Size Exclusion Chromatography for LNP Analysis: Strategies for Dealing with Method Development Challenges

Lipid-based nanoparticles, including liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs), have proven highly effective in delivering hydrophobic and hydrophilic therapeutics. Due to their exceptional biocompatibility, biodegradability, and entrapment efficiency, lipid nanoparticles (LNPs) have also emerged as a promising carriers for nucleic acids such as DNA, mRNA, and siRNA. The continued success of LNPs in treating various diseases underscores their enormous potential as a cutting-edge drug delivery system. The size of lipid nanoparticles plays a crucial role in determining their effectiveness as drug-delivery vehicles. Factors such as stability, encapsulation efficiency, drug release rate, bio-distribution, and cellular uptake are all influenced by the size of the particles. While analyzing size variants for lipid nanoparticles remains challenging, recent advances in low adsorption ultrawide pore size exclusion chromatography (SEC) columns have greatly improved sizing analyses. However, paying close attention to sample preparation, mobile phase considerations, and columns with optimal packing materials is essential to ensure reliable SEC separation. These can serve as tools to quickly and reliably monitor size-related changes that could indicate batch-to-batch variations, formulation-related changes, and sample stability. Our method showcases the ability of SEC to furnish reliable data on the LNP particle and formulation characteristics, given the proper conditions. The elution process is aided by using customized mobile phases with carefully chosen additives so as not to cause artificial changes to the intact LNP. This study underscores the effects of different mobile phase variables, including ionic strength, salt type, and the presence of organic solvents, surfactants, or stabilizing agents on LNP behavior. Moreover, the SEC methods outlined can be combined with other detectors like MALS, RI, and QELS (DLS) to obtain enhanced potency and safety readings.