James K. Smith

In Vitro Spontaneous Thrombin Generation in Human Factor-IX Concentrates

A series of in vitro studies designed to ascertain the potential in vivo thrombogenicity of human factor IX‐containing concentrates is described. Using concentrates obtained from several different Centres the fibrinogen clotting time with some preparations was less than 6 h and/or the recalcification time of normal plasma was shortened. In some preparations, however, the plasma recalcification time was lengthened.

A Therapeutic Concentrate of Coagulation Factors II, IX and X from Citrated, Factor VIII‐Depleted Plasma

Abstract. A simple procedure is described for the large‐scale absorption on to DEAE‐cellulose of coagulation factors II, IX and X from citrated, factor VIII‐depleted plasma. The coagulation factors are eluted frontally from the exchanger in a high yield and in a form suitable for therapeutic use, without further fractionation. The lyophilised concentrate is very stable without the addition of heparin and, when redissolved to isoosmolar solution, contains approximately 30 U/ml factors II, IX and X, 250–300 times purified from the starting plasma. The effectiveness of the concentrate in the treatment of haemophilia B is discussed.

Severely Heated Therapeutic Factor VIII Concentrate of High Specific Activity

Abstract. A new method for the manufacture of a heated factor VIII concentrate of high specific activity (2–6 IU factor VIII:C/mg protein) has been developed. Addition of heparin to cryoprecipitate extract at acid pH precipitated fibrinogen and fibronectin. Factor VIII was then recovered from the supernatant by precipitation with glycine and sodium chloride. After re‐solution and desalting on Sephadex G‐25, the concentrate was sterile‐filtered and lyophilised. The dried product was stable to heating in the final container at 80°C for 72 h. Data from 25 consecutive batches of concentrate prepared from 1,200‐1,500 kg plasma pools are presented. The mean final yield of heated product was 190 IU factor VIII:C/kg plasma. The concentrate has been found to be safe and effective in clinical use.

A Pasteurized Antithrombin III Concentrate for Clinical Use

Abstract. A method for large‐scale production of a pasteurized antithrombin III (AT III) concentrate for therapeutic use has been adapted from published methods. It includes the following steps: (1) batchwise adsorption onto heparin‐Sepharose from plasma depleted of cryoprecipitate and prothrombin complex; (2) chromatographic elution at high salt concentration; (3) pasteurization for 10 h at 60°C in the presence of added citrate ion; (4) desalting on Sephadex G‐25, and (5) sterile filtration and freeze‐drying.

Pilot Study for Large‐Scale Plasma Procurement Using Automated Plasmapheresis

Abstract. A pilot study for large‐scale automated plasmapheresis using the Haemonetics Model 50 machine was undertaken in the Yorkshire Region of the United Kingdom to determine the viability of such a programme for national self‐sufficiency in fresh plasma procurement for factor VIII concentrate production. The study was designed to resolve three areas of concern: donor safety and recruitment; a cost analysis, and the choice of anticoagulant for optimum factor VIII yields. The results show that large‐scale automated plasmapheresis could safely and economically produce high‐quality source plasma necessary for national self‐sufficiency.

Quality of plasma for fractionation—does it matter?

Many plasmapheresis techniques cause greater activation of platelets, complement and coagulation than does simple whole blood collection. Activation of the coagulation cascade has been particularly evident in tests for potential thrombogenicity in prothrombin complex concentrates made from apheresed plasma. Attempts to improve the factor VIII content and fractionation yield of source plasma have centred on rapid freezing, increased concentration of anticoagulants and the exploitation of anticoagulants optimal for the preservation of coagulant, rather than cellular, activities. The cumulative advantage is of the order of 10% over recovered plasma. There are potential pitfalls in applying low-citrate anticoagulation strategies to plasmapheresis. Recalcification of citrated plasma, under cover of an optimal concentration of heparin, might be explored more vigorously.

A small-scale model of factor VIII and factor IX fractionation from plasma

Abstract A small-scale model of factor VIII and factor IX fractionation from human plasma has been developed. Validation experiments demonstrate that it accurately reflects processing at pilot scale. Tests of reproducibility show that there is greater agreement between pools composed of plasma from the same donors than of random donors, but to predict the performance of pilot and manufacturing scale fractionation several pools from different donor panels must be tested.

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.

IMPROVED FACTOR VIII CONCENTRATE OF INTERMEDIATE PURITY

For some years until the end of 1976, the factor VIII concentrate prepared for the NHS in England, Wales and Northern Ireland was of the ‘intermediate purity’ type made, with minor modifications, by the method of Newnian et a1 (1971). This freeze-dried concentrate had replaced earlier products prepared as described by Kekwick & Wolf (1957) or Blomback (1958). It was issued by the Blood Products Laboratory, Lister Institute, Elstree, and the Plasma Fractionation Laboratory, Churchill Hospital, Oxford, in 400 ml bottles containing approximately 250 iu to be dissolved in 50 ml water giving a potency of about 5 iu/ml. A more concentrated solution was desirable and development work carried out independently in the two laboratories has now led to two modified procedures, each of which yields such an improved product. Although the details of the procedure differ, the final products have similar characteristics. Each vial contains 200-250 iu factor VIII and may be dissolved in 15-20 ml water giving a potency of approximately 11-15 iu/ml and offering greater convenience in use and economies in production. The improvements have been made, without reducing the yield of factor VIII, using fresh frozen plasma from the English and Welsh Regional Transfusion Centres. Potency was improved by reducing the volume of buffer used to extract the cryoprecipitate as described by James & Wickerhauser (1972), but the more concentrated extract was found to be difficult to sterilize by membrane filtration and after freeze drying dissolved only slowly. These drawbacks were overcome by introducing, in both new procedures, an additional purification stage in which contaminating proteins were precipitated from the factor VIII extract by manipulating pH and temperature. New ions or precipitants were not introduced. The dried products thus prepared, dissolve in approximately the same time as the earlier concentrate

Stability of normal human IgG in saline and glycine solutions.

Abstract. Human normal 15% IgG was prepared for therapeutic use in saline and in glycine solutions. The saline solution was more acceptable for both intravenous and intramuscular use. High anticomplementary activity was present in the IgG precipitate freshly prepared by ethanol fractionation, and increased by only a small factor after freeze‐drying.