Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1999-10-21
2002-10-15
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S413000, C530S350000, C530S300000, C514S002600, C514S021800, C514S012200, C260S66500B
Reexamination Certificate
active
06465624
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national phase of PCT/AT98/00034 filed Feb. 18, 1998, which claims priority from the Austrian application A 337/7 filed Feb. 27, 1997.
FIELD OF THE INVENTION
The invention relates to a method of recovering a purified von Willebrand factor (vWF) by means of cation exchange chromatography.
BACKGROUND
In plasma, vWF circulates at a concentration of from 5-10 mg/l, partly in the form of a non-covalent complex with factor VIII. vWF is a glycoprotein which is formed in various cells of the human body and later is liberated into the circulation. At this, a vWF dimer (primary vWF dimer) having-a molecular weight of approximately 450000 Da is synthesized in the cells, starting from a polypeptide chain having a molecular weight of approximately 225000 Da (vWF monomer) by forming several sulfur bonds. From the vWF dimers, further polymers of vWF with ever increasing molecular weights, up to approximately 20000000 Da, are in turn formed by forming links via sulfur bonds.
One important criterion for characterizing vWF is the multimer-structure analysis by agarose electrophoresis. It is assumed that particularly the high-molecular vWF polymers are of essential importance in the coagulation of blood. The functional activity of vWF usually is determined by the ristocetin-cofactor-activity (vWF:RistCoF). The ratio between activity and vWF antigen concentration (vWF:Ag) is determined as a characteristic for the purity and specific efficacy of vWF. The specific activity of a preparation increases with an increasing ratio of vWF:RistCoF to vWF:Ag.
vWF assumes an important function within hemostasis. It circulates in plasma partly as a complex with factor VIII. which aids the coagulation of blood as a cofactor. Factor VIII is stabilized by complex formation with vWF and protected from proteolytic degradation thereby. A further object of vWF is its participation in thrombocyte aggregation which makes an important contribution to primary hemostasis. At this, vWF binds to the glycoproteins Ib and IIb/IIIa of the surface receptors of the thrombocytes and thus cross-links the thrombocytes to a thrombocyte aggregate. What is furthermore important for primary hemostasis is the affinity of vWF to collagen, a component of the extracellular matrix which, in intact vessels, does not have a direct contact with blood, since it is shielded from the blood flow by a monolayer of endothelial cells. When blood vessels are injured, a local detachment of the endothelial cell layer occurs at the site of lesion, resulting in a direct exposure of the components of the extracellular matrix to the blood. By its affinity to collagen, vWF is capable of fixing the forming thrombocyte aggregate in the damaged vessel region to the exposed subendothelium. This results in a first, labile wound closure which will be further strengthened by the subsequent blood coagulation.
von Willebrand syndrome is characterized by a deficiency of a functional von Willebrand factor or by an abnormal spectrum in the multimer composition of the von Willebrand factor. On account of a deficient stabilization of factor VIII, patients afflicted with von Willebrand syndrome may develop a factor VIII deficiency in spite of the fact that usually the synthesis rate of factor VIII is normal, such factor VIII deficiency being a consequence of the greatly reduced plasma half-life of this coagulation factor. Therefore, patients suffering from von Willebrand syndrome may exhibit symptoms similar to those of hemophilia A patients (phenotypic hemophilia). The lack of functionally active vWF may also cause dysfunctions of thrombocyte aggregation and adhesion in patients afflicted with von Willebrand syndrome, which may lead to defects in primary hemostasis. On account of dysfunctions of these vWF-mediated procedures, patients afflicted with von Willebrand syndrome exhibit increased bleeding times.
For the treatment of von Willebrand syndrome, thus vWF preparations must be administered which balance out the lack of functionally active vWF. To this end, preparations may be used which are also used in the therapy of hemophilia A, such as cryoprecipitate or the factor VIII concentrates prepared therefrom, which contain complexes of factor VIII and vWF. However, for the treatment of hemophilia A, better purified factor VIII:C concentrates are always used which either do not contain vWF or contain merely traces thereof. Since supplementing patients afflicted with von Willebrand syndrome with factor VIII is not necessary, and since the factor VIII application harbours the risk of inducing inhibitory factor VIII antibodies in the patient, a vWF preparation as free as possible from contaminating factor VIII would be very desirable for the treatment of von Willebrand syndrome. Therefore, there is a demand for pure and virus-safe von Willebrand factor preparations having a high specific activity.
In the literature, various methods of purifying and recovering vWF have been disclosed.
EP 0 503 991 describes the purification of vWF from human cryoprecipitate by three successive chromatographic steps: 1) anion exchange chromatography on TSK-DEAE Fractogel, and elution of vWF by means of 0.15 M NaCl; 2) another anion exchange chromatography on TSK-DEAE Fractogel, and elution of vWF by means of 0.17 M NaCl; and 3) affinity chromatography on gelatin sepharose to separate the contaminating fibrinogen. There, amino acid- and calcium ion-containing buffers were used.
WO 89/12065 describes the separation of plasmatic vWF from factor VIII and further proteins by binding the proteins to an anion exchanger and step-wise elution by increasing the salt concentration. The vWF-containing fraction was chromatographed for a second time via an anion exchanger and recovered as a concentrate.
EP 0 469 985 discloses the purification of plasmatic vWF from cryoprecipitate, wherein factor VIII is selectively bound to an anion exchanger in a first step at a salt concentration of 250 mM, while vWF remains in the supernatant. After lowering the salt concentration of the vWF-containing supernatant to a salt concentration of between 100 mM and 150 mM, vWF is bound to a second anion exchanger and eluted at pH 6.6 with 300-350 mM NaCl. There, vWF having an activity of at least 50 U/mg is recovered, which contains a portion of factor VIII of <2%.
DE 39 04 354 describes the recovery of plasmatic vWF from cryoprecipitate, and the separation of vWF from factor VIII by selective adsorption of factor VIII on an anion exchanger, while vWF remains in solution. There, a solution containing 160 U/ml of vWF is recovered.
U.S. Pat. No. 5,006,642 describes the recovery of vWF from a solution of vWF and chaotropic agent, incurred as a by-product according to U.S. Pat. No. 4,361,509, by dialysis against a suitable buffer or by desalting the solution by means of a further chromatographic step.
EP 0 383 234 describes the production of a vWF concentrate by means of anion exchange chromatography, wherein a factor VIII/vWF-complex contained in a solution is dissociated by the addition of a calcium and amino-acid-containing buffer, and a vWF concentrate is recovered.
WO 96/10584 describes a method of recovering highly purified recombinant vWF by means of combined anion exchange/heparin affinity chromatography, and EP 0 705 846 describes the separation of high and low molecular fractions of recombinant vWF by means of heparin affinity chromatography.
To recover a purified vWF preparation having high specific activity, it has been necessary so far to combine several chromatographic steps. In particular, the production of preparations particularly containing high-molecular vWF multimers has so far been possible only via a heparin affinity chromatography. Heparin, however, is a relatively expensive chromatographic material.
SUMMARY
It is the object of the present invention to provide a method of recovering purified vWF having an improved specific activity, which method is suitable for a large technical application on an industrial scale. The method should be u
Dorner Friedrich
Eibl Johann
Fiedler Christian
Fischer Bernhard
Mitterer Artur
Baxter Aktiengesellschaft
Carlson Karen Cochrane
Robinson Hope A.
Townsend and Townsend / and Crew LLP
LandOfFree
Purification of von-Willebrand factor by cation exchanger... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Purification of von-Willebrand factor by cation exchanger..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Purification of von-Willebrand factor by cation exchanger... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2960782