Challenges and advances in analytical separation techniques for RNA-lipid nanoparticle therapeutics

Gene therapies are rapidly advancing as drug modalities, serving as a means to treat previously undruggable pathways. The use of small interfering ribonucleic acid (siRNA), messenger RNA (mRNA), and guide RNA (gRNA) in clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing, combined with lipid nanoparticles (LNPs), has demonstrated effective drug delivery. These complex delivery systems often require multiple analytical methodologies to achieve comprehensive characterization, some of which remain underdeveloped or inadequately adapted for RNA-LNP formulations. Commonly used batch-based methods, such as dynamic light scattering (DLS) or the modified RiboGreen assay, are frequently hindered by sample heterogeneity, a limitation that can be addressed through analytical separations. This review discusses the challenges limiting analytical separations for RNA-LNP therapeutics and highlights recent advances in separation science for reliable characterization and quality control. We focus on techniques for RNA, LNPs, and the RNA-LNP complex, emphasizing chromatographic, electrophoretic, and field-based separation techniques.