Compositions, methods, and kits for preparing plasminogen; and plasmin prepared therefrom

Abstract

Compositions and methods for preparing plasminogen, in particular recombinant plasminogen, and compositions and methods of utilizing same for preparing plasmin are provided.

We claim: 
     
         1 . A method for preparing recombinant plasmin, said method comprising:
 (a) contacting a composition comprising a recombinant plasminogen having a single kringle domain, a peptide linker, an activation site, and a serine protease domain, wherein said single kringle domain comprises at least 90% identity to kringle 1 or kringle 4 of SEQ ID NO:2; said peptide linker sequence between said single kringle domain and said serine protease domain consists of residues 91-94 of SEQ ID NO:1 with a cation-exchange medium under a cation-exchange condition that is sufficient for said cation-exchange medium to bind said recombinant plasminogen;   wherein said cation-exchange medium comprises a sulfopropyl ligand coupled to an agarose support; and   (b) converting said recombinant plasminogen to said recombinant plasmin.   
     
     
         2 . The method of  claim 1 , wherein said converting step comprises contacting said recombinant plasminogen with a plasminogen activator in an activation solution under an activation condition sufficient to convert said recombinant plasminogen to a recombinant plasmin. 
     
     
         3 . The method of  claim 2  further comprising: contacting said activation solution with an anion-exchange medium under an anion-exchange condition to obtain an anion-exchange medium flow-through comprising said recombinant plasmin, wherein said anion-exchange condition is such that said anion-exchange medium preferentially binds said plasminogen activator relative to said recombinant plasmin. 
     
     
         4 . The method of  claim 3 , wherein said anion-exchange medium flow through comprises an amount of said plasminogen activator or fragment thereof that is less than an amount of that which was present in said activation solution prior to contacting with said anion-exchange medium, and wherein said anion-exchange medium flow through comprises an amount of said recombinant plasmin that has a purity of at least about 50%. 
     
     
         5 . The method of  claim 3 , wherein said anion-exchange medium comprises a quaternary ammonium or a quaternary aminoethyl. 
     
     
         6 . The method of  claim 3  further comprising: contacting said anion-exchange medium flow-through with a second affinity medium under a second affinity condition sufficient to bind said recombinant plasmin. 
     
     
         7 . The method of  claim 3  further comprising: contacting a second affinity medium eluate comprising said recombinant plasmin with a hydrophobic interaction chromatography medium under a hydrophobic interaction condition sufficient such that said hydrophobic interaction chromatography medium preferentially binds said plasminogen activator relative to said recombinant plasmin. 
     
     
         8 . A method for preparing a recombinant plasmin, said method comprising:
 contacting a composition comprising a recombinant plasmin having a single kringle domain, a peptide linker, an activation site, and a serine protease domain, wherein said single kringle domain comprises at least 90% identity to kringle 1 or kringle 4 of SEQ ID NO:2; said peptide linker sequence between said single kringle domain and said serine protease domain consists of residues 91-94 of SEQ ID NO:1 with an anion exchanger whereby, if present in said composition, a proteinaceous material having an isoelectric point below that of said recombinant plasmin is separated from said recombinant plasmin.   
     
     
         9 . The method of  claim 8 , wherein prior to contacting said composition comprising said recombinant plasmin with said anion exchanger, a pH of said composition is adjusted to be less than said isoelectric point of said plasmin but greater than said isoelectric point of said proteinaceous material to be separated from said recombinant plasmin. 
     
     
         10 . The method of  claim 8 , wherein said anion exchanger comprises a quaternary ammonium. 
     
     
         11 . The method of  claim 8 , wherein said proteinaceous material is a streptokinase or a fragment thereof. 
     
     
         12 . The method of  claim 8 , further comprising an additional chromatography purification step comprising an affinity chromatography, an ion exchange chromatography, or a hydrophobic interaction chromatography.