Activated Factor VIII in Therapeutic Preparations: Analysis by Ultracentrifugation

Abstract

Activation of factor VIII is signaled by an increase in the 1-stage factor VIII activity and release from von Willebrand factor. Ultracentrifugation in 10—40% sucrose gradients was used to identify such activation in therapeutic concentrates. Plasma-derived factor VIII lots were examined and factor VIII sedimenting independently of von Willebrand factor was identified in some of the preparations. In addition there was slower sedimentation of factor VIII by the 1-stage assay than by the chromogenic assay. These results are consistent with a factor VIII cleaved at residue 1689, a site important for von Willebrand binding. This activated form leads to some of the assay discrepancies between the 1-stage assay and the chromogenic or the 2-stage assay. There is more rapid sedimentation of the factor VIII measured by the chromogenic assay than the von Willebrand factor in some manufacturers’ samples indicating that the method of fractionation may select a low molecular weight von Willebrand factor which does not bind factor VIII. Routine comparison between the 1-stage and chromogenic assays during fractionation may be able to identify such activated preparations. Other assay discrepancies may be due to structural differences between the standards and the tested product.

The influence of citrate concentration on the quality of plasma obtained by automated plasmapheresis: a prospective study

“…These findings indicate that the somewhat (approx. 5%) higher plasma levels found in plasma anticoagulated with 6‐percent sodium citrate are not due to partial activation of FVIII, as FVIII activated by thrombin yields markedly increased FVIII activities when the one‐stage assay is used, whereas the two‐stage chromogenic substrate assay results are not influenced by activated FVIII 14,15 . It was shown by others that FVIII recovery in frozen plasma increased with the rate of freezing and that the purity and recovery of FVIII in cryoprecipitates also increased with fast freezing of plasma 16 .…”

The impact of the intensity of serial automated plasmapheresis and the speed of deep‐freezing on the quality of plasma

“…As a consequence, FVIII:C/FVIII:CHR ratios were significantly greater in Group 2 plasmas. Because the one‐stage clotting assay unlike the CHR, overestimates FVIII activity in the presence of activated FVIII, 17,18 the FVIII:C/FVIII:CHR ratio reflects the degree of thrombin‐induced FVIII activation. This is supported by a significant correlation between FVIII:C/FVIII:CHR ratios and F1+2 levels, which were also significantly greater in Group 2 plasmas.…”

Continuous infusion of FVIII and FIX concentrates:in vitroanalysis of clinically relevant parameters

“…Manufacturers of coagulation factor concentrates have been called upon to provide data regarding the suitability of their products for application using continuous infusion [9–11, 30]. In preliminary reports we have demonstrated the in vitro stability of the coagulation activity in both the FVIII/vWF concentrate (IMMUNATE) and FIX concentrate (IMMUNINE) [15, 31].…”

“…Activation of the coagulation factors by trace amounts of copurified proteins is another point of worry for manufacturers and clinicians. It is generally agreed that higher results of FVIII:C, obtained by the 1‐stage as compared to the 2‐stage coagulation assays or chromogenic substrate assays, are indicative of the presence of FVIIIa in the sample [11, 28, 38]. To investigate this possibility, the FVIII/vWF concentrate was examined for the presence of FVIIIa generated during perfusion or incubation.…”

“…Due to increased awareness regarding the variables involved, clinicians as well as national registration bodies have called on the manufacturers for proof of safety, prior to use of factor concentrates in continuous infusion [9–12]. Validation results relevant to the safety and stability of one factor concentrate, intended for application by continuous infusion, cannot be transferred or interpolated to another [1, 4, 13].…”