THE PENROSE INQUIRY
Final Report

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Chapter 20

Haemophilia Therapy - The Period up to the Early 1980s

20.1 So far as is relevant to the Terms of Reference, the early history of the administrative and management structures set up for the provision of blood services in Scotland is discussed in Chapter 17, Blood and Blood Products Management. Collection procedures and the provision of manufacturing facilities are discussed in Chapter 18, Collection of Blood - General, and Chapter 19, Production of Blood Products - Facilities. The use of blood products in haemophilia therapy is discussed in Chapters 21, Haemophilia Therapy - Use of Blood Products, and 22, Haemophilia Therapy - Use of Blood Products 1985-1987. These are all inter-related aspects of the background against which products came to be in use that were associated with the transmission of hepatitis and HIV. This chapter deals with the development of the products in Scotland that were manufactured, prescribed and used in the material period when patients were at risk of infection.

20.2 The arrival of commercial concentrates in 1973 changed market conditions, and had a significant impact on the approach of the public sector producers in the UK as a whole to the production and distribution of NHS products. Coincidentally, plasma fractionation in Scotland was about to undergo very significant change with the opening of the new Protein Fractionation Centre (PFC) in Edinburgh.

20.3 This chapter deals with developments in technology up to 1982-83. Up to that point the risk of infection, so far as it was understood, was of transmission of hepatitis, first Hepatitis B and then non-A, non-B Hepatitis (NANB Hepatitis). After that point, the reports of AIDS in haemophilia patients treated with blood products, and with no other risk factors for AIDS, brought about a significant change in the approach to factor concentrate therapy in the treatment of haemophilia. It is appropriate to deal with the periods separately. The focus in this chapter is on the development of early blood products, and, so far as related to that, the steps taken to meet demand for products for clinical use. The historical context is of some importance.

Origins

20.4 The process of separation of whole blood into components for use, or further processing, already had a long history by the start of the reference period, reflecting the interaction of developing medical knowledge and technological progress. Blood is a complex mixture, including red cells, white cells and platelets, together with plasma which contains proteins, sugars, fats and a number of other smaller components such as hormones. The primary stage procedure of separation of whole blood into red cells, buffy coat (which contains white cells and platelets), and plasma depends particularly on the density differences between the corpuscular components and plasma.^[1] Centrifugation of blood results in layering of components according to their density.

20.5 From an early period, centrifugation was routinely performed at transfusion centres.^[2] Initially, storage, whether of whole blood or blood components, was hampered by the coagulation that inevitably follows collection of blood from the body. Once the collection procedure begins, the blood is removed from the body's natural metabolic sustaining environment, cools from body temperature, and is exposed to foreign substances.^[3] The effective and efficient collection and storage of blood became the focus of technological research and development. Early in the twentieth century, it was discovered that various salts, and in particular citrates, in an unphysiological preservative anticoagulant solution, could maintain the fluidity of blood stored in containers. Citrate-based anticoagulants, usually with the addition of sugar, and heparin anticoagulants were developed progressively thereafter.^[4] The blood collection procedures had to be rapid to avoid coagulation in the collection line. And the addition of the anticoagulant solution had to be prompt to prevent the development of foci of coagulation in the collection pack. Sterile disposable plastic pack assemblies introduced in the 1950s to replace glass bottles in the collection of blood donations provided for the easy introduction of the appropriate anticoagulant solution.^[5] Once bagged, the material had to be stored at the appropriate temperature, depending on the purpose for which the components were required.^[6] Centrifugation of the blood in plastic bags later provided the starting materials for further processing.^[7]

20.6 Three constituents of plasma were to become material to the development of therapeutic products: (i) the clotting factors, including fibrinogen and Factors VIII and IX; (ii) albumin, a normal protein in the blood which has oncotic properties and acts as a carrier protein for other substances; and, importantly, (though not directly relevant to the Terms of Reference) (iii) immunoglobulins (Ig), needed to boost antibodies in the blood of hypogammaglobulinemia patients, or patients who have suffered a needle stick injury, for example, to help fight off viral infections.^[8]

20.7 Refrigerated plasma was used clinically at or near the point of collection. Scientific developments in cryobiology enabled the storage of components for periods far greater than the point at which degradation would have occurred naturally in refrigerated materials. Typically, and with limited exceptions, plasma donations that were not required for immediate local clinical application were cooled rapidly and frozen. Fresh frozen plasma was retained for therapeutic application. Later, outdated stock was used for further processing. But at this early stage, it was used in the form in which it was separated immediately after the point of collection.

20.8 It is important to note that plasma, fresh or fresh frozen, was simply a component of the donor's blood as collected, untreated and unprocessed. It inevitably carried with it all of the proteins, including virus particles, circulating in the donor's blood. The risk of transmission of virus infection reflected the prevalence of infection in the donor population, unaffected by the processes by which it was extracted.

Early developments in process technology

20.9 Early technological developments did little to change that risk. Plasma filtration, introduced in August 1941, dealt with bacterial contamination.^[9] Freeze-drying, introduced at the Royal Infirmary of Edinburgh (RIE) in 1943, made plasma available as a powdered product that could be re-constituted at the point of use.^[10] The technology was to be important in the development of factor concentrates, but in isolation did not alter the risk of transmission of infection.

