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 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.

Immunoaffinity purification of factor VIII complex.

Murine monoclonal antibodies to human von Willebrand factor (vWf) were immobilised on Sephacryl S-1000. Various solutes were screened for their ability to elute 125I-vWf from the immobilised antibodies. The most effective solutions were then tested to determine which allowed retention of factor VIII procoagulant activity (VIII:C) and activity of vWf measured by platelet aggregation in the presence of ristocetin (Ristocetin cofactor activity R. cof.). Finally, F VIII complex was purified from both plasma and cryoprecipitate by immunoaffinity chromatography under the selected conditions. The product had a specific activity of 45 units of VIII:C per mg of protein and 60 units of R. cof. per mg representing a 4000-fold purification from plasma. The fibrinogen and fibronectin content were each less than 4% of the total protein with vWf accounting for 60% of the total protein in the final product. Multimer analysis of the product showed a similar pattern to normal plasma and contamination by murine monoclonal antibody was less than 300 ng per mg of protein. A novel product is thus obtained containing both clinically relevant VIII:C and R. cof. in a single vial whilst using only one specific monoclonal antibody.

The production and characterisation of a panel of ten murine monoclonal antibodies to human procoagulant factor VIII.

A panel of 10 murine monoclonal antibodies to procoagulant FVIII has been developed from the fusion of a single spleen. Balb/c mice were injected with a purified preparation of FVIII: Ag, and antibody production in sera and hybrid culture supernatants was monitored using a specific radiometric screening assay. The antibodies all inhibit FVIII clotting activity in normal plasma, and when immobilised on agarose retain their ability to recognise and bind the FVIII procoagulant protein. Studies on protein A-purified immunoglobulins demonstrate a range of properties within the panel of antibodies with regard to species cross-reactivity, clotting inhibition and immunoadsorption. The panel of antibodies has been used to screen heat-treated FVIII concentrates for the occurrence of heat-induced neoantigens.

Platelet production time in surgery.

A study of platelet production time in major surgery is described. This was equated to the time taken for renewal of maximum malondialdehyde (MDA) activity in blood platelets following suppression by a single dose of aspirin (Stuart et al 1975). The method was simplified by using EDTA disodium salt as an anticoagulant instead of citrate, resuspending platelets with small bar magnets, and reducing time of incubation with thiobarbituric acid reagent from 1 h to 15 min. Results for maximum platelet renewal taken to be the time to achieve a pre-aspirin baseline level were median in surgery 6.3 (3.8-9.2) d and controls 7.0 (6.2-12.5) d. Using the modified method, average baseline malondialdehyde (MDA) levels were 6.2 +/- 1.4 nmol/10(9) platelets in surgical patients and 6.34 +/- 1.2 nmol/10(9) platelets in controls. Platelet production time determination using this method is not strictly analogous compared with survival of 51Cr-labelled platelets and it however warrants further study.

Heparin-like activity in uterine fluid.

Uterine fluid was collected from a group of normal patients and a group of patients with menorrhagia. Heparin-like activity was detected in 34 out of 38 samples using an anti-Xa heparin assay. The heparin-like activity in uterine fluid was inhibited by adding the heparin antagonist hexadimethrine bromide to the assay. Concentrations of fibrinogen-fibrin degradation products (FDPs) were measured in five samples of uterine fluid. FDPs in the concentration detected had no effect on the anti-Xa assay. Heparin-like activity was higher in the group with menorrhagia, although the differences were not significant. Heparin-like activity increased throughout the menstrual cycle and decreased during menstruation, suggesting a possible cyclical variation in activity. There was no correlation between mast cell numbers in the endometrium and myometrium and heparin-like activity in uterine fluid and no correlation between the numbers and the stage in the menstrual cycle. In a few patients with intrauterine contraceptive devices (IUCDs) heparin-like activity was increased.