Asymmetrical Flow Field-flow Fractionation with Multi-angle Light Scattering for Separation and Characterization of Lipid Nanoparticles

The use of lipid nanoparticles (LNPs) for delivery of messenger RNA (mRNA)-based therapies has gained substantial attention in the field of drug development recently. Multiple attributes of such lipid nanoparticles have to be carefully characterized to ensure product safety and quality, whereas comprehensive characterization of these complex LNP formulations remains a challenging endeavor. Here, we present the development and application of an online isolation and characterization platform designed for the separation and in-depth analysis of mRNAs and mRNA-loaded LNPs using asymmetrical flow field-flow fractionation (AF4) coupled with multi-detectors, including multi-angle light scattering (MALS), dynamic light scattering (DLS) and dual concentration detectors (dRI/UV). The developed AF4 approach demonstrated exceptional resolution between mRNA-LNPs and mRNAs, excellent recoveries and exceptional repeatability. Notably, this platform allows for comprehensive and multi-attribute LNP characterization, including online particle sizing, morphology characterization, and determination of encapsulation efficiency, all within a single injection. Furthermore, real-time online sizing by synchronizing MALS and DLS presented higher resolution over batch-mode DLS, particularly in differentiating heterogeneous samples. Additionally, our approach proves to be a valuable tool for monitoring LNP stability under different stress conditions. Our work introduces a robust and versatile analytical platform using multi-detector AF4 that not only efficiently provides multi-attribute characterizations of mRNA-LNPs but also holds promise in advancing studies related to nanoparticle formulation screening, quality control and stability assessment.