title: Inactivation and clearance of viruses during the manufacture of high purity factor IX.
abstract: Haemophilia is a bleeding disorder characterised by a deficiency in Factor IX. Replacement therapy in the form of a Factor IX concentrate is a widely accepted practice. In this paper we describe a double virus inactivated chromatographic process for producing a high purity Factor IX product, MonoFIX((R))-VF. The process involves separation of the prothrombin complex by cryoprecipitation, fraction I precipitation and DEAE-cellulose adsorption, further ion-exchange chromatography of crude Factor IX, followed by solvent/detergent treatment. Heparin affinity chromatography is then used to further purify Factor IX. Final nanofiltration is sequential through 35 nm then 15 nm membrane filters. The principal virus inactivation/removal steps are solvent/detergent treatment and nanofiltration and the partitioning of relevant and model viruses provides further reduction in virus load through the production process.Solvent/detergent treatment was shown to achieve log reduction factors of 4.5 for HIV-1, 5.1 for Sindbis virus, 6.1 for vesicular stomatitis virus (VSV), 5.1 for bovine viral diarrhoea virus (BVDV) and 5.3 for pseudorabies virus (PRV). BVDV is a model for hepatitis C virus (HCV), and pseudorabies virus (PRV), like hepatitis B virus (HBV) is an enveloped DNA virus. Using scaled down models of the production process, we have also demonstrated the neutralization/partitioning of at least 6 logs of hepatitis A virus (HAV) during cryoprecipitation, Fraction I precipitation, and the DEAE adsorption and elution step, and a further 1.6 log reduction in HAV load as a result of heparin affinity chromatography. The log reduction factors for HAV as a result of the second ion-exchange chromatography step and as a result of enhanced neutralisation associated with solvent/detergent treatment were not significant. Nanofiltration was shown to contribute a further log reduction factor of 6.7 for HAV and 5.8 for BVDV indicating that log reduction factors of this order would be obtained with other viruses of a similar or larger size, such as HIV, HBV and HCV.Overall, these studies indicate that MonoFIX-VF is a product with an extremely high level of viral safety.
authors.affiliation: Research & Development, CSL Limited, Bioplasma Division, 189-209 Camp Road, Broadmeadows, Victoria, 3047, Australia. anna_johnston@csl.com.au
authors.forename: A
authors.initials: A
authors.lastname: Johnston
authors.forename: A
authors.initials: A
authors.lastname: Macgregor
authors.forename: S
authors.initials: S
authors.lastname: Borovec
authors.forename: M
authors.initials: M
authors.lastname: Hattarki
authors.forename: K
authors.initials: K
authors.lastname: Stuckly
authors.forename: D
authors.initials: D
authors.lastname: Anderson
authors.forename: N H
authors.initials: NH
authors.lastname: Goss
authors.forename: A
authors.initials: A
authors.lastname: Oates
authors.forename: E
authors.initials: E
authors.lastname: Uren
mesh_terms: D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004340:Drug Contamination / Q000517:prevention & control*; D005164:Factor IX / Q000302:isolation & purification* / Q000592:standards* / Q000627:therapeutic use; D005615:Freezing; D006467:Hemophilia A / Q000628:therapy; D006801:Humans; D011516:Prothrombin / Q000737:chemistry; D015203:Reproducibility of Results; D014462:Ultrafiltration; D014780:Viruses / Q000302:isolation & purification*
chemical_list: D011516:Prothrombin; D005164:Factor IX