20.10 Efforts to produce factor concentrates began in the 1950s and progressed during the 1960s.^[11] Before the reference period two procedures were developed in relation to the processing of plasma that became significant: fractionation and the preparation of cryoprecipitate. Two scientists achieved prominence for developing the fractionation technology applied in isolating specific proteins from blood plasma for clinical application, Professor Edwin J Cohn, a professor of biological chemistry at Harvard University Medical School, and Professor RA Keckwick of London. Both before and during the reference period Scottish scientists followed the Cohn methodology as it was developed from time to time, and this chapter therefore describes that methodology and its derivatives.

20.11 The processes adopted in fractionating plasma reflect in part the complexity of blood. The components of blood do not withstand heating, for example, to a common degree. Red cells are contained within a membrane that starts to disrupt at about 40°C, and the components then clot. Platelets and white cells are similarly susceptible to temperature increases. The fluid component of blood - the plasma proteins, fats and sugars - can be heated, but still if heated together are subject to denaturation: they fall apart. Clotting begins, but at different temperature ranges from the cellular components. It is not possible to treat whole blood with heat. The characteristics of each component require to be taken into account separately in developing a heat treatment strategy.^[12]

20.12 The manufacture of blood concentrates depends on the chemical and physical characteristics of proteins contained in blood plasma. Plasma proteins vary in solubility when exposed to differential conditions of pH, ethanol concentration, temperature, ionic strength, and protein concentration. Professor Cohn and his colleagues showed that plasma proteins could be separated and partially purified in a reaction medium in which hydrogen ion concentration, ionic strength, temperature, protein concentration, and the amount of added ethanol were all carefully controlled.^[13] A series of fractionation steps was devised for the major biological categories of plasma proteins: these were partitioned as precipitates or supernatants after each manipulative stage.

20.13 The method was known as cold ethanol fractionation. Cohn fractionation exploited the physical changes induced in the frozen plasma by thawing under controlled conditions. When frozen plasma was immersed in a water bath at 4-6°C, thawing produced a liquid component (the supernatant) which could be extracted, leaving an illiquid residue. The Cohn process resulted in five stages of precipitation, the plasma 'fractions', which produced a range of derivatives for clinical application. Fraction I contained fibrinogen and antihaemophiliac globulin (AHG, later known as Factor VIII), which was used to treat haemophilia, Fraction III contained most of the lipid bearing β-globulins, and Fraction V contained albumin, which was used as a plasma substitute.^[14] The plasma fractions were then removed by filtration or centrifugation.^[15]

20.14 With later discoveries concerning the various clinical states in which deficiency in one or other of the blood constituents was the causative pathological abnormality in patients, the possibilities of specific remedial therapy became evident. The development of the Cohn fractionation scheme, which demonstrated that plasma could be split into various different components (each of which had different clinical properties), provided the basis for manufacture of a wide range of human blood products.^[16] Process technology developed to exploit these characteristics of plasma.

20.15 In the period between 1940 and the reference period of this Inquiry, scientists pioneered the use of a range of chemical additives during the manufacturing process which modified the Cohn ethanol fractionation procedure, resulting in Factor VIII concentrates of varying purity, a function of the removal of other proteins such as fibrinogen and fibronectin from the original Fraction I. By 1970, depending on the chemical agent introduced, low purity, intermediate purity, high purity and very high purity concentrates of Factor VIII could be produced, although the composition and structure of Factor VIII were not then known.^[17]

20.16 By 1972, the Cohn fractionation process had undergone many modifications. So far as is material for the purposes of this Report, it had been discovered at a very early stage in Cohn's work that Fraction I contained fibrinogen and AHG. But alcohol precipitation alone did not provide the range, quantity and purity of concentrates of coagulation factors that scientific research was making available for clinical application.^[18]

20.17 Professor Alan Johnson of New York, with Dr Margaret Karpatkin and Dr Jack Newman published a method for large-scale production of concentrates in 1969.^[19] A further paper on the method was published in 1971.^[20] It came to be known as the 'Newman' method. The supernatant plasma from which precipitates had been prepared using this technology was a source of Factor IX.^[21] The Newman method was to become the basis of the processes for the production of protein factor concentrates adopted in Edinburgh.^[22]

20.18 Professor Johnson's 1971 paper described methods for the production of Factor VIII concentrates of intermediate and high purity. The paper traced the developments in technology based on increasingly sophisticated precipitation methods using chemical additives. They had published, in 1966 and later years, papers describing methods of producing clinically effective intermediate purity Factor VIII concentrate by simultaneous ethanol- and cryo-precipitation of Factor VIII from melting fresh-frozen plasma and adsorption of Factors II, VII, IX and X from the precipitate. The step forward in the 1971 paper was the introduction of polyethylene glycol (PEG) in the precipitation of intermediate purity concentrate, resulting in a concentrate purified 125 to 350-fold which was effective in the treatment of haemophilia patients. The technology could be adapted to large-scale production. The paper gave wide circulation to the methodologies involved.^[23]

