Identifying Key Factors Affecting mRNA-Lipid Nanoparticles Drug Product Formulation Stability

<i>Background</i>: The long-term stability of mRNA-lipid nanoparticles (LNPs), essential for mRNA vaccines and gene therapies, relies on managing physicochemical properties to preserve their integrity and effectiveness through optimized formulation components. This study systematically evaluated LNP formulations with varied compositions, e.g., Dlin-MC3-DMA and ALC-0315 as ionizable lipids, and DMG-PEG2k or ALC-0159 as polyethylene glycol (PEG)-lipids, stored at -80 °C, -20 °C, 5 °C, and 25 °C in Tris buffer (pH 7.4) for 12 months. <i>Methods</i>: Sixteen quality attributes were analyzed, including particle size, mRNA encapsulation, lipid oxidation, and transfection efficiency over different formulations and storage temperatures to mechanistically evaluate the long-term stabilities. <i>Results</i>: Formulations stored at -80 °C and -20 °C retained acceptable stability, while storage at 5 °C caused aggregation, reduced in vivo expression, and mRNA degradation. Storage at 25 °C led to complete loss of transfection within six months. Mechanistic studies identified oxidative and hydrolytic lipid degradation (e.g., DSPC) in ALC-0315 formulations and MC3 N-oxidation with subvisible particulates in MC3-containing LNPs as primary failure modes. Increasing Tris buffer concentration accelerated 5'-cap hydrolysis, emphasizing the importance of a low-ionic-strength buffer for LNP formulations. <i>Conclusions</i>: Findings re-emphasize the necessity of deep-cold storage (≤-20 °C) and optimized formulation components to preserve mRNA-LNP integrity, offering insights for designing next-generation LNPs with improved shelf-life.