Effects of mixing technique and ethanol removal on lipid nanoparticle physicochemical properties

Optimizing the production of lipid nanoparticle (LNP) therapeutics is necessary for drug delivery efficiency, stability, and scalability. A small but growing body of literature has begun to recognize that LNP properties (e.g., size, shape, and internal structure) depend on the flow conditions during mixing for antisolvent precipitation, in which LNPs are formulated. Here, we use different mixers, varying flow patterns (e.g., laminar or turbulent mixing) and flow rate ratios (FRR), i.e., 3:1 and 1:1, to prepare a standard LNP formulation. We then characterize the resulting formulations using small angle x-ray scattering (SAXS) to provide insights into particle shape/morphology, internal organization (L _α and H _II phases) of yeast RNA (yRNA), and structural differences/similarities that arise from the different mixing methods. The effect of ethanol removal on the LNPs’ structure, formulated from each mixing technique, is also discussed. We observed the 3:1 FRR mixers outperform the 1:1 configurations in certain desired LNP physiochemical properties. The differences observed in the LNPs produced across the two configurations are discussed. Furthermore, we use computational fluid dynamics to explain the turbulent mixing schemes among the 3:1 and 1:1 mixers.