Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1998-11-18
2001-10-23
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
C530S412000, C530S383000, C514S012200, C514S802000, C514S834000
Reexamination Certificate
active
06307032
ABSTRACT:
The present invention relates to a highly purified complex consisting of the components factor VIII (factor VIII:C or FVIII) and von Willbrand factor (vWF), a stable pharmaceutical preparation containing the highly purified complex as well as a method of preparing a highly purified factor VIII:C/vWF-complex.
Von Willebrand factor is a multimeric glycoprotein encoded by a gene on chromosome 12 and circulating in plasma at concentrations of from 5 to 10 &mgr;g/ml freely and as non-covalent complex with coagulation factor VIII, the protein which is encoded by a gene on chromosome 10 and which is impaired or is missing, respectively, in hemophilia A.
Among the two most important functions of vWF in haemostasis are:
1) the adhesion of the thrombocytes to an injured endothelium, vWF binding to the injured sub-endothelium and enabling a bridge between this surface and the platelets as well as an aggregation of the platelets among themselves. The first interaction between thrombocytes and sub-endothelium is effected via the glycoprotein Ib of the thrombocyte membrane and the collagen fibres of the injured endothelium. vWF binds to these two proteins and thus mediates the formation of a first layer of thrombocytes. Further cross-linking of the platelets among themselves is mediated by vWF by its binding to the glycoprotein complex IIb/IIIa. For these tasks of primary haemostasis, mainly the large multimers are responsible (Eller; Lab. Med. (1994); 18: 168-176).
2) by its binding site for FVIII, vWF also influences plasmatic coagulation. FVIII is present in plasma almost exclusively in a non-covalent complex with vWF, approximately every tenth vWF molecule carrying an FVIII molecule. Primarily dimers and small multimers are used as carriers. By this complexing with vWF, FVIII is protected against an increased proteolytic inactivation (e.g. by activated protein C).
Furthermore, FVIII is potentiated by complex formation in respect of its cofactor activity in the intrinsic coagulation (Eller; Lab. Med. (1994); 18: 168-176).
vWF is formed in the vascular endothelial cells, which are the main source of this plasma protein, by constitutive or stimulated liberation, but it is also synthetized in a smaller portion by the megakariocytes. (PNAS 92 (1995), 2428-2432).
The primary product of translation is comprised of 2813 amino acids. After splitting off the signal peptide (22 amino acids), dimerisation takes place. Further processing is effected in the Golgi apparatus, the dimers polymerizing under splitting off of the propeptide (741 amino acids). The propeptide plays an important role in the further linking of the dimers, where it catalyses the formation of disulfide bridges at the amino-terminal end. Thus, differently sized oligomers ranging in size from a dimer of 500,000 daltons to large multimers of up to 20 million daltons are developed. In addition to the proteolytic procedures, vWF is subject to other post-translational modifications, including glycosylation and sulfatizing. (Mancuso et al.; Hämostaseologie (1989); 9: 122-129).
Thus, due to the complexity of the biosynthesis, there is a large number of the most varying vWF molecules having the most varying tasks and properties.
As a consequence, von Willebrand factor may exhibit quite different binding activities to its natural binding partners. In particular it has been shown that the bindings of various vWF molecules to glycoprotein Ib, to collagen, to heparin, to glycoprotein IIb/IIIa-complex, to factor VIII and to the sub-endothelium may differ in strength.
This means, however, that each vWF preparation is composed of a mixture of these different vWF proteins or vWF aggregates, respectively, and thus will be heterogenous in terms of its properties, such as its essential binding strength to factor VIII.
The occurrence of various forms of vWF is also the cause of the complex and different phenotypes in the pathophysiology of von Willebrand Disease which, in certain cases, is due to an underproduction and in other cases to an overproduction of von Willebrand factor. Thus, e.g., an overproduction of vWF leads to an increased thrombosis tendency, whereas an undersupply of vWF results in an increased bleeding tendency or in increased bleeding times; this, however, is not always so, for it is decisive in which form von Willbrand factor is over- or underproduced.
