Prion protein: detection in 'spiked' anaerobic sludge and degradation experiments under anaerobic conditions.

The behavior of the transmissible spongiform encephalopathies (TSE) causing agent denominated "prion protein" in anaerobic sludge (biogas reactor) was assessed with incubation tests. A widely applied screening method for BSE in cattle on the basis of the Western blotting protocol was adapted to detect the Proteinase K resistant, scrapie-form prion protein (PrPSC). As PrPsc source homogenized TSE infected brain tissue of animals late in the clinical phase of disease was taken (301V/VM mouse-BSE; bovine BSE and 22A/SV mouse-scrapie). The incubation under mesophilic conditions did not show any significant reduction of the PrPsc titer. Under thermophilic conditions contradictory results were obtained. The reduction time of PrPsc in water was equal to or longer than the PrPsc reduction time in anaerobic sludge. In comparison, with sterilized (121 degrees C, steam pressure) or poisoned (sodium azide, 1% w/v) sludge used as incubation matrix a much shorter time resulted until no prion protein could be detected.

Distribution of a bovine spongiform encephalopathy-derived agent over ion-exchange chromatography used in the preparation of concentrates of fibrinogen and factor VIII.

BACKGROUND AND OBJECTIVES: The risk of haemophiliacs contracting variant Creutzfeldt-Jakob disease (vCJD) via treatment with factor VIII concentrates is not known. Therefore, in order to determine the extent to which the vCJD agent might be removed during the preparation of factor VIII concentrate, the partitioning of a bovine spongiform encephalopathy (BSE)-derived agent was measured over the main purification step used to prepare the Scottish National Blood Transfusion Service high-purity factor VIII concentrate (Liberate). MATERIALS AND METHODS: Murine-passaged BSE (strain 301V), in the form of a microsomal fraction prepared from infected brain, was used to 'spike' a solution of factor VIII of intermediate purity. The 'spiked' starting material was subjected to solvent-detergent treatment and then to anion-exchange chromatography with Toyopearl DEAE-650M. All fractions were tested for 301V infectivity using a murine bioassay, including the procedures used to clean the ion-exchange media after use. RESULTS: BSE 301V infectivity was reduced by 2.9 log(10) in the fibrinogen fraction and by 2.7 log(10) in the factor VIII fraction. Over 99% of the added 301V infectivity remained bound to the ion-exchange column after elution of factor VIII. A large quantity of infectivity was subsequently removed by washing the ion-exchange media with 2 m NaCl. No further BSE 301V infectivity was detected in column eluates after treatment with 0.1 m NaOH or a second wash with 2 m NaCl. CONCLUSIONS: Results using a BSE-derived agent suggest that vCJD infectivity would be substantially removed by the ion-exchange process used in the preparation of fibrinogen and factor VIII concentrate. Although 301V infectivity remained bound to the ion-exchange matrix following elution of factor VIII, this appeared to be eliminated by the procedure used for cleaning the ion-exchange media after each use.

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.