Scalable Production and Biophysical Characterization of High-Molecular-Weight Relaxed and Tense Quaternary State Polymerized Human Hemoglobin as Potential Red Blood Cell Substitutes

High-molecular-weight (HMW) (>500 kDa) glutaraldehyde-polymerized human hemoglobin (PolyhHb) is a promising hemoglobin-based oxygen carrier (HBOC) due to its decreased risk of vasoconstriction and oxidative tissue injury. Previously, HMW tense (T) quaternary state PolyhHb was synthesized at the pilot scale with tangential flow filtration (TFF) for the removal of low-molecular-weight species. However, T-state PolyhHb is limited to specific biomedical applications due to its low oxygen affinity, thus motivating the need to produce high oxygen affinity relaxed (R) quaternary state PolyhHb at the pilot scale. This study explored the pilot-scale synthesis and extensive biophysical characterization of both HMW T- and R-state PolyhHb. The resultant characterization demonstrated the successful synthesis of low and high oxygen affinity PolyhHb with increased molecular weight (∼1000-1500 kDa). Overall, T- and R-state PolyhHb provides a platform for manufacturing oxygen therapeutics with a diverse range of oxygen affinities and potential biomedical applications.