Lyophilized annelid mega-hemoglobin retains its’ quaternary structure and oxygen equilibrium properties after room temperature storage for over 6 months

The long-term storage stability and portability of hemoglobin (Hb)-based oxygen carriers are important design criteria in the development of these therapeutics. Lyophilization or storing proteins in a freeze-dried form is known to increase storage lifetime and reduce overall weight. In this study, we lyophilized the extracellular mega-hemoglobin of the annelid Lumbricus terrestris and tested the storage stability at different temperatures and oxygenation conditions. Storage in refrigerated conditions for over 6 months in the presence of N(2) reduced oxidation by 50% while storage at room temperature in the presence of N(2) reduced oxidation by 60%, all while maintaining the structural stability of the mega-hemoglobin. We also demonstrated a reliable strategy to freeze dry Hbs in the presence of a minimally non-reducing disaccharide sugar that could be easily re-solubilized in deionized water. Overall, this study made significant advances towards long term storage stability of oxygen therapeutics for direct applications in transfusion medicine.