Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1995-05-28
1997-12-23
Feisee, Lila
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530384, 530416, 530417, C07K 322, C07K 328, A61K 3516
Patent
active
057009147
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 of PCT/DK94/00122 filed Mar. 24, 1994, published as WO94/22905 Oct. 13, 1994, which is incorporated herein by reference.
TECHNICAL FIELD
The present invention is related to a method for controlled activation and degradation of Factor VII.
BACKGROUND ART
Coagulation Factor VII (FVII) is a vitamin K dependent serine protease playing a key role in the extrinsic pathway of blood coagulation. It is synthesized in the liver and secreted into the blood where it circulates as a single-chain glycoprotein (zymogen) with a molecular weight of about 50,000. In its activated form, FVIIa, the protease catalyzes the activation of two other vitamin K dependent coagulation facors of the serine protease family, Factor IX (FIX) and Factor X (FX).
Activated FX (FXa) will then convert prothrombin into thrombin. Thrombin will then convert fibrinogen into fibrin, the major constituent of the clot.
Factor VII can be purified from plasma and activated into Factor VIIa by the methods described by Broze and Majerus, J.Biol.Chem. 255 (4) (1980) 1242-1247 and Hedner and Kisiel, J.Clin.lnvest. 71 (1983) 1836-1841.
Factor VIIa may also be produced by recombinant DNA-technology by culturing in an appropriate medium mammalian cells transfected with a DNA-sequence encoding Factor VII, isolating the protein produced and activating said protein to Factor VIIa (European patent application No. 86302855.1).
Factor VIIa may be used in treating patients who have developed inhibitors to Factor VIII (Hedner, U. and Kisiel, W, J.Clin.lnvest, 71 (1983) 1836-1841) and for the treatment of patients suffering from bleeding disorders such as platelet disorders including thrombocytopenia, von Willebrand's disease and others typically present in association with severe tissue damages (European patent application No. 86309197.1).
During purification of the plasma-derived bovine protein (Radcliffe & Nermerson, J.Biol.Chem. 250 (1975) 338-395) and the human recombinant protein (Thim et al., Biochemistry 27 (1988) 7785-7793), FVII was activated into the two-chain form by hydrolysis of the Arg.sub.152 -lle.sub.153 bond. The activation of FVII is greatly enhanced by adsorption on anion exchangers (diethylaminoethyl or trimethylaminoethyl modified polymeric gel matrices) (A. H. Pedersen et al., Biochemistry 28 (1989), 9331-9336). The mechanism by which the FVII activation is enhanced is not known. In addition to the activation, a fraction of the FVIIa molecules is cleaved primarily at positions 290 and/or 315 by autodegradation (E. M. Nicolaisen et al., FEBS Lett. 317 (1993) 245-249). Such degradation products are inactive molecules and their occurrence in the Factor VIIa preparation will lead to a lower specific activity of the final preparation. Furthermore, the amount and nature of the degradation products may vary from one production batch to another giving rise to preparations with a variable content of biologically active Factor VIIa. A content of degradation products in the final preparation may trigger the immune system of the patient. Readministration may then result in allergic reactions, which in severe cases may have a lethal course. Patients may also develop high titers of antibodies against Factor VIIa rendering subsequent treatment difficult or ineffective.
In order to prepare a purified FVIIa product with a low content of degradation products it is essential to control the activation and impede degradation during the purification process and subsequent processing. In contrast to FVIIa the single chain form, FVII, is resistant to or much less prone to cleavage in the heavy chain. It might therefore be an advantage to purify FVII in its single chain form.
It is therefore the purpose of the present invention to provide a purification process for FVII by which activation and degradation is avoided or kept at an acceptable low degree with the purpose of providing a homogeneous product of high purity which can then be activated into FVIIa in a further step in high yields to give a
REFERENCES:
Radcliffe 1975 J. Biol Chem vol. 250(2) 388-395.
Broze et al 1980 J. Biol Chem vol. 255(2) 1242-1247.
Thim et al 1988 Biochemistry vol. 27: 7785-7793.
Pedersen et al (1991 Thrombosis & Haemostasis vol. 65(5):528-534.
Pusey et al 1985 Thromosis Res. vol. 39: 571-585.
J.o slashed.rgensen Tony
Pedersen Anders Hjelholt
Feisee Lila
Lambiris, Esq. Elias J.
Novo Nordisk A S
Reeves Julie E.
Zelson Esq. Steve T.
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