Removal and inactivation of viruses during the manufacture of a high-purity antihemophilic factor IX from human plasma

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 (GreenNine 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 (TCID 50 ), 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 log 10 TCID 50 , 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. © KSBB hÉóïçêÇëW=~åíáÜÉãçéÜáäáÅ=Ñ~Åíçê=fuI=îáê~ä=áå~Åíáî~íáçå=~åÇLçê=êÉãçî~äI=äçÖ=êÉÇìÅíáçå=Ñ~ÅíçêI=îáêìë=ë~ÑÉíó= = = = =

Influence of peptide ligand surface density and ethylene oxide spacer arm on the capture of porcine parvovirus

“…The acetylated resin bound none of the PPV in solution, as shown in Figure 3. The amino control resin, which has amine groups on the surface and is a weak ion exchange resin, was used because ion exchange resins may have inherent virus removal properties (24, 25); this allowed for comparison of peptide ligands to an anion exchange resin. The amino resin removed more virus from solution in the later column volumes than in the earlier column volumes (Figure 3).…”

Identification of Trimeric Peptides That Bind Porcine Parvovirus from Mixtures Containing Human Blood Plasma

“…This is not surprising, as anion exchange columns are often tested for their ability to clear viruses (35). It has been shown that a Q‐Sepharose column was able to clear 3 log of PPV when loaded at pH 6.5 (36) and as high as 5 log of MVM when loaded in Tris buffer at pH 8.0 (14). This follows the trend that increasingly basic solutions will make the virus surface more negatively charged, which would cause increasingly stronger binding of the virus to an anion exchange column.…”

The Simultaneous Human FVIII/vWF Purification and Virus Inactivation Combined in Chromatographic Column

“…On the other hand, it's well known that any chromatography purification step leads to the reduction of virus contamination not less than 0.5-3.0 log 10 per step [17,21]. This phenomenon can be explained by the fact that the chromatographic adsorbent, while firmly retaining the target protein, allowed washing out unrelated or weakly bound viruses, demonstrating a "filtering" model.…”