Validation of a Procedure for Potency Assessing of a High Purity Factor VIII Concentrate -Comparison of Different Factor VIII Coagulant Assays and Effect of Prediluent

Summary

The assessment of factor VIII coagulant activity (FVTII: C) in recently available highly purified and concentrated FVTII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVTII :C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVTII :C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVTII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVTII-deficient plasma used as prediluents. In order to validate our “in vitro” data we performed 6 “in vivo” analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.

• References

• 1 Langdell RD, Wagner RH, Brinkhous KM. Effect of anti-haemophilic factor on one-stage clotting tests. A presumptive test for hemophilia and a simple one-stage antihemophilic assay procedure. J Lab Clin Med 1953; 41: 637-647
• 2 Soulier JP, Larrieu MJ. Nouvelle méthode de diagnostic de l’hémophilie. Dosage des facteurs antihemophiliques A et B. Sang 1953; 24: 205-215
• 3 Biggs R, Eveling J, Richards G. The assay of antihemophilic globulin activity. Br J Haematol 1955; 1: 20-34
• 4 Seghatchian MJ, Miller-Anderson M. A colorimetric evaluation of factor VIII :C potency. Med Lab Sci 1978; 35: 347-354
• 5 Rosen S. Assay of factor VIII : C with a chromogenic substrate. Scand J Haematol 1984; 33 (supp) (Suppl. 40) 139-145
• 6 NIBSC unpublished report. 1989
• 7 Dawson NJ, Kemball-Cook G, Barrowcliffe TW. Assay discrepancies with highly purified factor VIII concentrates. Haemostasis 1989; 19: 131-137
• 8 Chantarangkul V, Ingram GI C, Thom MB, Darby SC. An artificial haemophilic plasma for one-stage factor VIII assay. Br J Haematol 1978; 40: 471-488
• 9 Kirkwood TB L, Barrowcliffe TW. Discrepancy between one-stage and two-stage assay of FVIII : C. Br J Haematol 1978; 40: 333-338
• 10 Barrowcliffe TW, Thomas DP. Factor VIII standardization. Lancet 1981; II 1342
• 11 Barrowcliffe TW. Comparisons of one-stage and two-stage assays of factor VIII :C. Scand J Haematol 1984; 33 (supp) (Suppl. 41) 39-54
• 12 Mikaelsson M, Oswaldsson U. Standardization of VIII :C assays: a manufacturer’s view. Scand J Haematol 1984; 33 (supp) (Suppl. 41) 79-86
• 13 Barrowcliffe TW, Kirkwood TB L. An international collaborative assay of factor VIII clotting activity. Thromb Haemostas 1978; 40: 260-71
• 14 Barrowcliffe TW, Curtis AD, Thomas DP. Standardization of factor VIII - IV. Establishment of the 3rd international standard for factor VIII :C concentrate. Thromb Haemostas 1983; 50: 697-702
• 15 Lee ML, Maglalang EA, Kingdom HS. An effect of predilution on potency assays of factor VIII concentrates. Thromb Res 1983; 30: 511-9
• 16 Frandsen H, Stender S, Scheibel E. An effect of predilution on VIII: C determination by the one-stage assay. Thromb Res 1987; 48: 195-203
• 17 Allain JR. Principles of in vivo recovery and survival studies. Scand J Haematol 1984; 33 (supp) (Suppl. 41) 123-45
• 18 Hellstem P, Kiehl R, Miyashita C, Schwerdt H, Von Blohn G, Kohler M, Biittner M, Wenzel E. Factor VIII :C recovery and half-life after infusion of steam-treated high purity factor VIII concentrate in severe hemophilia A - Comparison of one-stage assay, two-stage assay and a chromogenic substrate assay. Thromb Haemostas 1986; 56: 353-9
• 19 Messori A, Longo G, Morfini M, Cinotti S, Filimberti E, Giustarini G, Rossi-Ferrini PL. Multi-variate analysis of factors governing the pharmacokinetics of exogenous factor VIII in haemophiliacs. Eur J Clin Pharmacol 1988; 35: 663-8
• 20 Longo G, Matucci M, Messori A, Morfini M, Rossi-Ferrinie PL. Pharmacokinetics of a new heat-treated concentrate of factor VIII estimated by model independant methods. Thromb Res 1986; 42: 471-6
• 21 Mazurier C, De Romeuf C, Parquet-Gemez A, Jorieux S, Goudemand M. In vitro and in vivo evaluation of a factor VIII concentrate heat-treated to inactivate HTLV-III/LAV viruses. Vox Sang 1987; 52: 265-71
• 22 Bemtorp E, Nilsson IM. Biochemical and in vivo properties of commercial virus-inactivated factor VIII concentrates. Eur J Haematol 1988; 40: 205-14
• 23 Mazurier C, De Romeuf C, Parquet-Gemez A, Goudemand M. “In vitro” and “in vivo” characterization of a high-purity, solvent/ detergent-treated factor VIII concentrate. Evidence of its therapeutic efficacy in von Willebrand’s disease. Eur J Haematol 1989; 43: 7-14