Investigation on a Freeze-Drying Process for Long-Term Stability of mRNA-LNPs

Background: Thermostability remains a key bottleneck for equitable access to mRNA–LNP vaccines, largely due to cold-chain requirements. Objectives and methods: Here, we optimized freeze-drying formulations by screening excipients (sugars, sugar-alcohols, and proteins) and buffers to preserve mRNA–LNP physicochemical (size, polydispersity index -PDI and encapsulation efficiency -EE) and fluorescence intensity-based, functional integrity assay (in vitro transfection) during long-term storage of up to 6 months. Results: In a preliminary screening study, different sugars (sucrose, trehalose), sugar alcohol (mannitol), protein (gelatin) and different buffers (Tris, PBS, histidine) were evaluated. The preliminary result showed that sucrose and trehalose, along with Tris and histidine buffers, had a positive effect on maintaining the physicochemical properties during freeze-drying, while mannitol, gelatin and PBS buffer had a negative effect. Based on these findings, the optimized formulations containing sucrose/Tris, sucrose/histidine, trehalose/Tris and trehalose/histidine were chosen, and a stability study was performed at −80, −20, 4, and 20 °C for six months. Conclusions: Overall, except for the samples maintained at 20 °C, no significant changes in the physicochemical quality of the freeze-dried mRNA-LNPs were observed over six months at −80, −20, and 4 °C. The in vitro stability study demonstrated stability at 4 °C for four months across all formulations, while a formulation with sucrose/Tris maintained satisfactory stability even at 20 °C for the same duration. The main results of this study demonstrate the feasibility of storing mRNA drug products as solid formulations at non-freezing temperatures (≤ 4 °C).