Uncovering the influence of lyoprotection on stability of mRNA-LNPs after lyophilization and high temperature storage.

Lyophilization has the potential to enhance mRNA-LNP stability at elevated temperatures. The present study aimed to comprehensively evaluate the influence of lyoprotectants on critical quality attributes (CQAs) and function of mRNA-LNPs post-lyophilization and upon storage at 4 °C, 25 °C, and 40 °C. Twelve formulations containing sucrose, trehalose, and polyvinylpyrrolidone (PVP) and their combinations at three total lipid-to-lyoprotectant weight ratios (1:5, 1:10, and 1:20) were studied. The CQAs of lyophilized mRNA-LNPs depended on the lyoprotectant composition and the lipid-to-lyoprotectant weight ratio, with the 1:20 ratio performing best. At this ratio, mRNA-LNPs containing sucrose, sucrose: trehalose 1:1, and sucrose: trehalose: PVP 1:1:1 showed limited change in particle size, mRNA encapsulation efficiency, and in-vitro transfection efficiency compared to other formulations. Interestingly, a lipid-to-sucrose 1:10 ratio provided stabilization of particle size and encapsulation post-lyophilization but resulted in reduced transfection efficiency relative to the 1:20 ratio. Thus, similarities in physicochemical properties did not assure equivalent biological function. After one month of storage at up to 40 °C, lyophilization with sucrose: trehalose: PVP = 1:1:1 at a 1:20 ratio enabled mRNA-LNPs to maintain particle size distribution, encapsulation efficiency, mRNA integrity, and in-vitro transfection. The enhanced stability of the formulation is likely due to its higher glass transition temperature of the lyoprotectant combination. In contrast, at 40 °C, when sucrose was used as the sole lyoprotectant it crystallized, reducing its protective effect. The results highlight a strategy for high-temperature stability of mRNA-LNPs as a reference for formulation development.