Microfluidic mixing is a widely used technology for producing lipid nanoparticles (LNPs) that encapsulate messenger RNA (mRNA). One major challenge in advancing LNP-based RNA therapeutics consists of developing formulations that can be manufactured consistently across various scales of drug development. Even though microfluidics allows LNP preparation with precisely defined properties, manufacturing at a large scale is very limited and often achieved by parallelizing microfluidic devices (numbering up). Here, we demonstrate the use of a fluidic oscillator as a novel micromixing principle (FDmix) for the preparation of mRNA-LNPs. In this study, we investigated the feasibility of upscaling the production of eGFP mRNA-LNPs by increasing the size of the fluidic oscillator geometry and by adjusting flow parameters. We evaluated the comparability of mRNA-LNP batches, which were either produced using FDmiX S for low- (< 0.8 L/h) or FDmiX M for high-output production (< 4.8 L/h), via physicochemical properties (Z-Average, PDI, Cryo-TEM, encapsulation efficiency, lipid composition analysis via HPLC). Additionally, mRNA-LNPs were formulated using a T-junction under the same flow parameters to assess potential differences. FDmiX samples were tested for short- and long-term storage stability under different conditions (8 °C, -80 °C). Small LNPs (< 80 nm) with narrow distribution (PDI < 0.1) and high encapsulation efficiency (> 96%) were obtained with FDmiX. The biological functionality was shown in vitro by transfection experiments of colorectal adenocarcinoma cells DLD-1 using fluorescence imaging, flow cytometry, and Western Blot. Our results indicate the high potential of the FDmix technology for the production of mRNA nanoparticles. Already at a small scale, high volumes can be processed.
mRNA Technology