Towards the Rational Design of Biopharmaceutical Drugs: Formulation and Stability of mRNA and mRNA Lipid Nanoparticles

The COVID-19 pandemic brought about a rapid surge in the advancement of mRNA technology as an effective vaccine against the disease. The advantages of mRNA vaccines such as safety, potency and ease of production has led to further research about potentially using this technology to treat diseases that are otherwise difficult to control. The two FDA-approved vaccines are manufactured in the form of lipid nanoparticles, in which the mRNA in encapsulated within a mixture of lipids. However, these formulations must be stored at ultra-cold temperatures to maintain long-term stability. Being a novel technology in the field of biologics, there is still much to be learned about the stability of mRNA and mRNA lipid nanoparticles (mRNA-LNPs). Researchers from around the world are currently exploring methods to improve stability by employing various drying methods. One such technique that shows promise is lyophilization or freeze-drying. This thesis explores in detail the physical, chemical and thermal stability of mRNA-LNPs. It starts with the application of two simple analytical techniques to characterize RNA structure and its interactions with numerous excipients. Separately, three analytical methods for mRNA quantification are compared. A detailed study of the process of lyophilization and the impact of each stage of the cycle on the physical stability of RNA-LNPs is also carried out. This is then followed by a deeper investigation into the freezing stage of the cycle by comparing the effects of varying cooling rates. The influence of mRNA on the lipid nanoparticle is also assessed. Finally, stability studies are conducted, and the effects of higher temperatures are analysed. A variety of analytical techniques including dynamic light scattering, RiboGreen assay, UV-Vis spectroscopy, scatter-free UV-Vis spectroscopy, circular dichroism spectroscopy, capillary gel electrophoresis, and imaging techniques such as freeze-drying microscopy and cryo-transmission electron microscopy have been used as stability indicating methods. The results obtained highlight lyophilization as a potential technique to improve long-term stability of mRNA therapeutics.