Method for separating unmodified hemoglobin from cross-linked hemoglobin

Abstract

A method for separating unmodified hemoglobin from cross-linked hemoglobin in a hemoglobin solution. The method involves contacting the hemoglobin solution with a least one dissociating agent to form a dissociation solution wherein unmodified tetrameric hemoglobin is dissociated to form hemoglobin dimers. The hemoglobin dimers are then separated from the dissociation solution, while retaining the cross-linked hemoglobin in the dissociation solution.

What is claimed is:  
     
         1 . A method for separating unmodified hemoglobin from cross-linked hemoglobin in a hemoglobin solution, comprising the steps of: 
 a) contacting the hemoglobin solution with a least one dissociating agent to form a dissociation solution wherein unmodified tetrameric hemoglobin is dissociated to form hemoglobin dimers; and    b) separating the hemoglobin dimers from the dissociation solution, while retaining the cross-linked hemoglobin in said dissociation solution.    
     
     
         2 . A method of  claim 1  wherein at least a portion of the hemoglobin in the hemoglobin solution is polymerized.  
     
     
         3 . A method of  claim 1  wherein the dissociation agent is a water soluble inorganic salt.  
     
     
         4 . A method of  claim 3  wherein the inorganic salt is a sodium salt.  
     
     
         5 . A method of  claim 3  wherein the inorganic salt includes a multivalent cation.  
     
     
         6 . A method of  claim 5  wherein the multivalent cation is selected from the group consisting of Ca +2 , Mg +2 , Zn +2  and combinations thereof.  
     
     
         7 . A method of  claim 1  wherein the dissociation agent is a water soluble organic salt.  
     
     
         8 . A method of  claim 7  wherein the organic salt is a salt of an amine.  
     
     
         9 . A method of  claim 1  wherein the dissociation agent is a water soluble organic amine.  
     
     
         10 . A method of  claim 9  wherein the amine is guanidine.  
     
     
         11 . A method of  claim 1  further comprising the step of mixing the dissociation agent and the hemoglobin solution under low shear conditions.  
     
     
         12 . A method of  claim 1  further comprising the step of contacting the hemoglobin solution with a buffer.  
     
     
         13 . A method of  claim 12  wherein the Hb solution is buffered with 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol.  
     
     
         14 . A method of  claim 1  further comprising the step of contacting the Hb solution with a stabilizing agent.  
     
     
         15 . A method of  claim 14  wherein the stabilizing agent is ethylenediaminetetraacetic acid.  
     
     
         16 . A method of  claim 1  wherein the unmodified hemoglobin is separated from the dissociation solution by filtering the dissociation solution.  
     
     
         17 . A method of  claim 1  further comprising the step of washing the cross-linked hemoglobin with a physiologically acceptable buffer, after separating the unmodified hemoglobin from the dissociation solution, to produce a physiologically acceptable hemoglobin blood-substitute.  
     
     
         18 . A method for separating unmodified hemoglobin from cross-linked in a hemoglobin solution to produce a physiologically acceptable blood-substitute, comprising the steps of: 
 a) contacting the hemoglobin solution with a dissociating agent to form a dissociation solution, wherein unmodified tetrameric hemoglobin dissociated to form hemoglobin dimers;    b) filtering the hemoglobin dimers from the dissociation solution while retaining cross-linked hemoglobin in the retentate; and    c) washing the cross-linked hemoglobin with a physiologic buffer to produce a physiologically acceptable blood-substitute.    
     
     
         19 . A method of  claim 19  wherein the buffer contains sodium lactate and N-acetyl-L-cysteine.  
     
     
         20 . A method for separating unmodified hemoglobin from cross-linked hemoglobin in a hemoglobin solution, comprising the steps of: 
 a) mixing the hemoglobin solution with a dissociation agent, a buffer and a stabilizing agent to form a dissociation solution, wherein mixing is with low shear, and wherein the unmodified hemoglobin dissociates to form hemoglobin dimers within the dissociation solution;    b) filtering the dissociation solution to separate the hemoglobin dimers from the cross-linked hemoglobin; and    e) washing the cross-linked hemoglobin solution with a deoxygenated solution containing sodium lactate, N-acetyl-L-cysteine and physiologic electrolytes.