Peter R. Foster

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.

Removal of TSE agents from blood products

Transmissible Spongiform Encephalopathies (TSEs) are fatal neuro-degenerative disorders. Creutzfeldt-Jakob disease (CJD) in humans is divided into classical CJD (cCJD), of which there are a number of forms (sporadic, familial, GerstmannSträussler-Scheinker (GSS) syndrome), and variant CJD (vCJD), the latter probably transmitted by food contaminated with bovine spongiform encephalopathy (BSE). cCJD has been transmitted by medical procedures in which tissues with a high level of infectivity were involved [1] but transmission by blood products has not been observed [2] possibly because infectivity in blood is very low. By contrast, vCJD has probably been transmitted by transfusion of whole blood [3] consistent with experimental transmissions of BSE between sheep [4]. The prevalence of cCJD is 0·5–1·0 per million inhabitants per annum world-wide [5]. About 150 cases of vCJD have been recorded, but the subclinical prevalence of infection in the human population is not known. BSE has been discovered in over 20 countries and it is conceivable that large numbers of people have been exposed to infection. Without a suitable diagnostic test, the extent to which CJD agents may be present in blood donations is not known. It is therefore important to establish the extent to which TSE agents can be eliminated during the preparation of blood products. TSE diseases are associated with conversion of prion protein (PrP) to a pathogenic conformation (PrPSc) that accumulates in the brain causing degeneration. TSE agents have been found to be highly resistant to physical and chemical treatments and methods for their inactivation [6] are too severe to be applied to blood products. Attention has therefore concentrated on removal using separations technologies. PrPSc has a number of properties which could be exploited to separate it from other biological substances; including a low solubility in aqueous solution, the ready formation of aggregates and a tendency to adhere to surfaces [7]. Experimental approaches

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).

Prions and blood products

The transmission of Creutzfeldt-Jakob disease (CJD) by human pituitary-derived growth hormone has led to concerns that blood products might also provide a route for the iatrogenic transmission of CJD. A number of actions have been implemented by regulatory authorities to address such concerns, and numerous studies have been undertaken to determine whether or not there is a risk of CJD being transmitted in this manner. To date, no excess risk has been identified, leading to a growing consensus that there is little or no risk of long established forms of CJD being transmitted to recipients of blood products. This opinion does not extend to new variant CJD (vCJD) which is found predominantly in the UK and is believed to have resulted from the transmission of bovine spongiform encephalopathy (BSE) to humans. Unlike that of CJD, the prevalence of vCJD is not known. In addition, the detection of abnormal prion protein in the tonsils of vCJD-infected individuals has led to speculation that blood infectivity may be greater than in patients with CJD. A number of precautionary measures have been taken to address the possibility that vCJD may be transmissible by blood products; however, further scientific advances are needed to enable this risk to be defined. A suitable screening test is required to identify any infected blood donors, particularly where cellular blood components are being derived from populations believed to be at risk from BSE infection. Recent experimental data suggest that process operations used in the manufacture of plasma products may be capable of removing prion agents to a significant extent. However, further work is required to confirm these observations and to determine whether or not all potential vCJD infectivity would be removed by these means.

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®).

Control of large-scale plasma thawing for recovery of cryoprecipitate factor VIII.

Abstract. Cryoprecipitation is commonly used as the primary step in the preparation of clinical factor VIII concentrates; yet recovery is usually very low. Much of this loss is due to poor temperature control and a process of continuous plasma thawing has been designed to overcome this. A substantial improvement has resulted, with an increase in both yield and purity of factor VIII: C of over 50% in comparison to a conventional batch thaw process.

Studies on the Stability of VIII:C during the Manufacture of a Factor VIII Concentrate for Clinical Use

Abstract. The stability of VIILC was investigated by monitoring samples taken at different points from a routine process for the manufacture of factor VIII concentrate and by examining the stabilising influence of a number of product formulations. Loss of VIII:C over process‐finishing procedures (formulation, 0.22 μm filtration, dispensing) was associated with a citrate‐induced inactivation which could be prevented by controlling the ionised calcium concentration of the solution. These results were obtained using a one‐stage clotting assay but were not observed using a two‐stage assay. No evidence for activation was found in vitro (e.g. by FPA generation and VIII:C stability) and the yield increase suggested by the one‐stage assay was supported by results from a controlled clinical evaluation.

The effect of fluid-jet mixing on protein precipitate growth during low-frequency conditioning

The mixing effects of low-frequency conditioning during protein precipitate aggregation are investigated. The conditioning process is described on the basis of a jet-mixing phenomena with aggregation being enhanced by the mixing effect of a fluid jet formed during vibration. The mixing performance is shown to correlate with the frequency of formation and the amplitude of the fluid jet. This amplitude is determined by the response of the conditioning system to a pulse or disturbance and is, for a particular suspending fluid, a function of the geometry of the outlet pipe of the conditioning chamber. The fluid-jet acceleration determines the break-up or prevention of formation of large precipitate aggregates. Improvements in the precipitate particle size distribution are related to the extent of fluid-jet mixing which promotes the removal of fine particles by aggregation with larger particles. Maxima are observed in particle size enhancement as a function of the fluid-jet acceleration with up to 40% increase in particle size at the fine end of the cumulative size distribution.

Studies on the effect of heat treatment on the thrombogenicity of factor IX concentrates in dogs

Summary. This study examines the effects of heat treatment for 72 h at 80°C on the potential thrombogenicity of lyophilized human coagulation factor IX concentrates. Since heating generated minor amounts of thrombin, concentrate was prepared with antithrombin III addition prior to heat treatment. Changes in coagulation parameters were followed prior to and after infusion of 100 iu/kg of heated and unheated concentrates to dogs. All batches produced a transient fall in platelet count during infusion and a delayed rise in plasma fibrinopeptide A, accompanied by a minor prolongation of the activated partial thromboplastin time. Such changes were less marked for heated batches.

Hepatitis C and haemophilia

EDITOR,--In her editorial on hepatitis C and haemophilia Christine A Lee states that recombinant factor VIII “cannot transmit bloodborne viruses.”1 Although this belief is widely held, it is not correct. All biological substances can harbour infectious agents, and consequently all biopharmaceutical …