Separation of polymerized hemoglobin from unpolymerized hemoglobin on hydroxyapatite using HPLC

A method for producing an ultrapure stable polymerized hemoglobin blood-substitute, wherein said blood-substitute can be administered to humans or other vertebrates, comprising the steps of:

• a) mixing a mammalian blood with an anticoagulant to form a blood solution;
• b) diluting the blood solution with an isotonic solution;
• c) filtering said diluted blood solution to separate smaller plasma components from the red blood cells;
• d) centrifuging said filtered blood solution to separate white blood cells from the red blood cells;
• e) directing the red blood cells against a surface to tear the cell membranes of at least a portion of the red blood cells to form a hemoglobin solution;
• f) ultrafiltering the hemoglobin solution to remove large cell debris from the hemoglobin solution;
• g) ultrafiltering the hemoglobin solution to remove small cell debris from the hemoglobin solution;
• h) chromatographically treating said filtered hemoglobin solution by high performance liquid chromatography employing a pH gradient elution and a packing for the ion-exchange affinity separation f endotoxin from hemoglobin to form an ultrapure hemoglobin eluate;
• i) deoxygenating said ultrapure hemoglobin eluate to form a deoxygenated hemoglobin solution;
• j) mixing said deoxygenated hemoglobin solution with a sulfhydryl compound, to form an oxidation-stabilized, deoxygenated hemoglobin solution;
• k) mixing said oxidation-stabilized, deoxygenated hemoglobin solution with a cross-linking agent to form a polymerization reaction mixture;
• and
• l) polymerizing the polymerization reaction mixture, thereby forming a polymerized hemoglobin solution;
• m) contacting the polymerized hemoglobin solution with an alkaline solution, whereby the polymerized hemoglobin solution is basified;
• n) contacting the basified polymerized hemoglobin solution with sodium borohydride, whereby unstable bonds are reduced, thereby forming a stable polymerized hemoglobin solution;
• and
• o) contacting the stable polymerized hemoglobin solution with a hydroxyapatite HPLC column, whereby polymerized hemoglobin is separated from non-polymerized hemoglobin, thereby forming said ultrapure stable polymerized hemoglobin blood-substitute.