20.19 The Cohn/Newman method was used throughout the USA and in parts of Europe.

20.20 Viewed as a whole, the method produced progressively depleted plasma by extracting intermediate materials, in each case a solid, suitable for the specified final products, and a liquid supernatant.^[24] At the end stage of the original Cohn process a residue was left from the progressive thawing of frozen plasma, and the adsorption of proteins from the supernatant. The residue was slow to re-dissolve, and was initially discarded.^[25]

20.21 In 1959, before the refinements to the Cohn methodology described above had been introduced, Dr Judith Pool and her US colleagues had discovered that the residue left by Cohn fractionation, which remained at low temperature after drawing off the liquid produced in the thawing process, contained a high concentration of fibrinogen and Factor VIII, antihaemophilic activity. The residue also contained von Willebrand's Factor and other proteins, including fibronectin.^[26] In 1964, Dr Pool described a method of producing these concentrated factors from plasma by freezing which was quite independent of the need for Cohn fractionation.^[27] This was followed in 1965 by the publication by Pool and Shannon of further developments in the technology.^[28] The product was named cryoprecipitate.

20.22 The process devised by Dr Pool and her colleagues separated plasma from the red cells in whole blood donations by centrifugation at 4°C as soon as possible after collection, in the normal way, using standard compartmented plastic bags. The tubing connecting the compartments of the bag was clamped. The satellite bag containing the plasma was fast frozen. The whole bag was then refrigerated for cold-thawing of the frozen plasma. When thawed to only a few degrees above zero, and typically to 4°C, fibrinogen precipitated as a 'sludge' containing much of the Factor VIII content of the plasma.^[29] The temporary clamps were removed, and the supernatant plasma was allowed to return to the red cell bag. The bags were separated, and the cryoprecipitate frozen for storage pending use. The process did not produce mutually exclusive components. Red cells used in clinical practice contained very small amounts of plasma. Platelets were suspended in plasma. Each had the potential to transmit infection.^[30] However, the Pool and Shannon method produced a cryoprecipitate that was high in Factor VIII content, and that was stable and soluble.^[31] This provided a relatively purified form of Factor VIII for haemophilia therapy. Attempts had been made to isolate Factor VIII for clinical use in the treatment of haemophilia in the 1930s.^[32] But only now was there a relatively straightforward and effective procedure.

20.23 Cryoprecipitation of Factor VIII from single units of fresh-frozen plasma was viewed as a simple, practical procedure that could be carried out by any blood bank.^[33] It was used by blood transfusion centres in many countries and throughout Scotland. The single cryoprecipitate units might thereafter be pooled for further processing,^[34] but typically were used in multiples to make up a dose for Haemophilia A therapy. The product had lower coagulant activity than the material produced by Cohn Fraction I. But it was inexpensive to produce and the deficiency in coagulant activity could be made up by processing extra plasma.^[35] However, in this application, the method depended on prompt processing after collection, when coagulant activity was high, and new technology was required to process plasma on a large scale. This became the principal approach to the research and development of plasma processing in Glasgow in the early years of the reference period.

20.24 When cryoprecipitate from 10-15 individual plasma donations was combined and given to the patient it was possible to raise the Factor VIII level sufficiently to stop haemorrhage. During the late 1960s this treatment became available to haemophilia patients at hospitals on an out-patient basis. This was a major therapeutic advance for the treatment of Haemophilia A. Because cryoprecipitate does not contain very much Factor IX it was unsuitable for the treatment of Haemophilia B.^[36]

Haemophilia B

20.25 Treatment of patients with Haemophilia B was initially with fresh-frozen plasma. Until 1967 that was the only treatment available for correction of deficiencies in coagulation Factors II, VII, IX and X. In 1967, the PFC began making PPSB,^[37] a plasma derivative first produced in 1959 by Jean-Pierre Soulier in Paris for treatment of Haemophilia B patients. The demand for PPSB, which proved to have wide-ranging application, prompted research in Edinburgh into new methods of recovering Factor IX from the normal citrated plasma used in Cohn fractionation. The research, by Middleton, Bennett and Smith of the PFC, led to development of a Factor IX product, based on ion exchange purification.^[38]

20.26 In a similar fashion to Factor VIII, it had as its aim the production of a finished product with a specified amount of Factor IX activity per vial which would be suitable for home therapy and which would comply with the specifications of the British Pharmacopoeia (the UK standards for medicinal products).^[39] The product known as 'DEFIX' was produced at the PFC from 1972.

20.27 In the fractionation process, Factor IX was extracted downstream of Factor VIII, and it is appropriate to postpone discussion of Factor IX at this stage.

Blood product development and production in Scotland

20.28 The general trends in use of blood donations were followed in Scotland. Red cell preparations, including concentrates, were isolated. Platelet and leukocyte concentrates were derived from processing the buffy coat isolated in the primary separation procedure. As the twentieth century progressed, these operations superseded the use of whole blood for therapeutic purposes. The further processing of plasma became the principal downstream procedure of interest for present purposes. Developments in the production and use of cryoprecipitate were pursued in Glasgow and the west of Scotland, latterly centred on laboratories at Law Hospital. In Edinburgh and the South East of Scotland Region of the Blood Transfusion Service the emphasis came to be on fractionation.

