Analysis of lipid nanoparticles using two-dimensional chromatography: Simultaneous determination of encapsulation efficiency, nucleic acid integrity, and size of LNP formulations

Abstract

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. LNPs provide many benefits, such as protection of payloads from enzymatic degradation, enhanced cellular uptake, and controlled release, making them promising candidates for therapeutic applications. The complex composition and inherent instabilities of LNPs present significant challenges for their characterization, especially in determining encapsulation efficiency, payload integrity, and size distribution. This study presents a two-dimensional chromatography (2D) method to simultaneously assess crucial parameters of LNP formulations. The method employs a switching chromatography system comprising two distinct analytical columns, independent pumps, and two detectors, including an ultraviolet-visible detector (UV–Vis) and a multi-angle light scattering detector (MALS). A key benefit of this advanced configuration is the direct analysis of LNPs without the need for sample preparation. The 2D technique enables accurate quantification of both encapsulated and non-encapsulated nucleic acids, evaluation of payload integrity, and determination of LNP size and size distribution. The results demonstrate that this method provides a comprehensive and robust analytical solution, enabling further development and understanding of lipid-based drug delivery systems. The validation of the technique confirms its sensitivity (LOD 15 ng mRNA, LOQ 45 ng mRNA), linearity (R² > 0.99), precision (RSD < 15 %), accuracy (< 15 %), and potential to streamline and optimize process development, in-process control, and quality control of LNP formulations.