(438b) Impact of Formulation Process on Efficacy and Biophysical Attributes of mRNA-Lipid Nanoparticles

With several RNA-LNP therapeutics achieving regulatory approval in recent years, this versatile biologics delivery technology has proven its utility as a platform for an array of applications. The formulation of RNA-LNPs typically involves mixing of drug substance, often mRNA or siRNA, in an acidic buffer with a mixture of lipids dissolved in an organic phase. Formulation process parameters, such as flow rates, flow rate ratios, mixing device, lipid composition, ratios of lipid components to drug substance, and temperature, as well as buffer/salt composition and buffer exchange processes, play a key role in the optimization of lipid nanoparticle formulations. Herein, we examine the effects of several of these variables, independently and in combination, on the biophysical characteristics and in vivo efficacy of mRNA-LNPs via two different routes of administration in mouse models. For multiple ionizable lipid types and compositions, we observe consistent trends between formulation processes and key quality attributes, including particle size, encapsulation, morphology, and in vivo protein expression, while also noting key differences between them. We also present additional characterizations toward a mechanistic rationalization of how these process parameters impact the biological function of these LNPs. From this work, we have expanded upon the field’s collective understanding of the effects of LNP formulation processes on LNP structure and function, and provide deeper insights into process optimization considerations for biomedical applications.