Challenges and achievements in the implementation of the prokaryotic pilot plant

Introduction: An important factor in developing mRNA vaccines is the role of temperature during the manufacturing process.Temperature can influence the stability of mRNA and the lipid nanoparticles, which are crucial for effective delivery.Further research in this area could lead to more efficient manufacturing processes and improved vaccine quality, ultimately accelerating the development of mRNA-based therapeutics.Objectives: This study investigates the influence of temperature on the physical properties and fluorescence of LNPs loaded with self-amplifying mRNA.Additionally, we compared self-amplifying and conventional mRNA at the same temperature.Methodology: For this purpose, the same formulation was encapsulated with two different mRNAs (selfamplifying and conventional).Afterward, the LNPs were placed in dialysis, and the self-amplifying mRNA samples were incubated at two different temperatures (4 C and 35 C) overnight and the conventional only 4 C.Following this, the LNPs were used for transfection of cells, and indirect immunofluorescence was performed.Additionally, a DLS analysis was conducted.Results: Formulations were evaluated at 4 C and 35 C, in the case of self-amplifying mRNA, and 4C to the conventional mRNA.The average particle size of self-amplifying decreased from 143.3 nm at 35C to 118.4 nm at 4 C, with a corresponding reduction in polydispersity index (PDI) from 0.251 to 0.177, indicating improved homogeneity at lower temperatures.In contrast, the conventional mRNA displayed smaller particle sizes overall (95.9 nm) with a PDI of 0.150 in 4 C.Fluorescence microscopy revealed notable differences in mRNA expression.While self-amplifying formulations demonstrated fluorescence in cells under both temperature conditions, the conventional formulation showed no fluorescence, indicating a lack of mRNA expression or cell uptake. Conclusion:These results highlight the critical role of temperature in modulating the physical characteristics of LNPs and underscore the necessity of optimizing mRNA formulations for efficient expression.