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

Abstract

The assessment of factor VIII coagulant activity (FVIII:C) in recently available highly purified and concentrated FVIII 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 FVIII: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 FVIII:C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVIII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVII-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.