Thin-film freeze-drying of an influenza virus hemagglutinin mRNA vaccine in unilamellar lipid nanoparticles with blebs

Messenger RNA (mRNA) vaccines have revolutionized the fight against infectious diseases and are poised to transform other therapeutic areas. Lipid nanoparticles (LNP) represent the most successful delivery system for mRNA. While the mRNA-LNP products currently in clinics are stored as frozen suspensions, there is evidence that freeze-drying mRNA-LNP into dry powders can potentially enable their storage and handling at non-freezing temperatures. Previously, we successfully applied thin-film freeze-drying (TFFD) to transform a polyadenylic acid [poly(A)]-LNP formulation from a liquid suspension to dry powders. The poly(A)-LNP were structurally multilamellar spheres without blebs, but the mRNA vaccines in clinics are comprised of mRNA-LNP that are structurally spheres surrounded by a unilamellar lipid bilayer, with some containing blebs, and it was reported that the presence of blebs increases the sensitivity of mRNA-LNP to freeze-drying-induced stress. In the present study, using an influenza A virus hemagglutinin (HA) mRNA in LNP that were structurally similar to that in the COVID-19 mRNA vaccines currently in clinic, we studied the effect of TFFD on the physical properties, internal structure, as well as immunogenicity of the HA mRNA-LNP vaccine. We concluded that TFFD can be utilized to prepare dry powders of the HA mRNA-LNP, but a sufficient amount of excipients were needed to minimize changes in the physical properties, structure, and immunogenicity of the HA mRNA-LNP vaccine.