Methods for preparation of a heat stable hemoglobin based oxygen-carrier-containing pharmaceutical compositionsuch that beta-beta cross-linking is favored are provided. Using the methodsof the present invention, the oxygen affinity of the resultantmolecule can be controlled so that hemoglobin based oxygen carriers tailoredfor specific applications can be produced. Loweroxygen affmity crosslinked hemoglobin is useful for applications requiringrapid tissue oxygenation (e.g. hemorrhagic shock) whilehigher oxygen affinity cross-linked hemoglobin is useful for applicationsrequiring a slower rate of oxygenation (e.g. cancer adjuncttherapy). A highly purified and heat stable crosslinked non-polymerictetrameric hemoglobin having beta- beta cross-linking ofgreater than 40-60% and suitable for use in mammals without causing renalinjury and vasoconstriction is produced. A high temperatureand short time (HTST) heat processing step is performed to effectively removeany undesired dimeric hemoglobin, non- cross-linkedtetrameric hemoglobin, and plasma protein impurities.