20.29 The development of the first Factor VIII concentrate by the SNBTS was based on information obtained from Dr Cohn's laboratory in the early 1950s by Dr Drummond Ellis, then Head of the Regional Blood Transfusion Centre and Blood Products Unit (known until 1970 as the 'BPU'), at the RIE, where Edinburgh and South East Scotland Blood Transfusion Service was based.^[40] Dr Ellis later moved to the Blood Products Laboratory (BPL), Elstree, and was succeeded by Mr John Watt.^[41]

20.30 The early history of development work in the east of Scotland was described in an article published in 1965: Red Cell Banking and the Production of a Factor VIII Concentrate by Dr Cumming, Dr Ellis and Mr Grant of the BPU.^[42] Scotland's first fractionated plasma product was normal immunoglobulin for the prevention of measles, produced in 1952 at the RIE laboratory.^[43] Production of fibrinogen followed in 1956. Experimental quantities of Cohn Fraction I were made between 1952 and 1956.^[44] Routine production of an early version of Factor VIII known as antihaemophilic factor or AHF (from Cohn Fraction I) followed in 1956 and albumin in 1965.^[45] At this early period, the production of immunoglobulins was a significant part of the BPU's operations. A new pilot plant for fractionation was established at the BPU at the RIE in 1968.^[46] Cohn Fraction I, relatively rich in Factor VIII (antihaemophilic globulin) activity was produced there until production moved to the new facility built at the PFC, Liberton.

20.31 The fractionation process developed at BPU was initially very small-scale, as was the equivalent NHS process in England, and Factor VIII concentrates manufactured by the public service providers were available in very limited quantities. In Edinburgh, each bottle of Cohn Fraction I product was derived from six bottles of fresh plasma, the number of bottles that could be accommodated in one centrifuge load. Only one batch could be processed in a week. Dr Cumming and his colleagues reported, however, that it appeared from their results that it was possible to prepare a safe and reasonably active Cohn Fraction I from plasma, provided that suitable precautions were taken during processing.^[47] The similar Factor VIII preparation used in England and Wales during the 1960s was referred to as 'NHS freeze dried factor VIII concentrate' by Dr Rosemary Biggs.^[48]

Product range in 1973

20.32 So far as plasma products are concerned, the discussion in this report necessarily focuses on the production of factor concentrates and their use in haemophilia therapy. But it is important to note that, especially up to the beginning of the reference period, this bias gives a false impression of the scope of operations of the Blood Transfusion Services and of the manufacturing facilities. Dr James Smith said of the period before he moved to Oxford, in 1975:

This Inquiry focuses on haemophilia but at no time during these years were we able to neglect the many, many more patients who required immunoglobulins, albumin and other products, which we did not have the right to interfere with too much. These patients were more diffuse in their needs and the clinicians who used these products were scattered. So there was no, if I can call it, pressure group from patients with immunodeficiencies, for instance .... We all had to take account, equal account, of all the users of our products.^[49]

20.33 In a personal assessment of needs dated 12 June 1973, Mr Watt analysed the demand for plasma, drawing on a wide range of information. He wrote:

Discussion on the probable need for plasma for fractionation indicates that, of all fractions prepared, the main limiting consideration is the need for Plasma Protein Solution.^[50] The need for specific immune globulin and salt poor albumin will create errors in calculation but, in a coherent policy of overall balanced use of blood and its fractions, these errors practically cancel each other out to make a net error factor of less than 1% in the total estimate.^[51]

20.34 In his view, at that time, if plasma requirements for plasma protein solution (PPS) or stable plasma protein solution (SPPS) could be met, the supply of plasma would be sufficient for other fraction production. In particular, the amount of plasma, 200,000 litres or 1 million donations, required for AHG (antihaemophilic globulin) preparation was 'of no account in consideration of overall need'.^[52] The critical figure was the 400,000 litres required for PPS. At that stage the PFC had process potential to handle up to 300,000 litres of plasma per year, but could not finish PPS at equivalent rates.^[53]

20.35 Leaving aside questions of projected demand in numerical terms, the balance between AHG and PPS needs reflects Mr Watt's assessment that the principal driver of demand for plasma products in 1973 was the need for albumin, specifically PPS.

20.36 It is possible that the paper may have been, in part, an attempt by Mr Watt to respond to controversy that had developed within the SNBTS relating to developing technologies. There were some significant differences of opinion over the development of plasma products. As already noted, two processes for the production of Factor VIII products had developed, resulting in different products each of which sought to compensate for low Factor VIII levels in the patient's blood. Each allowed reliable Factor VIII treatment, when applied appropriately. Some experts favoured the use of cryoprecipitate, as for example in Glasgow. From around 1968, refinements in Cohn fractionation led to a product of comparable potency in Factor VIII activity which was easier to use, but which involved increased demand on the scarce resource of plasma.

20.37 The controversy was explicit at a joint symposium held on 4 February 1972 by the Royal Society of Edinburgh and the Royal College of Physicians. Professor Cash (then Deputy Director of the Edinburgh and South East Scotland BTS) reflected one view:^[54]

One of the disquieting trends in the last few years has been the energetic activities of the protein chemists. On the basis of the clinical desirability for a small-volume high factor VIII content product, techniques have been developed which go a long way towards this end ....The serious drawback in this work is the high production losses. The shortage of raw material for the treatment of all haemophiliacs at the present time is such that until comparable yields are obtained the production of this type of product should be actively discouraged, or at least strictly controlled and its use limited to a small group of patients, such as those with acquired inhibitors.

