Process for the production of a reversibly inactive acidified plasmin composition

Abstract

Disclosed is both a process for producing a reversibly inactive acidified plasmin by activating plasminogen and a process for producing a purified plasminogen. The produced plasmin is isolated and stored with a low pH-buffering capacity agent to provide a substantially stable formulation. The purified plasminogen is typically purified from a fraction obtained in the separation of immunoglobulin from Fraction II+III chromatographic process and eluded at a low pH. The reversibly inactive acidified plasmin may be used in the administration of a thrombolytic therapy.

What is claimed is:  
     
         1 . A method for purifying plasmin comprising: 
 cleaving a plasminogen in the presence of a plasminogen activator to yield an active plasmin;    substantially removing the plasminogen activator from the active plasmin to form a plasmin solution; and    buffering the plasmin solution with a low pH-buffering capacity agent to form a reversibly inactive acidified plasmin.    
     
     
         2 . The method of  claim 1 , wherein the step of substantially removing the plasminogen activator includes the steps of: 
 binding the active plasmin to an active plasmin-specific absorbent material to form a bound plasmin; and    eluting the bound plasmin with a low pH solution to form the plasmin solution.    
     
     
         3 . The method of  claim 2 , wherein the active plasmin-specific absorbent material comprises benzamidine.  
     
     
         4 . The method of  claim 2 , wherein the plasminogen activator is further removed by hydrophobic interaction.  
     
     
         5 . The method of  claim 1 , further including cleaving the plasminogen in the presence of stabilizers comprising omega-amino acids and glycerol.  
     
     
         6 . The method of  claim 1 , wherein the plasminogen is cleaved using a catalytic concentration of a plasminogen activator that is selected from the group consisting of immobilized, soluble and combinations thereof of plasminogen activators.  
     
     
         7 . The method of  claim 6 , wherein the plasminogen activator is selected from the group consisting of streptokinase, urokinase, tPA and combinations thereof.  
     
     
         8 . The method of  claim 7 , wherein the plasminogen activator is soluble streptokinase.  
     
     
         9 . The method of  claim 6 , wherein the plasminogen activator is immobilized on a solid support medium comprising SEPHAROSE.  
     
     
         10 . The method of  claim 1 , wherein the low pH-buffering capacity agent comprises a buffer comprising an amino acid, a derivative of at least one amino acid, an oligopeptide which includes at least one amino acid, or a combination thereof.  
     
     
         11 . The method of  claim 1 , wherein the low pH-buffering capacity agent comprises a buffer selected from acetic acid, citric acid, hydrochloric acid, carboxylic acid, lactic acid, malic acid, tartaric acid, benzoic acid, serine, threonine, methionine, glutamine, alanine, glycine, isoleucine, valine, alanine, aspartic acid, derivatives thereof, or combinations thereof.  
     
     
         12 . The method of  claim 1 , wherein the buffer is present in the reversibly inactive acidified plasmin at a concentration at which the pH of the acidified plasmin is raised to neutral pH by adding no more than about 5 times the volume of serum to the acidified plasmin.  
     
     
         13 . The method of  claim 1 , wherein the reversibly inactive acidified plasmin solution has a pH between about 2.5 to about 4.  
     
     
         14 . The method of  claim 1 , further including stabilizing the reversibly inactive acidified plasmin by adding a stabilizing agent selected from a polyhydric alcohol, pharmaceutically acceptable carbohydrates, salts, glucosamine, thiamine, niacinamide, or combinations thereof.  
     
     
         15 . The method of  claim 14 , wherein the salts are selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride and combinations thereof.  
     
     
         16 . The method of  claim 1 , further including stabilizing the reversibly inactive acidified plasmin by adding a sugar or sugar alcohol selected from glucose, maltose, mannitol, sorbitol, sucrose, lactose, trehalose, or combinations thereof.  
     
     
         17 . A method for purifying plasmin comprising: 
 cleaving a plasminogen to yield an active plasmin;    binding the active plasmin to an active plasmin-specific absorbent material to form a bound plasmin; and    eluting the bound plasmin with a substantially neutral amino acid to form a final plasmin solution which is substantially free of degraded plasmin.    
     