To differentiate and characterize the properties of vWF and of vWF syndrome, a number of analytical methods are used.
Thus, the ristocetin cofactor activity determination is essential for diagnostical purposes. In doing so, the thrombocyte aggregation in the presence of the antibiotic ristocetin is assayed which is reduced or not present at all in patients afflicted with vWF syndrome. (Macfarlane et al.; Thrombosis et Diathesis Haemorrhagica 1775; 34: 306-308).
Moreover, the collagen binding activity of vWF can be used for differentiating the vWF syndrome (Thomas et al.; Hämastaseologie (1994); 14: 133-139).
The binding dissociation constant between vWF and FVIII can be determined according to the method of Vlot et al. (Blood 83 (11) (1995); 3150-3157).
The molecular structure of vWF is determined by analysis of the multimer structure by means of SDS electrophoresis in 1.2% agarose gels (Ruggeri et al.; Blood (1981); 57: 1140-1143).
To determine the total amount of vWF antigen, various commercially available ELISA test kits are used.
The preparation of an optimal FVIII/vWF complex should aim at providing a stable product which, above all, is free from undesired accompanying proteins, since any unnecessary protein load harbors the risk of undesired side effects.
Thus, any concentration of vWF which is not necessary for stabilizing FVIII is a load on the hemophiliac.
In the course of methods employed for producing von Willebrand preparations, in particular from plasma pools, hitherto it has not been possible to eliminate the risk of a heterogenous composition due to the various forms of von Willebrand factor present; in the prior art it has not been possible so far to obtain a vWF preparation having uniform properties, e.g. with regard to its binding activity to a certain ligand.
It had been known to purify factor VIII complex by various anti-vWF-monoclonal antibodies both from plasma and also from cryoprecipitate (Thromb. Haemostas. 57 (1987), 102-105). In doing so, also the stability of the FVIII/vWF complex in various buffers at pH 6.5 was tested, among them buffers containing glycols, amino acids, chaotropic substances, amines, other salts or organic solvents and/or detergents. The addition of lysin to buffer solutions containing chaotropic substances at high concentrations, such as, e.g., 3M urea, resulted in a protection of factor VIII:C/vWF:Ag relative to denaturing effects. The activities of factor VIII:C and of vWF R.cof. after incubation with 20% v/v ethylene glycol+1 M KJ amounted, e.g., to 72% and 48%, respectively, after treatment with 3M urea to 88% and 77%, respectively.
The specific activity of factor VIII:C in an end product, eluted with 1 M KJ+1 M lysin+20 mM imidazole+5 mM CaCl
2
, pH 6.5, was 45 I.U./mg total protein, that of vWF was 60 I.U./mg.
It has also been reported to chromatographically purify factor VIII/vWF-complex from cryoprecipitate after adsorption on Al(OH)
3
and carrying out a virus inactivation measure by using anti-vWF monoclonal antibodies (Biotechnol. Blood Prot. 227 (1993), 109-114). Elution of the adsorbed complex was effected at pH 6.5 by the addition of the chaotropic agent KJ (1 M). The ratio factor VIII:C/vWF:AG was 0.8, the specific activity of factor VIII:C was 38 I.U./mg.
Finally, it has been known from EP-0 295 645-A2 to purify factor VIII complex from heterogenous biological liquids by means of affinity chromatography, using specific peptides directed against vWF. In doing so, the complex was eluted using pH gradients or buffers of high ionic strength (cf. Example 5 of EP-0 295 645-A2).
In EP 0 416 983 A1, a preparation with factor VIII/vWF-complex is described which was prepared by anion exchange chromatography.
Eibl Johann
Linnau Yendra
Schönhofer Wolfgang
Weber Alfred
Baxter Aktiengesellschaft
Carlson Karen Cochrane
Heller Ehrman White & McAuliffe
Schnizer Holly
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