Physicochemical and structural insights into lyophilized mRNA-LNP from lyoprotectant and buffer screenings

The surge in RNA therapeutics has revolutionized treatments for infectious diseases like COVID-19 and shows the potential to expand into other therapeutic areas. However, the typical requirement for ultra-cold storage of mRNA-LNP formulations poses significant logistical challenges for global distribution. Lyophilization serves as a potential strategy to extend mRNA-LNP stability while eliminating the need for ultra-cold supply chain logistics. Although recent advancements have demonstrated the promise of lyophilization, the choice of lyoprotectant is predominately focused on sucrose, and there remains a gap in comprehensive evaluation and comparison of lyoprotectants and buffers. Here, we aim to systematically investigate the impact of a diverse range of excipients including oligosaccharides, polymers, amino acids, and various buffers, on the quality and performance of lyophilized mRNA-LNPs. From the screening of 45 mRNA-LNP formulations under various lyoprotectant and buffer conditions for lyophilization, we identified previously unexplored formulation compositions, e.g., polyvinylpyrrolidone (PVP) in Tris or acetate buffers, as promising alternatives to the commonly used oligosaccharides to maintain the physicochemical stability of lyophilized mRNA-LNPs. Further, we delved into how physicochemical and structural properties influence the functionality of lyophilized mRNA-LNPs. Leveraging high-throughput small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM), we showed that there is complex interplay between mRNA-LNP structural features and cellular translation efficacy. We also assessed innate immune responses of the screened mRNA-LNPs in human peripheral blood mononuclear cells (PBMCs), and showed minimal alterations of cytokine secretion profiles induced by lyophilized formulations. Our results provide valuable insights into the structure-activity relationship of lyophilized formulations of mRNA-LNP therapeutics, paving the way for rational design of these formulations. This work creates a foundation for a comprehensive understanding of mRNA-LNP properties and in vitro performance change resulting from lyophilization.