Developability assessments with four mRNA-LNP vaccine formulations comparing mouse immunogenicity, structural attributes, and stability profiles.

A standardized developability workflow to rank-order the stability profiles of mRNA-LNP vaccine formulations is described. As proof-of-concept, three experimental mRNA-LNP formulations with varying lipid compositions were benchmarked against a Spikevax® mimic via mouse immunogenicity studies and various stability studies using a "routine analytical toolbox" to monitor (1) chemical integrity of the mRNA and lipid components, (2) structural integrity of the assembled nanoparticles, and (3) functionality (i.e., cell-based transfection assays). Although the four mRNA-LNP formulations displayed overall similar in vivo immunogenicity in mice (mRNA encoding Rabies glycoprotein G antigen), their in vitro stability profiles varied substantially (mRNA encoding luciferase). Functionality losses (i.e., decreasing luciferase expression) upon exposure to different environmental stresses in a common liquid formulation buffer correlated with distinct physicochemical mechanisms (i.e., mRNA degradation at elevated temperatures, and LNP disruption upon agitation and freeze-thaw). Heat-map analysis enabled rank-ordering of the mRNA-LNP formulations' overall stability profiles. Interestingly, further characterization of the four mRNA-LNPs by cryo-EM and ¹H-NMR identified specific structural attributes (i.e., bleb formation and lipid surface topology, respectively) at time zero that correlated with their observed stability profiles. Results are discussed in the context of how a standardized mRNA-LNP developability platform enables identification of mRNA-LNP vaccine candidates with optimized pharmaceutical properties.