Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-05-08
2004-12-14
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S412000, C530S350000, C435S007100, C514S021800
Reexamination Certificate
active
06831159
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method of purifying factor VIII/vWF-complex from a biological starting material by means of cation exchange chromatography and step-wise elution, as well as purified factor VIII/vWF-COMPLEX complex which particularly comprises high-molecular vWF multimers.
BACKGROUND
von Willebrand factor circulates in plasma at a concentration of from 5 to 10 mg/l, 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 there is effected by using a Ca-containing buffer having 0.3 M NaCl 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.
SUMMARY
Thus, it is the object of the present invention to provide a factor VIII/vWF complex having improved specific activity and stability.
It is a further object to provide a method of recovering such a factor VIII/vWF-complex. The method should be usable for the purification of both, a recombinant and a plasmatic factor VIII/vWF complex.
According to the invention, this object is achieved in that a method of recovering factor VIII/vWF-complex is provided, in which factor VIII/vWF-complex from a protein solution is bound to a cation exchanger, and factor VIII/vWF-complex having an improved specific vWF activity is recovered by step-wise fractionated elution. The recovery and enrichment of factor VIII/vWF having improved activity and stability is particularly effected in that factor VIII/vWF complex is bound at a low salt concentration, that by a step-wise raising of the salt concentration, fractions containing factor VIII/vWF-complex with low-molecular vWF multimers, inactive vWF degradation products and unspecific accompanying proteins are separated at a medium salt concentration, and fractions containing factor VIII/vWF-complex that particularly contains high-molecular vWF multimers are recovered at a higher salt concentration.
On account of its acidic isoelectric point (IEP=5.5 to 6) and its negative net charge resulting therefrom, factor VIII/vWF-complex usually is purified in a weakly acidic to basic environment via positively charged anion exchangers. Thus, on account of the methods described so far of purifying factor VIII/vWF-complex by means of positively charged anion exchangers, it could not be expected for factor VIII/vWF -complex to bind also to a negatively charged gel matrix of a cation exchanger at a pH lying above the IEP of the complex and at a low salt concentration, and to be selectively elutable therefrom by raising the salt concentration. Neither could it be expected that by a step-wise elution at a salt concentration of approximately between ≧250 mM and ≦300 mM, unspecific accompanying proteins, inactive vWF degradation products, complex components having a low specific activity, factor VIII/vWF-complex containing low-molecular vWF multimers, non-complexed or merely weakly bound factor VIII and free factor VIII are eluted, and that at a salt concentration of ≧300 mM in particular factor VIII/vWF-complex with high-molecular vWF multimers is obtained.
It has been found within the scope of the present invention that with the method according to the invention, departing from an impure biological material, purified fractions are obtained which are substantially free from contaminating nucleic acids. Thereby also nucleic acids are removed from protein preparations by this method. This effect cannot be demonstrated with conventional methods by means of anion exchangers, since nucleic acids, on account of their negative charge, bind to the anion exchanger, detach from the anion exchanger again by increasing the salt concentration, and get into the eluate.
When purifying the factor VIII/vWF complex, particular attention must be paid that, on account of the size of vWF ranging from 500 000 to several millions, only such carrier materials which do not impede the diffusion and distribution of the factor VIII/vWF complex in the carrier materials used will result in good purification and good yields. When carrying out the method according to the invention of purifying factor VIII/vWF-complex with a high specific activity by means of cation exchanger, a gel matrix is used which has not only a high loading capacity, is robust to handle and has a clear elution profile, but which also can be used economically on an industrial scale. Thus, the method according to the invention is particularly interesting for the recovery of purified factor VIII/vWF-complex on a large technical scale.
Every known cation exchanger can be used for carrying out this method, cation exc
Dorner Friedrich
Eibl Johann
Fischer Bernhard
Mitterer Artur
Schönberger Öyvind L.
Baxter Aktiengesellschaft
Low Christopher S. F.
Robinson Hope A.
Townsend and Townsend / and Crew LLP
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