Purification of Lumbricus terrestris Mega-Hemoglobin for Diverse Oxygen Therapeutic Applications

Oxygen therapeutics are being developed for a variety of applications in transfusion medicine. In order to reduce the side-effects (vasoconstriction, systemic hypertension, and oxidative tissue injury) associated with previous generations of oxygen therapeutics, new strategies are focused on increasing the molecular diameter of hemoglobin obtained from mammalian sources via polymerization and encapsulation. Another approach towards oxygen therapeutic design has centered on using naturally occurring large molecular diameter hemoglobins (i.e. erythrocruorins) derived from annelid sources. Therefore, the goal of this study was to purify erythrocruorin from the terrestrial worm Lumbricus terrestris for diverse oxygen therapeutic applications. Tangential flow filtration (TFF) was used as a scalable protein purification platform to obtain a >99% pure LtEc product, which was confirmed by size exclusion high performance liquid chromatography and SDS-PAGE analysis. In vitro characterization concluded that the ultra-pure LtEc product had oxygen equilibrium properties similar to human red blood cells, and a lower rate of auto-oxidation compared to human hemoglobin, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultra-pure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (~86% purity). In summary, we purified an LtEc product with favorable biophysical properties that performed well in an animal model using a reliable and scalable purification platform to eliminate undesirable proteins.