Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1996-04-29
1999-10-05
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435188, C12N 948, C12N 996
Patent
active
059622997
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for stabilizing protein C or activated protein C which is derived from plasma or is prepared by using the genetic recombination technique. More particularly, the present invention relates to a method for stabilizing protein C or activated protein C when it is stored or subjected to procedures such as isolation and purification, lyophilization, treatment by heating, etc. and to a preparation stabilized by said method.
TECHNICAL BACKGROUND
Protein C (hereinafter also referred to as "PC") is a kind of a vitamin K dependent protein, i.e. a protein containing .gamma.-carboxyglutamic acid, and is activated to activated protein C (hereinafter also referred to as "APC") by thrombin in the presence of thrombomodulin present on the surface of the vascular endothelial cell. Activated protein C is a kind of a serine protease and exhibits a strong anti-coagulant activity by inactivating cofactors of the blood coagulation system such as Factor Va (FVa) and Factor VIIIa (FVIIIa). It is also known that activated protein C releases a plasminogen activator from the vascular wall to accelerate the fibrinolytic system. Furthermore, it is known that a defect in protein C causes a severe thrombosis. Thus, it has been established that activated protein C is the most important factor which regulates the blood coagulation and fibrinolytic system. Therefore, protein C or activated protein C is expected to be exploited as a novel anti-coagulating agent or profibrinolytic agent.
It has hitherto been known that the amount of protein C present in a plasma or expressed in a tissue culture system is extremely low. Accordingly, in order to use protein C or activated protein C as an anti-coagulant agent or a profibrinolytic agent widely and safely, isolation and purification of protein C or activated protein C is important. In addition, storage as a solution or in frozen form for long periods of time, lyophilization or procedures for inactivation of contaminating viruses such as heating are indispensable to the process when protein C or activated protein C is industrially prepared on a large scale. However, storage, freezing or freeze-drying, or heat treatment of a highly pure protein C or activated protein C extremely lowers the activity thereof. There has also been no report on the stability of highly purified protein C or activated protein C. Under such circumstances, it is impossible to provide a highly pure protein C or activated protein C efficiently and stably on an industrial scale.
Under such circumstances, the present inventors have earnestly studied the stability of protein C or activated protein C, and as a result, have found that the activity of protein C or activated protein C can be maintained even after storage for a significant period of time or after procedures such as isolation and purification, lyophilization, heating, etc. by adding, to protein C or activated protein C, a salt buffer such as phosphate or citrate buffer containing sodium ion supplemented with at least one amino acid, and further adding either one or a combination of albumin and a non-ionic surfactant, and thereby, the present invention has been completed.
DISCLOSURE OF THE INVENTION
The present invention relates to a method for stabilizing protein C or activated protein C which comprises adding, to a salt buffer containing protein C or activated protein C and sodium ion, at least one amino acid, and further either one or a combination of albumin and a non-ionic surfactant. More particularly, the present invention relates to a method for stabilizing protein C or activated protein C which comprises dissolving protein C or activated protein C in a salt buffer such as phosphate or citrate buffer containing sodium ion, and adding to said buffer at least one of amino acids which constitute a protein, e.g. glycine, alanine, lysine, arginine, aspartic acid, glutamic acid, etc. and a polypeptide having a protein-stabilizing activity such as albumin, globulin, etc. and, in a prefera
REFERENCES:
patent: 5549893 (1996-08-01), Eibl et al.
McIntosh, R. and Foster, P., The Effect of Solution Formulation on the Stlity and Surface Interactions of Factor VIII During Plasma Fractionation, Transfus, Sci., 11:55-66; 1990.
Akimoto Yoshinori
Miyata Kazumasa
Nakagaki Tomohiro
Ogata Yoichi
Juridical Foundation The Chemo-Sero-Therapeutic Research Institu
Lankford , Jr. Leon B.
Teijin Limited
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