Introduction and Objectives: Ionizable lipid nanoparticles (iLNPs) are effective delivery vehicles for mRNA vaccines and therapeutics. A major challenge in this field is to address the storage conditions of mRNA-iLNPs, which are typically stored frozen and have a short shelf life. One strategy to address this is lyophilization to remove the water from the formulation that facilitates degradation of the mRNA. The objective of this study was to empirically test common buffers and cryoprotectants for the lyophilization of mRNA-iLNPs.
Materials and Methods: Shelf lyophilization of mRNA-iLNPs was evaluated by varying the lyophilization protocol, buffer, salt content and cryoprotectant content. The lead lyophilization conditions were applied to eGFP expression in vitro and an in vivo vaccination setting.
Results: The lead lyophilization protocol resulted in minimal changes to the physicochemical properties of the particles after drying and mRNA integrity was maintained after 3 months of storage at 4 °C. eGFP expression in HeLa cells showed transfection efficiency was similar after lyophilization, but the total eGFP expression was approximately 30% of the freshly prepared control. There was no difference in the number of antigen-specific antibodies generated by a lyophilized and fresh vaccine.
Conclusion: A lyophilization procedure for mRNA-LNPs was empirically determined that could be stored at 4 °C for months. The physicochemical properties of the particles, transfection efficiency and immune response was unaffected by lyophilization.