Precipitation was performed at varied pH (pH 5 15, 5 25 and 5 45)

Precipitation was performed at varied pH (pH 5.15, 5.25 and 5.45) to test pH values above and below the target pH for their effect in removing PPV and MEV. Of these variations, pH changes were the only parameters that had a significant effect on virus removal. The minimum virus removal values at pH 5.15 are reported in Table 1. Non-enveloped viruses, i.e. PPV and

MEV are removed by ≥4.0 log10 at the worst case conditions tested. As shown in Table 2, pH can influence the efficacy of virus removal by this step. For S/D treatment, validation studies were performed using the lower limit of S/D concentration in IVIG manufacturing. The pH in validation studies was elevated to pH 6.20 to avoid any inactivation by low pH. The temperature was reduced to 25.5 °C, to below the minimum used in production. S/D inactivation was stopped by treating the test sample with C-18 Sepharose beads to remove PARP inhibitor the S/D reagents. Viruses were rapidly inactivated by S/D to below the limit of detection (Fig. 2). Inactivation of all test viruses was in the range of >4 to >7 log10 and the results are summarized in Table 1. Virus filtration studies were performed at high and low pH and with

an excess of test sample with respect to filter surface area. Values at high pH were reported in Table 1. The data show that 35 nm filtration removed significant titers of all the enveloped viruses tested (HIV, PRV and BVDV) and also of non-enveloped viruses (BPV and SV40), depending on the size of the virus particles. IOX1 molecular weight Although BPV is small (18–24 nm), BPV virus particles are complexed with antibodies to Parvovirus B19 and their effective diameter is

increased. During plasma fractionation, classes of proteins are precipitated and separated from proteins in solution by centrifugation or filtration. Viruses are distributed into the fibrinogen precipitate (Cohn–Oncley fraction I) and the IgG fraction (Cohn–Oncley Pyruvate dehydrogenase fractions II+III). The most effective virus removal step during cold ethanol fractionation to produce IgG is precipitation of fraction III [4]. This step was investigated at worst case conditions, i.e. reduced concentration of ethanol (15% instead of 17%), elevated temperature (−4 °C instead of −5 °C), reduced amount of filter aid and reduced filter area per volume. The virus reduction data observed in this study showed that fraction III precipitation and removal by centrifugation when combined with the clarification by depth filtration in the presence of a filter aid was effective in removing two non-enveloped viruses that were used as models for B19 parvovirus and hepatitis A virus, i.e. PPV and MEV. Of these variations, pH changes were the only parameters that had an effect on virus removal. For each virus tested, the reduction in virus levels in the fraction III supernatant at pH 5.45 was greater than at pH 5.15.

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