• Journal of Microbiology
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• v.38 no.3
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• pp.187-191
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• 2000

A validation study was conducted to determine the efficacy of solvent/Detergent (S/D) inactivation and Q-Sepharose column chromatographic removal of the human immunodeficiency virus (HIV) during the manufacturing of a high purity antihemopilic factor VIII (GreenMono) from human plasma. S/D treatment using the organic solvent, tri (n-butyl) phosphate, and the detergent, Trition X-100, was a robust and effective step in eliminating HIV-1. The HIV-1 titer was reduced from an initial titer of 8.3 log10 TCID50 to undetectable levels within one minute of S/D treatment, HIV-1 was effectively partitioned form factor VIII during Q-Sepharose column chromatography with the log reduction factor of 4.1 . These results strongly assure the safety of GreenMono From HIV.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.1
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• pp.19-25
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• 2006

With particular regards to the hepatitis A virus (HAV), a terminal dry-heat treatment ($100^{\circ}C$ for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor IX concentrate. The loss of factor IX activity during dry-heat treatment was of about 3%, as estimated by a clotting assay. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor IX compared with those of the factor IX before dry-heat treatment. The dry-heat-treated factor IX was stable for up to 24 months at $4^{\circ}C$, The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV and murine encephalomyocarditis virus (EMCV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Porcine parvovirus (PPV) and bovine herpes virus (BHV) were potentially sensitive to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were ${\ge}5.60$ for HAV, ${\ge}6.08$ for EMCV, 2.64 for PPV, and 3.59 for BHV. These results indicate that dry-heat treatment improves the virus safety of factor IX concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.997-1003
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• 2008

Viral safety is a prerequisite for manufacturing clinical antihemophilic factor VIII concentrates from human plasma. With particular regard to the hepatitis A virus (HAV), a terminal dry-heat treatment ($100^{\circ}C$ for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor VIII concentrate. The loss of factor VIII activity during dry-heat treatment was of about 5%. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor VIII compared with those of the factor VIII before dry-heat treatment. The dry-heat-treated factor VIII was stable for up to 24 months at $4^{\circ}C$. The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV, murine encephalomyocarditis virus (EMCV), and human immunodeficiency virus (HIV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Bovine herpes virus (BHV) and bovine viral diarrhea virus (BVDV) were potentially sensitive to the treatment. However porcine parvovirus (PPV) was slightly resistant to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were ${\geq}5.55$ for HAV, ${\geq}5.87$ for EMCV, ${\geq}5.15$ for HIV, 6.13 for BHV, 4.46 for BVDV, and 1.90 for PPV. These results indicate that dry-heat treatment improves the virus safety of factor VIII concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid-enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.497-503
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• 2001

The purpose of this study was to examine the efficacy and mechanism of the cryo-precipitation, solvent/detergent (S/D) treatment, monoclonal anti-FVIIIc antibody (mAb) column chromatography, Q-Sepharose column chromatography, and lyophilization involved in the manufacture of antithemophilic factor VII(GreenMono) from human plasma, in the removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses, including the bovine viral diarrhoea virus (BVDV), bovine herpes virus (BHV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. BHV and EMCV were effectively partitioned from a factor VII during the cryo-precipitation with a log reduction factor of 2.83 and 3.24, respectively. S/D treatment using the organic solvent, tri(n-butyl) phosphate (TNBP), and the detergent, Triton X-100, was a robust and effective step in inactivating enveloped viruses. The titers of BHV and BVDV were reduced from the initial titer of 8.85 and $7.89{log_10} {TCID_50}$, respectively, reaching undetectable levels within 1 min of the S/D treatment. The mAb chromatography was the most effective step for removing nonenveloped viruses, EMCV and PPV, with the log reduction factors of 4.86 and 3.72, respectively. Q-Sepharose chromatography showed a significant efficacy for partitioning BHV, BVDV, EMCV, and PPV with the log reduction the log reduction factors of 2.32, 2.49, 2.60, and 1.33 respectively. Lyophilization was an effective step in inactivating g nonenveloped viruses rather than enveloped viruses, where the log reduction factors of BHV, BVDV, DMCV, and PPV were 1.41, 1.79, 4.76, and 2.05, respectively. The cumulative log reduction factors of BHV, BVDV, EMCV, and PPV were ${\geqq}$11.12, ${\geqq}$7.88, 15.46, and 7.10, respectively. These results indicate that the production process for GreenMono has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.

• KSBB Journal
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• v.23 no.1
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• pp.37-43
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• 2008

The use of antihemophilic factor IX complex has been associated with a variety of thrombotic complications, the major cause of which was the contamination of thrombogenic proteins such as vitamin K-dependent clotting factors II, VII, and X. In order to produce a commercial factor IX (GreenNine VF) free from thrombogenic potential, industrial-scale production process for high-purity factor IX from human plasma has been developed. The purification process contains cryo-precipitation, DEAE-sephadex A-50 anion-exchange chromatography, DEAE-toyopearl 650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and CM-sepharose FF cation-exchange column chromatography. Also the process includes two viral inactivation and removal procedures, solvent/detergent treatment and nanofiltration using Viresolve NFP filter. The purification yield was 35.4%. The specific activity in the purified concentrate was 190.8 IU/mg which exceeded that in the factor IX complex (FacNine) by a factor of 48. The activities of factor II, VII, and X were not detected in GreenNine VF. SDS-PAGE analysis showed that GreenNine VF had the highest purity in comparison with commercially available high purity factor IX concentrates, Mononine, Octanyne, Berinin HS, and Immunine STIM plus 600. One batch size of the production was 2,400 vials of 250 IU product or 1,200 vials of 500 IU product from 1,600 L cryo-poor plasma.