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AssigneesJuridical Foundation The Chemo-Sero-Therapeutic Research Institute, Teijin Limited
InventorsKazumasa Miyata, Yoshinori Akimoto, Yoichi Ogata, Tomohiro Nakagaki
Estimating the term…
A method for stabilizing protein C or activated protein C and a preparation obtained by said method are provided, said method and preparation being applicable during procedures such as isolation and purification, lyophilization, heating, etc. or when stored. To a salt buffer containing protein C or activated protein C and sodium ion are added at least one amino acids, and further either one or a combination of albumin and a non-ionic surfactant.
A method for stabilizing protein C or activated protein C which comprises adding a salt buffer, a sodium salt for a final concentration of 50 to 100 mM of sodium ion, at least one amino acid for a final concentration of 0.005M to 0.1M, and a compound selected from the group consisting of albumin for a final concentration of 0.5 to 10% w/v, a non-ionic surfactant for a final concentration of 0.0005 to 0.1% w/v, and a mixture thereof, to protein C or activated protein C to form a protein C or activated protein C-containing composition in which protein C or activated protein C is stable at 37.degree. C. for 24 hours.
An aqueous buffer solution containing protein C and/or activated protein C which contains 100 to 2500 U/ml of protein C and/or activated protein C, 50 to 200 mM of sodium ion, 5 to 100 mM of an amino acid, and further either one or a combination of 0.5 to 10% (w/v) of albumin and 0.0005 to 0.1% (w/v) of a non-ionic surfactant, wherein said protein C and/or activated protein C is stable in said aqueous buffer solution at 37.degree. C. for 24 hours.
A composition containing protein C and/or activated protein C which contains 1.times.10.sup.5 to 2.5.times.10.sup.6 U of protein C and/or activated protein C, 50 to 200 mg of sodium ion, 5 to 100 millimoles of an amino acid, and further either one or a combination of 5 to 100 g of albumin and 0.005 to 1 g of a non-ionic surfactant, wherein said protein C and/or activated protein C is stable in said composition at 37.degree. C. for 24 hours.
A method for stabilizing protein C or activated protein C which comprises adding a salt buffer, sodium ion, at least one amino acid, and a compound selected from the group consisting of a non-ionic surfactant and a mixture of a non-ionic surfactant and albumin to protein C or activated protein C to stabilize protein C or activated protein C.
Dependencies are inferred from the claim text.
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Serine endopeptidases: protein C
Serine endopeptidases: protein C
Drugs for disorders of the blood or the extracellular fluid: antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
Enzymes; Proenzymes; Compositions thereof ; Processes for preparing, activating, inhibiting, separating or purifying enzymes: stabilising an…
Term ended 29 Apr 2016 (estimated)
USPTO file history isn’t available for this patent, so only indexed dates are shown.
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.
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.
The present invention is illustrated in more detail by means of the following Examples but should not be construed to be limited thereto.
Effect of Various Counterions on Stability of APC:
To a solution containing human activated protein C with the activity of 500 U/ml was added 2.5% human serum albumin (hereinafter also abbreviated as "HSA"). The solution was then dialyzed against a solution of Na citrate, Na phosphate and Na sulfate (each 20 mM), containing 0.7% NaCl and 0.067 M glycine. After dialysis, each solution was left to stand at 37.degree. C. for 24 hours and the activity was measured. The results are shown in Table 1. All the counterions tested, Na citrate, Na phosphate and Na sulfate, showed a similar satisfactory stability.