Studies on the removal of a bovine spongiform encephalopathy-derived agent by processes used in the manufacture of human immunoglobulin.

BACKGROUND AND OBJECTIVES: There is still uncertainty over how the agent of variant Creutzfeld-Jakob disease (vCJD) would partition during the manufacture of plasma derivatives. In this study, a BSE-derived agent was used as a vCJD model to determine the extent to which infectivity could be removed by selected steps used in the manufacture of intravenous immunoglobulin (IVIG). MATERIALS AND METHODS: Murine-passaged BSE (strain 301V), in the form of a microsomal fraction prepared from infected brain, was used to "spike" the starting material in three experiments. The partitioning of BSE infectivity was measured over Fraction I+III precipitation, borosilicate microfibre depth filtration and Seitz depth filtration, with these steps being examined individually and in series. RESULTS: Most 301V infectivity partitioned into Fraction I+III (log reduction 2.1). Infectivity remaining in Supernatant I+III was reduced by AP20 glass-fibre depth filtration (log reduction 0.6) and subsequently removed to below the limit of detection by Seitz KS80 depth filtration, giving an overall log reduction of > or = 2.9 for the three steps in series. By contrast, glass-fibre depth filtration gave a log reduction of 2.4 when challenged directly with "spiked" feedstock. Seitz KS80 depth filtration gave a log reduction of > or = 3.1 when challenged directly with 'spiked' feedstock and also removed residual infectivity to below the limit of detection when applied as the final step in series. CONCLUSIONS: Results using a BSE-derived agent suggest that vCJD infectivity should be substantially removed from immunoglobulin G (IgG) solutions by Fraction I+III precipitation and Seitz KS80 depth filtration. The three different process steps examined acted in a complementary manner to one another when operated in series. However, the data demonstrated that it would be inappropriate to add together the reduction factors that had been derived for each step in isolation.

Studies on the removal of abnormal prion protein by processes used in the manufacture of human plasma products.

BACKGROUND AND OBJECTIVES: To identify if any process steps used in plasma fractionation may have a capability of removing agents of human transmissible spongiform encephalopathy (TSE). MATERIALS AND METHODS: Sixteen fractionation steps were investigated separately by adding a preparation of hamster adapted scrapie 263K to the starting material at each process step and determining the distribution into resultant fractions of protease-K-resistant (abnormal) prion protein by Western blot analysis. RESULTS: A number of process operations were found to remove abnormal prion protein to the limit of detection of the assay. These were cold ethanol precipitation of fraction IV (log reduction, LR, >/=3.0) and a depth filtration (LR >/=4.9) in the albumin process; cold ethanol fraction I+III precipitation (LR >/=3.7) and a depth filtration (LR >/=2.8) in the immunoglobulin processes and adsorption with DEAE-Toyopearl 650M ion exchanger (LR >/=3.5) in the fibrinogen process. In addition, a substantial degree of removal of abnormal prion protein was observed across DEAE-Toyopearl 650M ion exchange (LR = 3.1) used in the preparation of factor-VIII concentrate; DEAE-cellulose ion exchange (LR = 3.0) and DEAE-sepharose ion exchange (LR = 3.0) used in the preparation of factor-IX concentrates and S-sepharose ion exchange (LR = 2.9) used in the preparation of thrombin. CONCLUSIONS: Plasma fractionation processes used in the manufacture of albumin, immunoglobulins, factor-VIII concentrate, factor-IX concentrates, fibrinogen and thrombin all contain steps which may be capable of removing causative agents of human TSEs.