Method and Apparatus for Preparing an Acellular Red Blood Cell Substitute

Abstract

A process is disclosed for the preparation of an essentially tetramer-free, substantially stroma-free, polymerized, pyridoxylated hemoglobin. Also disclosed is an essentially tetramer-free, substantially stroma-free, polymerized, pyridoxylated hemoglobin product capable of being infused into human patients in an amount of up to about 5 liters.

1 . An aqueous solution of pyridoxylated, polymerized hemoglobin, where the hemoglobin solution is capable of being infused into a human patient in an amount of up to about 1.5 liters without causing a decrease in kidney performance. 
     
     
         2 . A solution of hemoglobin according to  claim 1 , wherein the solution has a half-life in a human patient of about 15 hours. 
     
     
         3 . A solution of hemoglobin according to  claim 1 , wherein the solution has a half-life in a human patient of about 24 hours. 
     
     
         4 . A solution according to  claim 1 , wherein the solution may be infused in an amount of up to about 5 liters. 
     
     
         5 . A solution of hemoglobin according to  claim 1 , wherein the solution is capable of being infused into a human patient in an amount of up to about 3 liters without causing a decrease in kidney performance, and the solution has a half-life in a human patient of about 24 hours. 
     
     
         6 . A process for preparing a solution of pyridoxylated, polymerized hemoglobin capable of being infused into a human patient in an amount of up to about 3 liters, comprising:
 (a) removing leucocytes by filtering a mixture containing red blood cells through a filter having a minimum average pore size sufficient prevent the passage of leucocytes;   (b) lysing the red blood cells;   (c) adding carbon monoxide to and heating the product of (b) to a temperature of about 60-62° C. for about 10 hours to yield a heat-treated hemoglobin solution;   (d) filtering the heat treated hemoglobin solution to remove stroma and stromal contaminants precipitated by the heating;   (e) degassing the heat-treated hemoglobin solution by sparging oxygen and then nitrogen through the heat-treated solution at a temperature of about 10° C. to produce a foam to yield a degassed, heat-treated hemoglobin solution;   (e) pyridoxylating the degassed solution to yield a solution of pyridoxylated hemoglobin;   (f) polymerizing the solution of pyridoxylated hemoglobin to produce a solution of pyridoxylated, polymerized hemoglobin;   (g) oxygenating the solution;   (g) purifying the solution to remove tetrameric hemoglobin and collecting purified pyridoxylated, polymerized hemoglobin, where the solution contains less than 0.8% based on the total weight of hemoglobin of tetramer;   (h) deoxygenating the purified pyridoxylated, polymerized hemoglobin; and   (i) adjusting the pH and electrolyte levels in the solution of purified pyridoxylated, polymerized hemoglobin to physiological levels.   
     
     
         7 . A process according to  claim 6 , wherein the degassed solution is pyridoxylated by contacting the degassed solution with pyridoxal-5-phosphate at a molar ratio of pyridoxal-5-phosphate to hemoglobin of from about 1:1 to 3:1. 
     
     
         8 . A process according to  claim 6 , wherein the degassed solution is pyridoxylated by contacting the degassed solution with pyridoxal-5-phosphate at a molar ratio of pyridoxal-5-phosphate to hemoglobin of about 2:1. 
     
     
         9 . A process according to  claim 7 , wherein the pyridoxal-5-phosphate is contacted with the hemoglobin and borohydride for about one hour. 
     
     
         10 . A process according to  claim 9 , wherein the hemoglobin in the pyridoxylated solution is polymerized by contacting the pyridoxylated solution with glutataldehyde at a glutaraldehyde to hemoglobin molar ratio of from about 24:1. 
     
     
         11 . A process according to  claim 10 , wherein the pyridoxylated solution is contacted with glutaraldehyde for about 18 hours and then quenched. 
     
     
         12 . A method of transfusing a human patient in need of a transfustion, comprising administering to the patient up to about 1.5 liters of a pyridoxylated, polymerized hemoglobin solution. 
     
     
         13 . A method of transfusing a human patient in need of a transfustion, comprising administering to the patient up to about 3.0 liters of a pyridoxylated, polymerized hemoglobin solution. 
     
     
         14 . A method of transfusing a human patient in need of a transfustion, comprising administering to the patient up to about 5.0 liters of a pyridoxylated, polymerized hemoglobin solution. 
     
     
         15 . A method according to  claim 14 , wherein the hemoglobin is a glutaraldehyde-polymerized hemoglobin. 
     
     
         16 . A method according to  claim 15 , wherein the hemoglobin has the molecular weight distribution shown in  FIG. 4 . 
     
     
         17 . A solution according to  claim 1 , wherein the hemoglobin is a glutaraldehyde-polymerized hemoglobin having the molecular weight distribution shown in  FIG. 4 .