20.38 At that stage, in 1972, Mr Watt and colleagues from the PFC (as the BPU had now been re-named) had reservations about the stage technological development had reached. At the symposium they commented that the Newman method was promising, but that it lacked the clinical data necessary to support its effectiveness.

20.39 However, opinion was to change rapidly. Mr Watt had met Dr Johnson in Australia in 1966, and as a result the PFC was provided with advice from Dr Johnson when Mr Watt joined the PFC the following year.^[55] Dr Johnson's 1971 paper was not referred to in the symposium presentation by Mr Watt and his colleagues. It was to provide the methodology for Factor VIII preparation adopted in Scotland.^[56] In the course of the reference period, close collaboration developed between the SNBTS and Dr Johnson's team.^[57] For present purposes, the development of the new PFC at Liberton, Edinburgh, and the adoption of the Newman method there, marked the move towards commercial-scale production of factor concentrates in Scotland.

20.40 The incentive to produce factor concentrates was described by Dr Peter Foster:

They were more potent, defined and purified than cryoprecipitate; they could be filtered to remove bacterial contaminants and had a lower incidence of allergic reactions than cryoprecipitate. In contrast to cryoprecipitate they were also amenable to large volume manufacture compliant with good pharmaceutical manufacturing practice (GMP). The fact that they were freeze dried also made them easier, quicker and more convenient to use than cryoprecipitate, which had to be stored frozen. Crucially, they enabled patients to treat themselves at home, giving people with haemophilia access to education and employment which had not previously been possible.^[58]

20.41 Mr Watt wrote a report (with the assistance of Dr Smith) on the 'Development of Factor VIII concentrates' in December 1973.^[59] The paper focused on the transition from the production of Fraction I, antihaemolytic factor, into the start of the new era of production of more potent concentrates inspired by Johnson and Newman.^[60] He described recent developments. In the latter part of 1972, laboratory-scale batches of plasma, 2-10 litres, were fractionated by the method of Newman and Johnson to intermediate potency Factor VIII. In February 1973, they progressed to the 10-60 litre scale. By the date of his report, a product of intermediate type had been expanded to 100-litre scale and was obtaining 30-40% yield. The report stated:

Large scale crushing and thawing equipment was commissioned in early September 1973, and is functioning adequately on a load of 100 [litres] plasma. It is expected that with minor improvements the batch size may be increased to 180 [litres].

20.42 Cohn Fraction I was produced until the quarter ended 27 September 1974. In the quarterly report for that period it was noted:

This is the last occasion on which A.H.F. (Cohn Fraction I) will appear in these reports. The ... old item (Cohn Fraction I) will not appear after this quarter.^[61]

20.43 By this time, the PFC employed a small volume computer-controlled continuous fractionation process invented by Mr Watt. This contributed to the increased throughput possible at the RIE in the last phase of operation of the facility there, and it was to promote considerably larger-scale production of concentrates after the move to Liberton. Until the move, however, factor concentrate production in Edinburgh remained a small-scale operation, and already exposed the market to imported products as discussed in Chapter 21 Haemophilia Therapy - Use of Blood Products.

20.44 A report to Area Health Boards set out the position as at 6 January 1975:

It is not possible to overlap production at the Royal Infirmary and Ellen's Glen as the computer has been moved to the latter and hence there will be an interim period, as the new plant is tested and brought into production, when the supply of blood products will be reduced. The length of this interim period will depend on the rapidity with which the new plant can be brought into full production; there are many novel features in its design and all must be thoroughly tested.^[62]

20.45 In the Inquiry's Preliminary Report an attempt was made to reflect trends in production by reference to a selection of data from annual reports of the SNBTS which appeared to show that the production of anti-D immunoglobulin and SPPS was more significant than the production of AHF as the PFC at Liberton came on stream (consistent with Mr Watt's approach to calculating production targets for the new facility^[63]). In response to the Preliminary Report, the SNBTS observed that it was unclear what the figures represented as no units had been given. That criticism is accepted. However, with limited exceptions, the source material, SNBTS data, did not specify the units applicable to the several products listed.^[64] The disruptive effect of the move on concentrate production is illustrated in Chapter 21, Haemophilia Therapy - Use of Blood Products, Figure 21.5

20.46 The production of intermediate Factor VIII fell from its 1972-73 level as preparations were made to transfer to the PFC facility. Leaving aside comparisons between products, Anti-D and SPPS production fell to a more limited extent in 1973-75, the construction and commissioning phase. The rapid build-up of production of Factor VIII concentrate after commissioning of the PFC reflected a change of emphasis in production towards meeting the demand for products for haemophilia therapy.

Technology after the Protein Fractionation Centre moved to Liberton

20.47 There were major changes in the technology employed in the PFC at Liberton at or about the beginning of the reference period and continuing throughout the period dealt with in this chapter. They were generally related to increasing process capacity and efficiency, but included work aimed at the removal of virus from concentrates. Although the PFC's Factor VIII concentrate processes were based on Dr Johnson's work, scientists at the PFC contributed to the development of process technology over the period covered in this chapter, internally and in collaboration with Dr Johnson.