     
         18 . The method of  claim 17 , wherein the plasminogen is cleaved using a catalytic concentration of a plasminogen activator.  
     
     
         19 . The method of  claim 17 , wherein the activated plasmin solution is stabilized by the addition of omega-amino acids and sodium chloride.  
     
     
         20 . The method of  claim 17 , wherein the substantially neutral amino acid comprises an omega-amino acid.  
     
     
         21 . The method of  claim 20 , wherein the omega-amino acid selected from the group consisting of lysine, epsilon amino caproic acid, tranexamic acid, poly lysine, arginine, analogues thereof and combinations thereof.  
     
     
         22 . The method of  claim 17 , further including filtering out the substantially neutral amino acid from the final plasmin solution.  
     
     
         23 . The method of  claim 17 , further including adding a low pH-buffering capacity agent to the final plasmin solution to form a reversibly inactive acidified plasmin.  
     
     
         24 . The method of  claim 23 , further including adjusting the pH of the reversibly inactive acidified plasmin to a pH between about 2.5 to about 4.  
     
     
         25 . The method of  claim 17 , further including adding a stabilizer to the final plasmin solution.  
     
     
         26 . The method of  claim 25 , wherein the stabilizer is selected from the group consisting of amino acids, salts or combinations thereof.  
     
     
         27 . A process for the purification of plasminogen from a plasma source comprising: 
 adding the plasminogen containing solution to a plasminogen-specific absorbent material;    eluting the plasminogen from the plasminogen-specific absorbent material at a pH of between about 1 to about 4; and    collecting the plasminogen containing eluate.    
     
     
         28 . The process of  claim 27 , wherein the plasma source is derived from Fraction II+III of Cohn plasma fractionation process.  
     
     
         29 . The process of  claim 27 , further comprising the steps of: 
 extracting plasminogen from a plasma paste fraction with a buffer solution at a pH in a range from about 3.5 to 10.5 and collecting the plasminogen containing solution;    adding polyethylene glycol or ammonium sulfate to the plasminogen containing buffer solution to precipitate impurities;    separating the precipitated impurities from the effluent containing plasminogen; and    adding the effluent containing plasminogen to a lysine affinity resin.    
     
     
         30 . The process of  claim 29 , wherein the plasminogen extraction solution is at a pH in a range from about 7.0 to about 10.5.  
     
     
         31 . The process of  claim 29  wherein about 1 to about 10% w/w polyethylene glycol or 80 to 120 g/L ammonium sulfate is added.  
     
     
         32 . The process of  claim 29 , further including adding a plasminogen solubility enhancer.  
     
     
         33 . The process of  claim 32 , wherein the plasminogen solubility enhancer is selected from the group of omega-amino acids consisting of lysine, epsilon amino caproic acid, tranexamic acid, poly lysine, arginine, combinations thereof and analogues thereof.  
     
     
         34 . The process of  claim 32 , further including removing the plasminogen solubility enhancer  
     
     
         35 . The process of  claim 27 , wherein the eluted plasminogen is treated at a pH between about 3 and about 4.  
     
     
         36 . The process of stabilizing plasminogen during pH adjustment from about 3 to neutral by adding omega-amino acids prior to pH adjustment.  
     
     
         37 . The process of  claim 27 , further including removing pathogens.  
     
     
         38 . The process of  claim 37 , wherein the removing pathogens includes inactivating viral pathogens and removing TSE pathogens.  
     
     
         39 . The process of  claim 38 , wherein viruses are inactivated or removed by the steps selected from the group consisting of heat treatment, caprylate addition, solvent detergent addition, nanofiltration and combinations thereof.  
     
     
         40 . The process of  claim 38 , wherein TSE are removed by the steps selected from the group consisting of PEG precipitation, depth filtration and nanofiltration.  
     
     
         41 . The process of  claim 27 , wherein the plasminogen-specific absorbent material comprises a lysine affinity resin.