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 (Hb) obtained from mammalian sources via polymerization and encapsulation. Another approach toward oxygen therapeutic design has centered on using naturally occurring large molecular diameter Hbs [i.e., erythrocruorins (Ecs)] derived from annelid sources. Therefore, the goal of this study was to purify Ec from the terrestrial worm Lumbricus terrestris for diverse oxygen therapeutic applications. Tangential flow filtration was used as a scalable protein purification platform to obtain a >99% pure Lumbricus terrestris Ec (LtEc) product, which was confirmed by size exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In vitro characterization concluded that the ultrapure LtEc product had oxygen equilibrium properties similar to those of human red blood cells and a lower rate of auto-oxidation compared to human Hb, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultrapure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (similar to 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.