20.48 As at April 1975, Dr Foster, then Head of Research and Development at the PFC, wrote a summary report on research and development work in progress.^[65] A wide range of projects, begun on various dates from 1970, were described. The report indicated that there was about to be a step change in the volume of production of Cohn fraction products. A basic continuous fractionation unit, with semi-automatic computer control, would be commissioned at the new facility. It would give a processing capability of at least 2000 litres a week for SPPS. Extension of the system to other PFC products was being evaluated and was expected to allow process optimisation in terms of yield, purity and daily work schedules and an increase in throughput. The development of Factor IX products was in hand. The evaluation and re-design of the Factor VIII systems and processes were also in hand for the production of intermediate Factor VIII concentrate. The use of sonic vibration for precipitate conditioning was being studied with a view to improving centrifugal separation, particularly in continuous processing.

20.49 Two significant aspects of this work were continuous processing, and precipitate conditioning. Dr Foster commented that precipitate conditioning had always formed an important part of fractionation, but that little information had been published on the subject, and the physico-chemical changes involved had not been identified. Significant advances in centrifugal separation in continuous processing were anticipated, along with gains in general knowledge of plasma fractionation and protein isolation.

20.50 The SNBTS followed up the topics: by early 1976 it was becoming apparent that there might be an increase in demand for fractionation. A report was prepared in January 1976.^[66] Dr Johnson had been involved in discussions in November of the previous year. Length of storage of frozen plasma for fractionation was considered. The use of polyethylene glycol to enhance Factor VIII recovery and of heparin to stabilise plasma were to be studied. The design of a continuous thawing system to produce more granular cryoprecipitate was in hand. The PFC's Factor VIII products were known as 'NY' between late 1979 and late 1984 to reflect the collaboration with New York University (and Dr Johnson in particular).^[67]

20.51 For the fractionator, yield of Factor VIII activity was important, and the balance between purity and yield was vital. When the Edinburgh scientist Dr Duncan Pepper^[68] applied for a research grant on 15 June 1978,^[69] the specified areas of interest were Factor VIII stability and yield. Edinburgh research had dealt with methods of maximising the rate of thaw of frozen plasma within the constraint imposed by Factor VIII solubility, and the use of sophisticated mixing and temperature control systems using a thaw-siphon technique. Blocks of frozen plasma were crushed to increase the surface area over which heat was applied. By continuously removing the thawed plasma, below the solubility temperature of the Factor VIII component, over a wide area of plasma 'snow' produced by crushing, dissolution of the Factor VIII was avoided and the degree of Factor VIII degradation was reduced. It was said that the surface area factor had been ignored by others. The design of processes for crushing and continuous thawing, using fluid removal for temperature control, became one of the defining features of research and development work at the PFC for a considerable time. Dr Foster published a poster presentation and abstract of their work at the Seventh International Congress of Thrombosis and Haemostasis, London, in July 1979. The emphasis within the PFC on techniques for large-scale plasma thawing for the recovery of cryoprecipitate Factor VIII continued.

20.52 In the end, not all of the developments proposed by Dr Johnson were taken up universally. Dr Smith commented that the higher purity concentrate using polyethylene glycol and glycine never gained wide use, and was not continued beyond initial experiments in Edinburgh.^[70]

20.53 It is not necessary to trace all of the developments in technology in this period for the purposes of this report. The manufacturing process became complex and highly defined. The emphasis was on technological improvements in processing raw materials to increase efficiency in the production of an intermediate purity concentrate, while meeting demand for other blood products such as immunoglobulins and albumin products, particularly SPPS.

20.54 A statement was provided by Dr Foster which includes a narrative of the various manufacturing steps along with simplified flow diagrams as at the end of 1983. (See Figures 20.1, 20.2 and 20.3 at the end of this chapter for the flow diagrams.) Dr Foster had also earlier supplied a floor plan of the ground floor of the PFC,^[71] a series of photographs detailing elements of the fractionation process^[72] and a film of the process made in 1995, which was viewed on day 41 of the public hearings.^[73]

20.55 A total of 17 steps were needed in order to achieve a finished product with a specified amount of Factor VIII activity per vial, starting from frozen plasma which the PFC received from the SNBTS. They were described in some detail by Dr Foster as at the end of 1983 in response to a request by the Inquiry^[74] and were discussed at length during Day 41 of the Inquiry's hearings.^[75] The aim was to create a product which would be suitable for home therapy and which would comply with the specifications of the British Pharmacopoeia.

20.56 The process began with 'plasma conditioning' by bringing the frozen plasma delivered to the PFC from minus 40°C to a temperature of about minus 15 to minus 10°C. Batches of 4000 donations each were processed at the rate of about two per week.^[76] The plasma was then stripped from its plastic containers, crushed in a hammer mill, and thawed as quickly as possible to recover cryoprecipitate particles for processing.^[77] The plasma had to be thawed at a temperature that avoided the particles of cryoprecipitate from being dissolved.

