PREPARATION OF HIGH SPECIFIC ACTIVITY PLASMA AHF BY ANTI-FVIIIc IMMUNOAFFINITY CHROMATOGRAPHY

Abstract

To reduce the risk of pathogenic virus transmission associated with the therapeutic administration of plasma derived AHF (FVIIIc) we have developed a process wherein anti-FVIIIc immunoaffinity chromatography is used to isolate FVIIIc from a cryoprecipitate solution which has been treated with an organic solvent/detergent mixture to inactivate 1-ipid-enveloped viruses. A final ion exchange chromatography step is used to further remove contaminants (eg. anti-FVIIIc antibody) eluted with FVIIIc during the immunoaffinity step. FVIIIc obtained in the process has a specific activity of 1500 to 2000 AHF units (one stage clotting assay) per mg of protein, representing a ≥120,000-fold purification from plasma. The purified FVIIIc is stabilized for lyophilization and storage by the addition of human albumin. Polyclonal anti-FVIIIc immunoblots reveal polypeptides with apparent mol wts between 90,000 and 230,000 (heavy chain) and 70,000 to 76,000 (light chain). The major လcontaminant” in the preparation is von Willebrand Factor, one mol (Mr = 250,000 subunit) per mol of FVIIIc. Fibrinogen and fibronectin are barely detectable in the final preparation at levels of 1.0 and 0.3 ng per AHF unit respectively, representing a 3 million-fold purification of FVIIIc relative to these proteins. Anti-FVIIIc antibody, used in the immunoaffinity step of the process, is not detectable in the preparation at levels of 2.0 ng/mL (i.e. ≤0.01 ng per AHF unit). The extent to which virus reduction is associated with the process was also evaluated. The addition of an organic solvent/detergent mixture to cryoprecipitate solution resulted in HIV (and other lipid-enveloped viruses) inactivation to levels below the level of assay sensitivity in <5 min; representing a measurable 4 to 5 log reduction in virus titer. In addition, substantial (nonenveloped and lipid-enveloped) virus reduction was associated with the immunoaffinity step used in the process (3,400 to 50,000-fold depending on the virus/experimental conditions studie). The overall process results in a high specific activity AHF preparation which also appears to be substantially improved over previous AHF preparations with respect to viral contamination.