Enabling mRNA-LNP Drug Products Through Advanced Engineering and Characterization

The theme of “turbocharging innovation and driving back to rational design” implies a solid understanding of molecular-level fundamentals that underpin pharmaceutical properties. The LNP is a complex nanostructured entity that serves to protect the delicate RNA molecule from the harshly degrading nuclease environment in vivo while facilitating intracellular delivery. One difficulty with the analysis of LNPs is that many biophysical techniques produce a globally averaged signal or have limited selectivity for the RNA molecule within the solid particle. Although cryo-EM is a powerful tool for visualizing LNPs, the mass density contrast alone is not distinctive enough to unambiguously resolve RNA from lipidic components. We found that a contrast enhancement approach could be used to elucidate the location of mRNA within the LNP providing striking new insights into LNP morphological structure and the physicochemical microenvironment of the encapsulated RNA. The RNA molecule itself is a polyanion with complex conformational and charge interaction behaviors in solution. Large RNA molecules in solution are generally considered to be more extended and diffuse than globular proteins but still may adopt diversely complex higher-order structures. This presentation will describe application of techniques to evaluate higher-order structures of mRNA both unencapsulated and encapsulated within the LNP.