Removal of plasmin(ogen) from protein solutions

Abstract

A method for specifically removing or isolating plasmin(ogen) or plasmin in presence of fibrinogen from a mixture containing plasmin(ogen) or plasmin by contacting the mixture with a rigid amino acid wherein the amino group of the amino acid and the carboxylic group of the amino acid are about 6-8 Angstroms, preferably about 7 Angstroms apart and the rigid amino acid is covalently bound to the support via the amino group of the amino acid.

1 . A method for specifically removing or isolating plasmin(ogen) or plasmin in presence of fibrinogen from a mixture containing plasmin(ogen) or plasmin by contacting the mixture with a rigid amino acid wherein the amino group of the amino acid and the carboxylic group of the amino acid are about 6-8 Angstroms, preferably about 7 Angstroms apart and the rigid amino acid is covalently bound to the support via the amino group of the amino acid.  
     
     
         2 . The method according to  claim 1  wherein the mixture is selected from the group consisting of body fluids such as blood; 
 blood fractions, cryoprecipitate, cell cultures, animal tissue extracts, such as bovine lungs, bovine intestines or animal bone extracts gelatin, bovine serum albumin as well as animal derived water immiscible fats, such as lanoline (PC-phosphatidyl choline).  
 
     
     
         3 . The method according to  claim 1  wherein the support is a chromatographic material.  
     
     
         4 . The method according to  claim 1  wherein to the support is bonded a rigid amino acid wherein the amino group of the amino acid and the carboxylic group of the amino acid are about 6-8 Angstroms, preferably about 7 Angstroms apart.  
     
     
         5 . The method according to  claim 3  wherein the chromatographic material is a hydrophilic material such as agarose, cellulose, controlled pore glass, silica gels, dextranes or an organic artificial polymer such as based on polyacrylamides polystyrens.  
     
     
         6 . The method according to  claim 3  wherein the chromatographic material is agarose or sepharose.  
     
     
         7 . The method according to  claim 1  wherein the chromatographic material is a particulate material or a monolithic block-material.  
     
     
         8 . The method according to  claim 1  wherein the rigid amino acid is bound to the support via a linker between the support and tranexamic acid.  
     
     
         9 . The method according to  claim 8  wherein linker is a bifunctional linker.  
     
     
         10 . The method according to  claim 9  wherein the bifunctional linker is selected from the group consisting of N-hydroxy succinimide, DAPA, CNBr, epoxy, diaminodipropylamine (DADPA), 1,6 diaminohexane, succinic acid, 1,3 diamino-2-propanol, ethylendiamine (EDA), TNB, pyridyldisulfide, iodoacetamide, maleimide activated support or combinations thereof.  
     
     
         11 . The method according to  claim 1  wherein the support is modified by a moiety which reacts with primary or secondary amino groups.  
     
     
         12 . The method according to  claim 1  wherein after contacting the mixture with the support having bound tranexamic acid the mixture is incubated with the support for a sufficient time period to bind plasminogen or plasmin and eluted with a neutral aqueous solution containing sodium salts, calcium salts, buffer salts.  
     
     
         13 . The method according to  claim 1  wherein subsequently after contacting the mixture the plasmin or plasmin(ogen) is eluted with an aqueous solution containing a sufficient amount of a ligand competing with plasmin(ogen) binding sites of the support bound rigid amino acid.  
     
     
         14 . The method of  claim 13 , wherein the ligand is lysine.  
     
     
         15 . A mixture derived from natural sources being substantially free of plasmin(ogen).  
     
     
         16 . The mixture of  claim 15  wherein the mixture is a blood derivative, blood or blood fraction, cryoprecipitate.  
     
     
         17 . The mixture of  claim 16  wherein the blood derivative is a plasma derived blood clotting factor or mixture of blood clotting factors, albumin, immunoglobulin, fibrinogen, fibronectin, α 1 -antitrypsin, anti-thrombin III, von Willebrand factor.  
     
     
         18 . A plasmin(ogen) and/or plasmin containing fraction obtainable by a method according to  claim 1 .  
     
     
         19 . A support having covalently bound a rigid amino acid wherein the amino group of the amino acid and the carboxylic group of the amino acid are apart about 6-8 Angstroms, preferably about 7 Angstroms.  
     
     
         20 . The support of  claim 19  wherein the amino acid is selected from the group consisting of tranexamic acid and 4-aminomethylbicyclo-[2.2.2.]-octane-1-carboxylic acid.  
     
     
         21 . The support of  claim 19  or 20 wherein the support is a chromatographic material.  
     
     
         22 . The support according to  claim 19  wherein the chromatographic material is a hydrophilic material such as agarose, cellulose, controlled pore glass, silica gels, dextranes or an organic artificial polymer such as based on polyacrylamides polystyrenes.  
     
     
         23 . The support according to  claim 19  wherein the chromatographic material is agarose or sepharose.  
     
     
         24 . The support according to  claim 19  wherein the chromatographic material is a particulate material or a monolithic block-material.  
     
     
         25 . The support according to  claim 19  wherein the tranexamic acid is bound to the support via a linker between the support and tranexamic acid.  
     
     
         26 . The support according to  claim 25  wherein the linker is a bifunctional linker.  
     
     
         27 . The support according to  claim 26  wherein the bifunctional linker is selected from the group consisting of N-hydroxy succinimide, DAPA, CNBr, epoxy, diaminodipropylamine (DADPA), 1,6 diaminohexane, succinic acid, 1,3 diamino-2-propanol, ethylendiamine (EDA), TNB, pyridyldisulfide, iodoacetamide, maleimide activated support or combinations thereof.  
     
     
         28 . The support according to  claim 19  wherein the support is modified by a moiety which reacts with primary or secondary amino groups.  
     
     
         29 . A mixture derived from natural sources being substantially free of plasmin(ogen) and plasmin.  
     
     
         30 . The mixture of  claim 29  wherein the natural source is blood, a blood derivative or blood fraction, cryoprecipitate, cell cultures, animal tissue extracts, such as bovine lungs, bovine intestines or animal bone extracts gelatin, bovine serum albumin as well as animal derived water immiscible fats, such as lanoline (PC-phosphatidyl choline).  
     
     
         31 . The mixture of  claim 30  wherein the blood derivative is in particular a plasma derived blood clotting factor or mixture of blood clotting factors, such as FVIII, FIX, fibrinogen, fibronectin, α 1 -antitrypsin, anti-thrombin III, von Willebrand factor, albumin, immunoglobulin.