EP 1 211 261 A2

[0329] The method further comprising the steps of:EP 1 211 261 A2510a) deoxygenating the hemoglobin in the hemoglobin eluate to form a deoxygenated hemoglobin solution;b) mixing the deoxygenated hemoglobin solution with a first reducing agent, to form an oxidation-stabilized, deoxygenatedhemoglobin solution;c) mixing said oxidation-stabilized, deoxygenated hemoglobin solution with a cross-linking agent to form a polymerizationreaction mixture;d) polymerizing the polymerization reaction mixture, thereby forming a stable polymerized hemoglobin solution; ande) purifying the polymerized hemoglobin solution from non-polymerized hemoglobin in the presence of a physiologicsolution and a second reducing agent, whereby the polymerized hemoglobin solution is made physiologicallyacceptable, and whereby the reducing agent scavenges oxygen, thereby forming a stable, polymerized, purifiedhemoglobin product.1520[0330] A method wherein the polymerized hemoglobin solution is purified from non-polymerized hemoglobin by diafiltrationusing an ultrafiltration membrane.[0331] A method wherein the polymerization reaction mixture is stabilized in its molecular weight distribution bychemically reducing the polymerization reaction mixture.[0332] A method wherein the red blood cell fraction is subjected to a chromatographic column that comprises apacking for ion-exchange affinity separation of endotoxin from hemoglobin.[0333] A method further comprising the steps of:2530a) mixing blood containing red blood cells 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 bloodcells to form a hemoglobin solution;f) ultrafiltering the hemoglobin solution to remove large cell debris from the hemoglobin solution; andg) ultrafiltering the hemoglobin solution to remove small non-hemoglobin components from the hemoglobin solution,thereby producing a purified red blood cell fraction containing hemoglobin.[0334] A method further comprising the steps of:3540455055a) deoxygenating the hemoglobin eluate to form a deoxygenated hemoglobin solution;b) mixing said deoxygenated hemoglobin solution with a reducing agent, to form an oxidation-stabilized, deoxygenatedhemoglobin solution;c) mixing said oxidation-stabilized, deoxygenated hemoglobin solution with a cross-linking agent to form a polymerizationreaction mixture;d) polymerizing the polymerization reaction mixture, thereby forming a polymerized hemoglobin solution;e) contacting the polymerized hemoglobin solution with an alkaline solution, whereby the polymerized hemoglobinsolution is basified;f) contacting the basified polymerized hemoglobin solution with sodium borohydride, whereby unstable bonds arereduced, thereby forming a stable polymerized hemoglobin solution;g) diafiltering the stable polymerized hemoglobin solution with a physiologic solution, whereby the polymerizedhemoglobin solution is made physiologically acceptable, thereby forming ultrapure, stable, polymerized hemoglobinproduct; andh) filtering said ultrapure stable polymerized hemoglobin product to separate tetrameric hemoglobin from saidultrapure stable polymerized hemoglobin product.[0335] A method for preserving the oxidation stability of a hemoglobin product comprising maintaining the hemoglobinproduct in an atmosphere substantially free of oxygen.[0336] A method for producing a polymerized hemoglobin solution from hemoglobin contained in a hemoglobin solution,comprising the steps of:a) mixing said hemoglobin solution with a reducing agent, whereby the hemoglobin solution is deoxygenated,thereby forming an oxygen-stabilized, deoxygenated hemoglobin solution;b) mixing said mixing said oxygen-stabilized, deoxygenated hemoglobin solution with a cross-linking agent, therebyforming a polymerization reaction mixture; and36