Hemoglobin-polysaccharide conjugates

Abstract

Hemoglobin conjugates useful as a hemoglobin-based oxygen carriers are prepared by reacting hemoglobin with oxidatively ring-opened polysaccharides such as hydroxyethyl starch or dextran, and storing the resultant conjugate under conditions which allow it to transform to a lower molecular weight product, after conjugation. The conjugate is then reductively stabilized to form secondary amino bonds between the hemoglobin and the polysaccharide, and formulated as an HBOC.

What is claimed is:  
     
         1 . A polysaccharide-hemoglobin conjugate useful as an oxygen transporter, comprising hemoglobin covalently linked through secondary amine linkages between amino groups on the hemoglobin and residues of aldehyde groups on the polysaccharide produced by oxidative saccharide ring-opening thereof, the secondary amine linkages having been formed by reaction of said amino groups and said aldehyde groups in a first stage to form Schiff base linkages, and in a second, subsequent stage by reduction to effect stabilization, the conjugate having no detectable residual unbound hemoglobin and no detectable residual amounts of components of molecular weight higher than about 500 kDa.  
     
     
         2 . The conjugate of claim l wherein the polysaccharide is hydroxyethyl starch or dextran  
     
     
         3 . The conjugate of  claim 2  wherein hemoglobin is human hemoglobin.  
     
     
         4 . The conjugate of  claim 3  herein the hemoglobin is deoxyhemoglobin.  
     
     
         5 . The conjugate of  claim 3  wherein the hemoglobin is intramolecularly cross-linked.  
     
     
         6 . The conjugate of  claim 3  wherein the polysaccharide is hydroxyethyl starch of molecular weight from about 70 to about 1000 kDa.  
     
     
         7 . The conjugate of  claim 6  wherein the hydroxyethyl starch has a substitution ratio from about 0.5 to 0.7.  
     
     
         8 . The conjugate of  claim 6  having a P50 from 4-50 at 37° C.  
     
     
         9 . A process of preparing a hemoglobin based oxygen carrier which comprises reacting an oxidatively ring-opened polysaccharide carrying aldehyde groups, with hemoglobin, to form a conjugate thereof, maintaining the conjugate under controlled conditions of aqueous solution with predetermined pH to effect controlled molecular weight reduction and molecular weight re-distribution of the conjugate, stabilizing the conjugate by reduction of the Schiff base linkages, between the polysaccharide and the hemoglobin to stable, secondary amine linkages, and recovering a solution of the polysaccharide-hemoglobin conjugate so formed which has no detectable unbound hemoglobin residue and no detectable product residue of molecular weight greater than about 500-600 kDa.  
     
     
         10 . The process of  claim 9  wherein the polysaccharide is dextran or hydroxyethyl starch(HES).  
     
     
         11 . The process of  claim 10  wherein the conjugate is maintained in aqueous solution under pH conditions from about 7.2-10, at temperatures from about 15-30° C. to effect molecular weight reduction and molecular weight re-distribution.  
     
     
         12 . The process of  claim 11  wherein the molecular weight reduction and molecular weight redistribution is terminated at a predetermined extent by said stabilization of the conjugate by reduction of the aldehyde-amine bonds to secondary amine linkages.  
     
     
         13 . The process of  claim 12  including a single stage of reduction, to effect said stabilization and substantially simultaneously to reduce residual aldehyde groups.  
     
     
         14 . The process of  claim 12  including two stages of reduction, the first to effect said stabilization and the second to reduce the aldehyde groups.  
     
     
         15 . The process of  claim 13  wherein the reduction is effected with a boron-based reducing agent.  
     
     
         16 . The process of  claim 15  wherein the reducing agent is borane dimethylamine.