An in vitro method for the preparation of a highly purified and heat stable oxygen carrier containing pharmaceutical composition is disclosed. The oxygen carrier-containing pharmaceutical composition including haemoglobin and the haemoglobin consisting essentially of non-polymeric crosslinked tetrameric haemoglobin having a beta-beta cross-linking of greater than 40% preferably greater than 50%. The method comprises a) providing a sample of mammalian whole blood including at least red blood cells and plasma. The red blood cells are then separated from the plasma in the mammalian whole blood and the red blood cells that were separated from the plasma are filtered to obtain a filtered red blood cell fraction. The filtered red blood cell fraction is washed to remove plasma protein impurities, resulting in washed red blood cells. The washed red blood cells is disrupted to create a solution comprising a lysate of disrupted red blood cells and a filtration is performed filtration to remove at least a portion of the waste retentate from the lysate. A first haemoglobin solution is extracted from the lysate and at least one purification process is performed to remove one or more of viruses, waste retentate, or protein impurities. The first haemoglobin solution is cross-linked by bis-3,5-dibromosalicyl fumarate to form crosslinked haemoglobin in an oxygenated environment. The crosslinked haemoglobin is non-polymeric crosslinked tetrameric haemoglobin having at least 40% beta-beta cross-linking. Any residual chemicals are removed from the solution and the crosslinked haemoglobin are heat treated in a deoxygenated environment to denature and precipitate any residual non-stabilized/ non-crosslinked haemoglobin, any dimeric haemoglobin and any other protein impurities such that the resulting heat stable crosslinked tetrameric haemoglobin has an undetectable concentration of dimer and consists essentially of nonpolymeric crosslinked tetrameric haemoglobin with a beta-beta cross-li
