Manufacturing mRNA-Loaded Lipid Nanoparticles with Precise Size and Morphology Control

Lipid nanoparticles (LNPs) are the leading platform for delivering nucleic acid therapeutics, produced by rapidly mixing lipids in ethanol with nucleic acid cargo in an aqueous buffer. LNP production is often approached with a mixing-focused mindset that reduces the entire self-assembly process to a single step, obscuring the relationship between the process inputs and LNP properties. Here, we present a method for producing mRNA-loaded LNPs, with independent and predictive control over both the size and morphology and without compromising other quality attributes. By decoupling particle design from mixing and formulation changes, this method enables the rational engineering of LNPs with defined properties. The method leverages mixing under high fusogenicity conditions, achieved by modulating the solvent composition, followed by timed postinjection of an aqueous buffer to kinetically arrest LNPs at the desired properties. We demonstrate the method using benchmark LNP formulations in an impinging jet mixer, a state-of-the-art technology for LNP manufacturing. The resulting LNPs exhibit up to an 8-fold increase in <i>in vitro</i> transfection efficacy compared to those produced by the conventional method. In addition, the method facilitates quality control and supports predictive modeling and rational process translation.