RA, Zimmerman TS. Synthetic factor VIII peptides with amino acids sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine. Blood 1990; 75: 1999-2004.
• 35 Josic D, Stadler M, Buchacher A, Schulz P, Schütz R, Wiry G, Schwinn H. Biochemical and immunochemical investigations of different plasma-derived and recombinant factor VIII products. Thromb Haemost 1997; suppl: 512.
• 36 Barrowcliffe TW, Dawson NJ, Kemball-Cook G. Activated factor VIII in concentrates prepared by monoclonal antibody and recombinant technology. Thromb Haemost 1989; 62 (1) (suppl): 198.
• 37 Tubbs JE, Kemball-Cook G, Barrowcliffe TW. Effects of von Willebrand factor and phospholipid on co-factor activity of factor VIII in concentrates. Thromb Haemost 1991; 65 (6) (suppl): 944.
• 38 Hill-Eubanks DC, Lollar P. von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain. J Biol Chem 1990; 265 (29) 17854-8.
• 39 Saenko EL, Scandella D. A mechanism for inhibition of factor VIII binding to phospholipid by von Willebrand factor. J Biol Chem 1995; 270: 13826-33.
• 40 Yee TT, Williams MD, Hill FGH, Lee CA, Pasi KJ. Absence of inhibitors in previously untreated patients with severe haemophilia A after exposure to a single intermediate purity factor VIII product. Thromb Haemost 1997; 78: 1027-9.