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

Chintan Savla,Andre F Palmer,Parag A Deshpande

doi:10.1371/journal.pone.0263996

Abstract

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 N2 reduced oxidation by 50% while storage at room temperature in the presence of N2 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.

Highlights

The storage stability of blood and blood products is a key area of concern for researchers and medical professionals
Human Hb was used as a control to test the effect of lyoprotectant concentration on the protein before and after the lyophilization process
Hb and LtEc samples were stored under different temperature and gas conditions post lyophilization for 6 months and the formation of metHb was monitored over time—ambient temperature (25 ̊C), cold storage (4 ̊C), and freezer storage (-80 ̊C) in the presence and absence of a N2 atmosphere

Summary

Introduction

The storage stability of blood and blood products is a key area of concern for researchers and medical professionals. We lyophilized LtEc and hHb, and monitored metHb formation, structural stability, and equilibrium oxygen binding properties after 6 months of storage under various temperature conditions and in the presence and absence of a deoxygenated environment.

Results

Conclusion