20.57 The crushed plasma 'ice' was discharged continuously into a cylindrical thawing vessel which heated the ice to just above its melting point, and released melted plasma, containing particles of cryoprecipitate, to drain by gravity into a holding vessel. From there, the material was pumped to a centrifuge where cryoprecipitate particles were accumulated on the walls of the vessel, and the clarified liquid supernatant drained into a collection vessel for further processing. The cryoprecipitate was used to make Factor VIII and the cryo-supernatant was used to make Factor IX.^[78]

20.58 Continuous thawing was a major advance on previous technology which had depended on thawing in small-volume batch tanks, and in changing temperature conditions. It enabled plasma throughput to be increased relatively easily.^[79] In comparison with the superseded batch thawing method, the yield of Factor VIII activity was increased by about 50%.^[80] Solubility was enhanced, and in due course this enabled the product (NY) to withstand dry heat treatment at 68°C for 2 hours without further process modification.

20.59 The use of continuous thawing was devised by the SNBTS and published in 1978.^[81] It was introduced for routine production in August 1979, and progressed to faster production with upgraded equipment in January 1981.^[82] The improvements were reported in 1982.^[83]

20.60 After centrifugation, the cryoprecipitate was rinsed in a 2% solution of ethanol at 2°C to remove any residual plasma which might contain potentially damaging substances. The rinsed cryoprecipitate was suspended in a buffer solution which was designed to protect the material from chemical shock as processing continued to dissolve most of the cryoprecipitate whilst excluding material that was poorly soluble.^[84] The pH of the solution was adjusted to pH 7.0, the optimum pH for the recovery of Factor VIII, by the slow addition of dilute hydrochloric acid.

20.61 At this stage a residue of unwanted coagulation factor proteins remained in the cryoprecipitate, including other coagulation factors (Factors II, VII, IX and X) which had not been removed by the thawing process, as well as impurities which could otherwise cause the Factor VIII to become unstable. These were known to bind preferentially to aluminium hydroxide [Al(OH)3] and a stable gel of that material was introduced to remove them. This occurred by adsorption of the unwanted materials, leaving the Factor VIII in solution. The aluminium hydroxide gel and the adsorbed materials were then separated from the main solution by centrifugation in bottles, and the supernatant solution was decanted from the bottles into a sterile pressure vessel for filtration through a series of successively finer filters.

20.62 An anticoagulant, tri-sodium citrate, was then added to prevent de-stabilisation of the Factor VIII by any residual activity from trace levels of coagulation factors other than Factor VIII. The pH was adjusted to pH 6.8 with dilute hydrochloric acid. Further filtration with even finer filters followed.

20.63 The final Factor VIII solution was then dispensed into sterile glass vials using an automated aseptic dispensing system. The amount of Factor VIII dispensed was less than the capacity of the glass vial so that patients could add distilled water to the final freeze-dried product. Each vial was fitted with a raised stopper with small grooves in the side to allow moisture to escape from the vials during freeze-drying. The products were frozen solid, and then dehydrated by freeze-drying.

20.64 Overall, the process to this stage took around one week. Three to four months were then needed for inspection, labelling and other procedures before the batch could be released for use.^[85]

Factor IX

20.65 The manufacture of DEFIX was also described in depth in Dr Foster's statement on manufacturing.^[86] The process was also discussed during Day 41 of the Inquiry's hearings.^[87] A total of 17 additional steps were involved after the removal of the cryosupernatant from the cryoprecipitate. Ion exchange technology was used to separate Factor IX and related proteins from a supernatant containing immunoglobulin and SPPS/albumin. The cryosupernatant was prepared for ion exchange by use of sterile, pyrogen^[88] free water at 4°C and adjustment of the pH to 6.9. Ion exchange gel was added and Factor IX and related proteins became attached to the gel. The separation was achieved by centrifugation. The ion exchange gel and adhering proteins were suspended in a buffer 'wash' solution for ease of pouring into a chromotography column.

20.66 The wash solution was allowed to drain from the column, leaving the ion exchange gel and its bound proteins as a squat column. In a process known as elution, the chromatography column was flushed with a buffer solution containing sodium chloride and other sodium compounds until the coagulation factors were observed to begin emerging from the column, detected by a sharp rise in the conductivity of the solution at the column's out-flow. Various different 'eluates' or fractions were collected in containers. The fractions which met the requisite specifications for Factor IX activity and non-thrombogenicity were then thawed, in sealed containers, at room temperature. When thawed, the containers were opened, the selected fractions pooled and samples taken of Factor IX activity. After that, the solution was diluted, if necessary, to achieve a target Factor IX potency of 34 IU/ml. Filtration to 0.22 micrometres followed, as in the case of Factor VIII, and the solution was dispensed aseptically into glass vials, which were then frozen in the same manner as outlined for Factor VIII above.

20.67 Two of the steps common to the processes were to become significant at a later period: plasma conditioning and continuous thawing.

Virus research at the Protein Fractionation Centre in the 1970s

20.68 The processes for Factor VIII and Factor IX production so far described were the result of extensive research and development, much of it involving innovative science and technology. The removal of unwanted proteins at successive stages of the programme probably removed virus particles incidentally in the preparation of the concentrates prepared for clinical use. But they did not provide for the inactivation of any residual virus particles remaining in the final product. The products remained potentially infective in clinical use. Subject to any parallel developments in virus inactivation that were achieved, increases in the efficiency of process technology, leading to increased production capacity and output, necessarily increased the exposure of patients to risk. Until 1975 at the earliest, the known risk was of transmission of Hepatitis B. Blood donations were screened with increasing efficiency, so that the plasma received for fractionation became less likely to carry virus. For immediate purposes, the focus is on fractionation technology and the steps taken to reduce risk in processing. Virus inactivation by heat treatment is discussed in greater detail in Chapter 23, Viral Inactivation of Blood Products for Haemophilia Therapy up to 1985.

