von Willebrand factor circulates in plasma at a concentration of from 5 to 10 mg/I, mainly in the form of a non-covalently bound complex with factor VIII. In the cryoprecipitate, factor VIII/vWF-complex is highly enriched and can be isolated therefrom or from plasma or from plasma fractions by means of known fractionation methods.
In hemophilia, blood coagulation is impaired by a deficiency of certain plasmatic blood coagulation factors. In hemophilia A, the bleeding inclination is based on a deficiency of factor VIII or of vWF, respectively (phenotypic hemophilia). Treatment of hemophilia A is mainly effected by substituting the missing coagulation factor by factor concentrates, e.g. by infusion of factor VIII or of factor VIII/vWF-complex.
A purified factor VIII, complexed with vWF, is desirable for utilization in the therapy of patients suffering from hemophilia A, but also for von Willebrand syndrome (Berntorp, 1994, Haemostasis 24:289-297). In particular, it has been emphasized repeatedly that in preparations lacking vWF or having only a low content thereof, an increased bleeding time and a low factor VIII:C half-life can be observed in vivo. Normalization of vWF in vivo is important so as to maintain a concentration of factor VIII in plasma both by reducing the factor VIII elimination rate and by aiding the release of endogenous factor VIII (Lethagen et al., 1992, Ann. Hematol. 65: 253-259).
DE 3 504 385 describes the execution of an ion exchange chromatography for the purification of factor VIII/vWF-complex, wherein the factor VIII complex is bound via sulfate groups and is eluted with citrated buffer, calcium chloride and NaCl gradient. Therein, the factor VIII/vWF-complex is eluted from the carrier at a concentration of 0.5 M NaCl.
EP 0 416 983 describes the recovery of the factor VIII/vWF-complex from human plasma by a combination of barium chloride- or aluminum hydroxide-precipitation and anion exchange chromatography on DEAE Fractogel.
Harrison et al. (Thrombosis Res., 1988; 50, 295—304) describes the purification of factor VIII/vWF-complex by chromatography on dextrane-sulphate-sepharose.
EP 0 600 480 describes a purification method for factor VIII/vWF-complex from whole plasma by means of combined anion exchange/cation exchange chromatography. The elution of the FVIII/vWF-complex adsorbed on the cation exchanger is accomplished using a Ca-containing buffer having 0.3 M NaCI in a pH range of between 6.6 and 7.0.
WO 96/10584 describes a method of recovering highly-purified recombinant vWF by means of a combined anion exchange/heparin affinity chromatography, and EP 0 705 846 describes the separation between high and low molecular fractions of recombinant vWF by means of heparin affinity chromatography.
The factor VIII preparations described in the prior art to the greatest part do contain the entire vWF multimer pattern, yet they vary as regards their portions of high-molecular vWF (HMW-vWF) and low-molecular vWF (LMW-vWF), and they also exhibit so-called triplet structures suggesting a proteolytic degradation, in particular of HMW-vWF. The stability of these preparations often is limited thereby.
It has been emphasized repeatedly that factor VIII/vWF preparations containing substantially HMW-vWF possibly might have a positive influence on the bleeding time, since they carry out the primary function of vWF, the platelet agglutination, and have a higher affinity to the platelet receptors glycoprotein IB and IIb/IIIa than low-molecular vWF multimers.
There has been a demand for a factor VIII complex having a sufficiently specific activity of factor VIII:C- and vWF-activity. One problem in the recovery of such a complex particularly is the separation of molecules containing low-molecular vWF multimers, and the enrichment of complexes with a high specific vWF activity.
USPTO Backgrounds
USPTO Backgrounds