Large‐scale production and properties of human plasma‐derived activated Factor VII concentrate

Background and Objectives An activated Factor VII (FVIIa) concentrate, prepared from human plasma on a large scale, has to date not been available for clinical use for haemophiliacs with antibodies against FVIII and FIX. In the present study, we attempted to establish a large‐scale manufacturing process to obtain plasma‐derived FVIIa concentrate with high recovery and safety, and to characterize its biochemical and biological properties. Materials and Methods FVII was purified from human cryoprecipitate‐poor plasma, by a combination of anion exchange and immunoaffinity chromatography, using Ca 2+ ‐dependent anti‐FVII monoclonal antibody. To activate FVII, a FVII preparation that was nanofiltered using a Bemberg Microporous Membrane‐15 nm was partially converted to FVIIa by autoactivation on an anion‐exchange resin. The residual FVII in the FVII and FVIIa mixture was completely activated by further incubating the mixture in the presence of Ca 2+ for 18 h at 10 °C, without any additional activators. For preparation of the FVIIa concentrate, after dialysis of FVIIa against 20 m m citrate, pH 6·9, containing 13 m m glycine and 240 m m NaCl, the FVIIa preparation was supplemented with 2·5% human albumin (which was first pasteurized at 60 °C for 10 h) and lyophilized in vials. To inactivate viruses contaminating the FVIIa concentrate, the lyophilized product was further heated at 65 °C for 96 h in a water bath. Results Total recovery of FVII from 15 000 l of plasma was ≈ 40%, and the FVII preparation was fully converted to FVIIa with trace amounts of degraded products (FVIIaβ and FVIIaγ). The specific activity of the FVIIa was ≈ 40 U/µg. Furthermore, virus‐spiking tests demonstrated that immunoaffinity chromatography, nanofiltration and dry‐heating effectively removed and inactivated the spiked viruses in the FVIIa. These results indicated that the FVIIa concentrate had both high specific activity and safety. Conclusions We established a large‐scale manufacturing process of human plasma‐derived FVIIa concentrate with a high yield, making it possible to provide sufficient FVIIa concentrate for use in haemophiliacs with inhibitory antibodies.