Lipid nanoparticles (LNPs) have emerged as the most advanced drug delivery system for gene therapies and vaccines, due to their ability to encapsulate a diverse range of payloads, including nucleic acids and proteins. They provide protection from enzymatic degradation, enhanced cellular uptake, and controlled release. The complex composition and inherent instability of LNPs pose significant challenges for their characterization, particularly in assessing encapsulation efficiency, payload integrity, and size distribution. To address these challenges, we have developed a novel two-dimensional chromatography (2D) method capable of simultaneously evaluating key parameters of the LNP formulations. The method utilizes a switching chromatography system with two distinct analytical columns, independent pumps, and multiple detectors including an ultraviolet-visible detector (UV-VIS) and a multi-angle light scattering detector (MALS). This configuration enables the direct injection and analysis of the LNPs without prior sample preparation. The 2D technique facilitates the accurate quantification of both encapsulated and non-encapsulated nucleic acids, the assessment of payload integrity, and measurement of the LNP size and size distribution. The findings indicate that this approach provides a comprehensive and efficient analytical platform, thereby advancing the understanding and development of robust lipid-based drug delivery systems.