20.69 In the 1970s, researchers at the PFC were active in exploring the physical removal of the Hepatitis B virus by precipitation of the virus using polyethylene glycol (PEG) as part of the Factor IX production process.^[89] The research was part of the 'Supernine Project', a collaborative exercise with Dr Johnson and other scientists at New York University, which aimed to replace the PFC's standard DEFIX product for Haemophilia B with a concentrate that would be three to five times more potent, and have a reduced risk of transmitting Hepatitis B.^[90] The US part of the project ultimately ran into funding difficulties when the USA National Institute of Health refused an application for further chimpanzee studies.^[91] These funding difficulties meant that research could not be continued to assess whether the process was successful in removing Hepatitis B infectivity.

20.70 Another part of the project involved an assessment of possible thrombogenic reactions connected to the new Supernine product, a known complication of the use of Factor IX therapy.^[92] A team at the PFC led by Dr Foster demonstrated that the PEG processing used in an advanced form of the product reduced the amount of thrombogenic material present.^[93] Supernine was ultimately never released for clinical use as the Medicines Control Agency were reluctant to issue a second Factor IX licence. But the work on thrombogenic reactions was of continuing benefit.

20.71 In February 1982, Dr Alex MacLeod, PFC, conducted a series of experiments in the pasteurisation of PFC's intermediate Factor VIII product with a view to inactivating NANB Hepatitis virus. It was found that if the standard product was diluted in its normal reconstituted volume, heating resulted in clotting. However, they found that if the product was diluted in the presence of certain stabilisers, the product could be heated at 60°C in a water bath and remain fluid, though becoming cloudy. It was concluded at that time that the ability to pasteurise Factor VIII concentrate was linked to purity, and that a high purity product would be required for effective treatment. The project did not progress further at that stage. But, once more, research had provided information that would prove to be of value later in the 1980s.

20.72 The PEG precipitation method was not applicable to Factor VIII because the sizes of the Factor VIII complex and the virus molecule were not sufficiently different for effective separation by the process technology developed at the time.^[94] In his report dated December 1973,^[95] Mr Watt included in his narrative of candidates for investigation the use of specific solid-phase polyelectrolytes in a procedure for the purification of Factor VIII. The procedure had been developed by Dr Johnson. It exploited a characteristic of Factor VIII which resulted in the protein attaching preferentially to the polyelectrolyte, while other substances (including viruses) were not attached and could, in theory, be separated by washing.^[96] The PFC's research had depended on proprietary polyelectrolytes supplied by Monsanto for research purposes only. The company had required that the project should be carried out under strict confidentiality and that all research reports should be destroyed or returned to them. Monsanto were unwilling to agree to license-out the reagent for production purposes, and the project was discontinued.^[97]

20.73 In view of the difficulties encountered, Dr Foster approached research groups, including groups already involved in research collaboration with the SNBTS, at a number of UK universities to encourage them to undertake fundamental research into ways of eliminating the risk of coagulation factor concentrates transmitting hepatitis.^[98] However, his attempts to set up collaborations with UK universities were unsuccessful.

20.74 Until 1981, and the publication of the work of Behring on the pasteurisation of Factor VIII,^[99] knowledge of the possibility that Factor VIII might be treated with heat to inactivate virus contamination was limited to those who were aware of the first public disclosure of the work at a symposium in Bonn in October 1980. Professor Cash attended that symposium and reported the information to Dr Foster among others.^[100] Dr Foster commented on his response to the information:

I was quite shocked when I heard this claim, as the notion that factor VIII might be able to be heat treated under conditions that would destroy hepatitis viruses was inconceivable to me.^[101]

20.75 Dr Foster had substantial reasons for his reaction. The view of Dr Webb, under whom he had studied at University College London, was that apart from albumin all fractions were heat labile. Dr Foster's own doctoral research made Factor VIII an implausible candidate for research on heat treatment; and experience of the PFC's experiments of filtration performed at 20°C and progressively higher temperatures had confirmed his view that Factor VIII was sensitive to an increase in temperature and that loss of Factor VIII activity was temperature-dependent. The view of others, including Dr Frank Boulton, was that Behring's claims could not possibly be true, and that eventually it would be discovered that it was a mistake.^[102]

20.76 On the eve of the outbreak of AIDS, therefore, there was a step change in perception of the possibilities of heat treatment to inactivate hepatitis viruses, but continuing scepticism among scientists. Meantime, research in England, led by Dr John Craske, was reaching the conclusion that all Factor VIII concentrates in production in the early 1980s, imported or NHS, were potentially infective for NANB Hepatitis. The scene was changing rapidly. Viral inactivation by heat treatment is discussed in the following chapters.

Figure 20.1: Simplified process flow-sheet for the fractionation of plasma at the pfc at the end of 1983

Figure 20.2: (A, B & C)

Figure 20.3: (A & B)

21. Haemophilia Therapy - Use of Blood Products >