Method for the preparation of a heat stable oxygen carrier-containing pharmaceutical composition and the use thereof

A method for the preparation of a highly purified and heat stable oxygen carrier-containing pharmaceutical composition packaged in a multilayer, flexible infusion package, the oxygen carrier-containing pharmaceutical composition including hemoglobin, the hemoglobin consisting essentially of nonpolymeric cross-linked tetrameric hemoglobin, the method comprising:

• a) providing mammalian whole blood including at least red blood cells and plasma;
• b) separating the red blood cells from the plasma in the mammalian whole blood;
• c) filtering the red blood cells that were separated from the plasma to obtain a filtered red blood cell fraction;
• d) washing the filtered red blood cell fraction to remove plasma protein impurities, resulting in washed red blood cells;
• e) disrupting the washed red blood cells by a precise and controlled hypotonic lysis for 2 to 30 seconds or a time otherwise sufficient to lyse the red blood cells in an instant cytolysis apparatus to create a solution comprising a lysate of disrupted red blood cells at a flow rate of 50-1000 L/hr;
• f) performing filtration to remove at least a portion of the waste retentate from the lysate;
• g) extracting a first hemoglobin solution from the lysate;
• h) performing a first ultrafiltration process, using an ultrafiltration filter configured to remove impurities having a higher molecular weight than hemoglobin to further remove any viruses and residual waste retentate from the first hemoglobin solution to obtain a second hemoglobin solution;
• i) performing flowthrough column chromatography on the purified hemoglobin solution to remove protein impurities;
• j) performing a second ultrafiltration process using an ultrafiltration filter configured to remove impurities and to concentrate the purified hemoglobin solution;
• k) cross-linking the hemoglobin in a bis-3,5-dibromosalicyl fumarate cross-linking solution to form cross-linked hemoglobin in a deoxygenated environment wherein the cross-linked hemoglobin is nonpolymeric cross-linked tetrameric hemoglobin;
• l) exchanging a suitable physiological buffer for the cross-linking solution;
• m) removing any residual chemicals by tangential flow filtration;
• n) heat treating the cross-linked hemoglobin in a deoxygenated environment to denature and precipitate any residual non-reacted hemoglobin, non-stabilized hemoglobin (dimer) and any other protein impurities such that the resulting heat stable cross-linked tetrameric hemoglobin has an undetectable concentration of dimer and consists essentially of nonpolymeric cross-linked tetrameric hemoglobin;
• o) adding N-acetyl cysteine immediately following heat treating the cross-linked tetrameric hemoglobin to maintain a low level of met-hemoglobin;
• p) removing precipitate by a centrifugation or a filtration apparatus to form a clear solution;
• q) adding the purified and heat stable cross-linked tetrameric hemoglobin to a pharmaceutically acceptable carrier and
• r) introducing the purified and heat stable cross-linked tetrameric hemoglobin and pharmaceutically acceptable carrier to a multilayer, flexible infusion package.