Patent ID: 8106011

Claim:
A method for the preparation of a highly purified and heat stable oxygen carrier-containing pharmaceutical composition, the oxygen carrier-containing pharmaceutical composition including hemoglobin, the hemoglobin consisting essentially of non-polymeric crosslinked tetrameric hemoglobin having a beta-beta cross-linking of greater than 40%, 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 to create a solution comprising a lysate of disrupted red blood cells; 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 at least one purification process to remove one or more of viruses, waste retentate, or protein impurities; i) cross-linking the first hemoglobin solution by bis-3,5-dibromosalicyl fumarate to form crosslinked hemoglobin in an oxygenated environment wherein the crosslinked hemoglobin is non-polymeric crosslinked tetrameric hemoglobin having at least 40% beta-beta cross-linking; j) removing any residual chemicals; k) heat treating the crosslinked hemoglobin in a deoxygenated environment to denature and precipitate any residual non-stabilized/non-crosslinked hemoglobin, any dimeric hemoglobin and any other protein impurities such that the resulting heat stable crosslinked tetrameric hemoglobin has an undetectable concentration of dimer and consists essentially of non-polymeric crosslinked tetrameric hemoglobin with a beta-beta cross-linking of at least 40% and an oxygen affinity greater than the oxygen affinity of native hemoglobin of the same species measured under substantially similar conditions; l) removing precipitate by a centrifugation or a filtration to form a clear solution; and m) adding the purified and heat stable crosslinked tetrameric hemoglobin to a pharmaceutically acceptable carrier.