Effect of transcript length, temperature, and lyophilization on mRNA-lipid nanoparticle stability

Lyophilization enhances the long-term stability of mRNA-lipid nanoparticles (LNPs), yet the full extent of the physicochemical and functional implications of freeze-drying remains underexplored. In the current work, the influence of key variables, such as mRNA length, formulation conditions, and lyophilization status, on the stability of mRNA-LNPs was investigated. mRNA constructs were synthesized encoding monomeric ( egfp )₁, dimeric ( egfp )₂, and tetrameric ( egfp )₄ sequences to vary mRNA length systematically. A suitable LNP composition and lyophilization conditions were identified to generate lyophilized mRNA-LNPs for a long-term stability study. The physicochemical properties of mRNA-LNPs changed initially after lyophilization but were stable during storage for both lyophilized and aqueous samples. However, these properties did not reliably predict functional stability. Aqueous ( egfp )₁-LNPs stored at room temperature showed a rapid 50-fold decrease in enhanced green fluorescent protein (EGFP) expression within 28 days. In contrast, lyophilized mRNA-LNPs underwent only a 6-fold loss in EGFP expression at 28 days, followed by a gradual decline to a 9-fold loss at 84 days. Lyophilization appears to result in two or more LNP subpopulations, leading to functional heterogeneity. Furthermore, inactivation rate constants were calculated using a first-order rate model as a function of temperature and mRNA length, which can serve as a basis for future mRNA-LNP stability modeling.