Removal and inactivation of viruses during the manufacture of a high-purity antihemophilic factor IX from human plasma |
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Authors: | In Seop Kim Jung Eun Bae Hark Mo Sung Yong Kang Yong Woon Choi |
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Institution: | 1. Department of Biological Sciences and Center for Biopharmaceuticals Safety Validation, Hannam University, Daejeon, 305-811, Korea 2. Graduate Program in BT-NT Fusion Science and Technology, Hannam University, Daejon, 305-811, Korea 3. Green Cross Corp., Yongin, 449-900, Korea 4. Department of Molecular Science and Technology, College of Engineering, Ajou University, Suwon, 443-749, Korea
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Abstract: | The purpose of this study was to evaluate the efficacy and mechanisms of the solvent/detergent (S/D) treatment, DEAE-toyopearl
650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and Viresolve NFP filtration
steps employed in the manufacture of high-purity antihemophilic factor IX (Green-Nine VF) from human plasma, with regard to
removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses,
including human immunodeficiency virus (HIV), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A
virus (HAV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. Samples
from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking
the manufacture of high-purity factor IX. Samples were collected at each step, immediately titrated using a 50% tissue culture
infectious dose (TCID50), and virus reduction factors were evaluated. S/D treatment using the organic solvent, tri (n-butyl) phosphate (TNBP), and
the detergent, Tween 80, was a robust and effective step in inactivation of enveloped viruses. Titers of HIV, BHV, and BVDV
were reduced from the initial titer of 6.06, 7.72, and 6.92 log10 TCID50, respectively, reaching undetectable levels within 1 min of S/D treatment. DEAE-toyopearl 650M anion-exchange column chromatography
was found to be a moderately effective step in the removal of HAV, EMCV, and PPV with log reduction factors of 1.12, 2.67,
and 1.38, respectively. Heparin-sepharose 6FF affinity column chromatography was also moderately effective for partitioning
BHV, BVDV, HAV, EMCV, and PPV with log reduction factors of 1.55, 1.35, 1.08, 1.19, and 1.61, respectively. The Viresolve
NFP filtration step was a robust and effective step in removing all viruses tested, since HIV, BHV, BVDV, HAV, EMCV, and PPV
were completely removed during the filtration step with log reduction factors of ≥ 5.51, ≥ 5.76, ≥ 5.18, ≥ 5.34, ≥ 6.13, and
≥ 4.28, respectively. Cumulative log reduction factors of HIV, BHV, BVDV, HAV, EMCV, and PPV were ≥ 10.52, ≥ 12.07, ≥ 10.49,
≥ 7.54, ≥ 9.99, and ≥ 7.24, respectively. These results indicate that the production process for GreenNine VF has a sufficient
virus reduction capacity for achievement of a high margin of virus safety. |
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