Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1996-04-29
1998-11-03
Achutamurthy, Ponnathapura
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
530413, 530344, 530830, 530856, 435 13, 435 681, A61K 3516, A61K 3816, C12Q 156, C07K 1714
Patent
active
058310258
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a 371 national stage application of PCT/JP94/01807, filed Oct. 27, 1994.
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a human Activated Protein C preparation having a high specific activity which is prepared by activation of human Protein C with thrombin or an equivalent protease, said Protein C being derived from plasma or prepared by using the genetic recombination technique. The present invention further relates to a method for activating human Protein C and a process for purifying human Activated Protein C a high purity.
TECHNICAL BACKGROUND
Protein C (hereinafter also abbreviated as "PC") is a kind of a vitamin K dependent protein synthesized in the liver and is an enzyme precursor having a molecular weight 62,000 consisting of two chains, i.e. L chain (molecular weight 21,000 ) and H chain (molecular weight 41,000 ). Protein C is partially degraded in vivo by a thrombin-thrombomodulin complex, thrombin bound to thrombomodulin occurring on the membrane surface of the vascular endothelial cells, and thereby a peptide comprising 12 amino acids is released from the amino terminal of H chain to form Activated Protein C (hereinafter also abbreviated as "APC"). APC is a kind of a serine protease which exhibits a strong anti-coagulant activity by specific degradation and inactivation of the blood coagulation Factor V and Factor VIII (primarily activated form Va, VIIIa) and promotes the release of a plasminogen activator from the vascular wall to accelerate the fibrinolytic system. Accordingly, APC is expected to be used as a therapeutic agent.
APC itself is well known in the art and includes those obtained by in vitro activation of protein C with thrombin or thrombin-thrombomodulin complex, said protein C being derived from plasma or prepared by using the genetic recombination technique (Blood, 63, p.115-121 (1984 ); J. Clin. Invest., 64, p.761-769 (1979); J. Clin. Invest., 79, p.918-925 (1987)); or those directly expressed as APC by the genetic recombination technique (Japanese Patent First Publication (Kokai) No. 61-205487, Japanese Patent First Publication (Kokai) No. 1-2338 and Japanese Patent First Publication (Kokai) No. 1-85084), and the like.
However, for preparation of APC, especially in the case where protein C is fractionated from plasma and then activated to produce the desired protein, there are various problems need to be overcome in order to efficiently remove contaminating proteins having physico-chemical properties quite similar to APC and to highly purify APC so that APC having a desired high specific activity is obtained. For example, there still remain a number of problems to be solved with regard to efficient activation of Protein C into APC, subsequent removal of an activating agent, and purification of APC.
Known methods for activation of Protein C include, for example, activation with trypsin, RVV-X, thrombin, thrombin-thrombomodulin, activation using gel wherein RVV-X is immobilized to cepharose, activation using gel wherein thrombin-thrombomodulin complex is immobilized and the like (J. Biol. Chem., 251, 3052-3056 (1976); Biochemistry, 15, 4893-4900 (1976); Biochemistry, 16, 5824-5831 (1977); J. Clin. Invest., 64, 761-769 (1977); Biochem. Biophys. Res. Commun., 94, 340-347 (1980); J. Clin. Invest., 77, 416-425 (1986)).
However, the methods mentioned hereinabove are not satisfactory in view of production of APC on industrial scale. For activation of a large amount of Protein C, it is preferable to activate Protein C at a high concentration with a small amount of an activating agent. The above methods also do not satisfy this requirement.
As to purification of Activated Protein C after activation of Protein C, a method is known wherein APC is developed in an eluent fraction by SP-SEPHADEX chromatography and thereby thrombin added during activation of Protein C is adsorbed and removed (Biochemistry, 16, 5824-5831 (1977); J. Clin. Invest., 64, 761-769 (1979); J. Biol. Chem., 251, 3052-3056 (1976); Biochemistry,
REFERENCES:
Miekka et al Thrombosis and haemostasis, vol. 69, No. 6, p. 719, Jun. 30, 93.
Orthner et al, Vox Sang, vol. 69, No. 4, pp. 309-318, 1995.
Nakahira Shinji
Nouchi Toshinobu
Ogata Yoichi
Achutamurthy Ponnathapura
Juridical Foundation The Chemo-Sero-Therapeutic Research Institu
Ponnaluri Padmashri
Teijin Limited
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