New national standards for factor VIII and factor IX must be manufactured to replace diminishing supplies of MEGA, the factor VIII standard, and FN2, a factor IX standard. The standards must be stabile, have assay characteristics that are compatible with all currently licensed preparations, and give equivalent one-stage plasma and chromogenic test results in order to harmonize with the recommendations of the European Pharmacopoeia. To meet these requirements, we evaluated source material supplied by four manufacturers. Factor VIII source material included two recombinant products, two monoclonal antibody-purified preparations, and two products purified by conventional chromatography. Factor IX bulks included one product purified by monoclonal antibody, two by chromatography, and one by fractional precipitation. Factor VIII was evaluated by plasma and chromogenic substrate tests; factor IX was assayed in plasma. The short term stability of both factors was measured after they underwent a thaw, freeze, thaw cycle, similar to conditions that will occur during the manufacture of the standards. The structural integrity of the factors was evaluated by Western blots. After rethrawing, 3 of the 6 factor VIII preparations had plasma potencies within 25% of their initial labeled values. One preparation lost 70% of its activity. Two of the three stabile preparations had chromogenic test results within 15% of their plasma values. After incubation at 30 degrees C for 150 hours, most samples maintained their potency within 10% of initial values. Western blot results were similar for all factor VIII products. Two candidate source materials for factor VIII were selected to be evaluated further in an international calibration study to choose the final standard. Eighteen laboratories in the US, Europe, Japan and China are participating in the study. The candidates are being assessed with respect to similarities in chromogenic and plasma test outcomes, and the coefficient of variation. Once the standard is chosen, another international calibration study will be undertaken to designate its potency. Regarding factor IX, the thaw-freeze-thaw cycle had little effect on activity. Two factor IX products were stabile when incubated at 30 degrees C for up to 75 hours, while the others lost activity. Only one factor IX product showed no degradation on a Western blot. This material was selected to be the standard; an international collaborative study was performed to assign its potency. CBER found that the potency of the current WHO international Factor IX reference standard was inconsistent with the previous WHO reference standard. In meetings with the European Pharmacopoeia (EU) and WHO, a compromise potency value for the FDA standard was agreed upon that was an average of the potencies derived from the two WHO standards. The FDA standard was accepted by the EU, and has become the first international coagulation working standard. WHO may also accept it as their reference or working standard.
