Conditions for Storage of Stroma-Free Hemoglobin Solutions for Fabricating Blood Substitutes
The stroma-free hemoglobin (Hb) solutions used for fabricating blood substitutes must be stable for a long period of time during storage and be available when massive clinical transfusions are required. One of the major problems in the long-term storage of Hb is its susceptibility to autoxidation, and thus becomes denatured (methemoglobin, metHb) and loses its capability to bind oxygen reversibly. In the study, porcine stroma-free Hb solutions were prepared by the aqueous two-phase system. The effects of varying conditions (temperature, pH, Hb concentration, cryoprotectant, and frequency of freeze-and-thaw process) on the extent of metHb formation during storage of Hb solutions were investigated. The results demonstrated that the stroma-free Hb solution obtained by the aqueous two-phase system was highly pure. The stroma-free Hb solutions can be safely stored at 4℃ or –20℃ without denaturation only for 2 months. Afterward, deterioration of Hb started to occur as evidenced by the increase in the metHb content. The extent of metHb formation increased significantly when the pH value of the storage solution is lower than the isoelectric point of Hb. Additionally, the extent of metHb formation increased with increasing the Hb concentration. A higher frequency of freeze-and-thaw process induced a greater extent of metHb formation. However, addition of cryoprotectant in the storage solution may inhibit the extent of metHb formation. In contrast, the stroma-free Hb solution can be safely stored at –80℃ without formation of metHb for at least 6 months.