Highly-purified soluble thrombomodulin and method for producing same

Abstract

Highly-purified soluble thrombomodulin which has a content of host cell-originated proteins being in a ratio of less than 10 ng of the proteins per 10,000 U of the soluble thrombomodulin, wherein the soluble thrombomodulin is produced by a transformant cell obtained by transfecting a host cell with a DNA containing a nucleotide sequence encoding the soluble thrombomodulin.

What is claimed is: 
     
         1 . Highly-purified soluble thrombomodulin which has a content of host cell-originated proteins being in a ratio of less than 10 ng of the proteins per 10,000 U of the soluble thrombomodulin, wherein the soluble thrombomodulin is produced by a transformant cell obtained by transfecting a host cell with a DNA containing a nucleotide sequence encoding the soluble thrombomodulin. 
     
     
         2 . The highly-purified soluble thrombomodulin according to  claim 1 , which is manufactured on an industrial scale. 
     
     
         3 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , which is used as a material for a medicament. 
     
     
         4 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , wherein purity of the highly-purified soluble thrombomodulin is 99% or higher based on the total proteins. 
     
     
         5 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , wherein the soluble thrombomodulin is produced by serum-free culture of the transformant cell. 
     
     
         6 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , wherein the host cell is a Chinese hamster ovary cell. 
     
     
         7 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , wherein the soluble thrombomodulin has the following properties (1) to (5):
 (1) an action of selectively binding to thrombin, 
 (2) an action of promoting activation of Protein C by thrombin, 
 (3) an action of extending thrombin clotting time, 
 (4) an action of suppressing platelet aggregation caused by thrombin, and 
 (5) anti-inflammatory action. 
 
     
     
         8 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , wherein molecular weight of the soluble thrombomodulin is in the range of 50,000 to 80,000. 
     
     
         9 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , wherein the highly-purified soluble thrombomodulin is produced by a method comprising the following steps:
 (a) the step of obtaining a transformant cell by transfecting a host cell with a DNA encoding soluble thrombomodulin; 
 (b) the step of obtaining a solution containing soluble thrombomodulin by culturing the transformant cell, and 
 (c) the step of bringing the solution containing soluble thrombomodulin into contact with nylon and/or polyethersulfone to obtain highly-purified soluble thrombomodulin having a content of host cell-originated proteins being in a ratio of less than 10 ng of the proteins per 10,000 U of soluble thrombomodulin. 
 
     
     
         10 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , wherein the soluble thrombomodulin is a peptide containing:
 (i) the amino acid sequence of the positions 367 to 480 in the amino acid sequence of SEQ ID NO: 9 or 11, and the amino acid sequence of (ii-1) or (ii-2) mentioned below, and the peptide is soluble thrombomodulin having the following properties (1) to (5): 
 (ii-1) the amino acid sequence of the positions 19 to 244 in the amino acid sequence of SEQ ID NO: 9 or 11, or 
 (ii-2) the amino acid sequence of (ii-1) mentioned above, further including substitution, deletion or addition of one or more amino acid residues, 
 (1) an action of selectively binding to thrombin, 
 (2) an action of promoting activation of Protein C by thrombin, 
 (3) an action of extending thrombin clotting time, 
 (4) an action of suppressing platelet aggregation caused by thrombin, and 
 (5) anti-inflammatory action. 
 
     
     
         11 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , wherein the soluble thrombomodulin is a peptide containing:
 (i-1) the amino acid sequence of the positions 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or 11, or 
 (i-2) the amino acid sequence of (i-1) mentioned above, further including substitution, deletion or addition of one or more amino acid residues, 
 and the peptide is soluble thrombomodulin having the properties (1) to (5) mentioned below: 
 (1) an action of selectively binding to thrombin, 
 (2) an action of promoting activation of Protein C by thrombin, 
 (3) an action of extending thrombin clotting time, 
 (4) an action of suppressing platelet aggregation caused by thrombin, and 
 (5) anti-inflammatory action. 
 
     
     
         12 . The highly-purified soluble thrombomodulin according to  claim 1  or  2 , wherein the DNA containing a nucleotide sequence encoding soluble thrombomodulin is a DNA encoding the amino acid sequence of SEQ ID NO: 9 or 11. 
     
     
         13 . A pharmaceutical composition comprising the highly-purified soluble thrombomodulin according to  claim 1  or  2  and a pharmaceutically acceptable carrier.