Effects of different mixing techniques on mRNA lipid nanoparticle physicochemistry and biological performance

Abstract Lipid nanoparticle (LNP) formulation requires a mixing step. Many studies, especially from academic groups, utilize either microfluidic mixers or hand mixing to prepare LNPs, but commercial-scale processes use turbulent-flow mixers. This discrepancy in mixing techniques has been underexplored, as LNPs made by different techniques may exhibit different performance, such that bench-scale results cannot be replicated using materials manufactured at scale. We here isolate and interrogate the effect of primary mixing. Lipid nanoparticles are produced from ten mixers (one used in two ways), holding all other formulation parameters constant, to directly compare across techniques. LNPs produced from the different mixers exhibit widely different physical properties and biological performance. Notably, manual pipetting common in academic practice yields particles that do not resemble those produces by turbulent-flow mixers. Findings are connected mechanistically to physicochemical characteristics that arise from the different flow regimes. Further establishing the relationship between mixing and LNP properties is critical. Graphical abstract