The Preparation and Clinical Use of a New Concentrate Containing Factor IX, Prothrombin and Factor X and of a Separate Concentrate Containing Factor VII

Abstract

A method of preparation for therapeutic use of a concentrate of factors IX, 11 and X and a separate concentrate of factor VII is described. The method is based upon adsorption of the factors on DEAE-cellulose from plasma or from the supernatant after removal of factor WI. Conditions of elution are selected so that, in a single step, the factors are obtained at a suitable salt concentration and potency to permit injection of a therapeutic dose by syringe. The yield of factor IX in vitro is about 5~7 5 % and the purification about 300-fold. The yield of factor VII is lower and variable. Experience in the use of 14 batches of factor-IX concentrate in the treatment of 29 patients and on the use of a batch of factor-VII concentrate in the treatment of one patient is described. The in viuo dose response to factor I X made by different methods is compared,The first reliable therapeutic concentrate of factor IX was prepared by a method requiring specially collected blood (Didisheim et al, 1959; Blatrix & Soulier, 1959). W e found that the residue from a large-scale fractionation of human plasma for factor VIII, 7-globulin and albumin, contained sufficient factor I X to make it an acceptable starting material (Biggs et al, 1961). The method in current use, which will subsequently be called the 'routine' method, was described by Bidwell et al(x967, 1972). The product (subsequently called the 'routine' material) is a white powder which dissolves readily giving a clear solution in which the factor-IX concentration is 4-10 x that of plasma and the purification 250-550 x . The overall yield by in vitro assay is c 20% of the factor IX assumed to be present in the original starting plasma, DEAE-celldose was used by Melin and co-workers (Tdlis et a!, 1965) for preparing a factor-IX-prothrombin compIex from plasma separated from blood decalcified by ion-exchange resin. Deggeller (1969) and Casillas et al (1969) showed that the same principle could be applied to ACD plasma from which factor VIII for therapeutic use had previously been separated. Both methods have practical disadvantages (see Table I).The establishment of the Plasma Fractionation Laboratory as part of the Oxford Haemophilia Centre presented an opportunity to deveIop a method of producing factor-IX concentrate from a starting material other than a large-scale fractionation residue. The criteria adopted for a method intended to supersede our present well-established procedure were : Correspondence: Mr G. W. R. Dike, Oxford Haemophilia Centre, Churchill Hospital, Oxford. G TABLE 11. Yield, purification and patients' responses for 14 batches of factor-IX concentrate Batch

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

“…On electrophoresis in polyacrylamide gel, the concentrate was found to contain all the zones present in the preparation of Dike et al [8] with some deferences in the relative intensities of the minor bands; our concentrate contained less pre-albumin, albumin and ctj-globulin than did that of Dike et al [8].…”

“…The relevance of immunoglobulin type to hetero agglutinin activity and pyrogenicity is being pursued in the light of these results. It has been found that IgM, and rather less IgG, is eluted coincidently with the factor IX-containing fractions, using either our standard procedure or the conditions of Dike et al [8]. The 'second eluate' from the control experiment discussed above contained less IgM than the 'first eluate'.…”

“…One experiment was carried out precisely according to the procedure of Dike et al [8] using a 10 X 60 cm column; in other experiments all our standard condi tions were used except for the substitution of the exchanger equilibrating conditions and elution buffers of Dike et al [8]. Differences in column dimensions had little effect on the distribution of proteins in the eluate, although the sharper elution made possible by a narrow column yielded a higher ratio of factor IX to salts.…”

“…Batches of concentrate were made according to the method of Dike et al [8], or using the latter's buffer system in preparations otherwise identical with our standard procedure. All proved as pyrogenic in rabbits as our routine batches, but those issued for clinical use were not pyrogenic in human patients.…”

“…The wide range of alternative methods for the production of factor IX concentrates has been well reviewed recently [8,16].…”