BACKGROUND: Therapeutics and vaccines involving mRNA have shown significant progress over the last five years. These lipid nanoparticle-based formulations introduce substantial analytical complexity, as multiple Quality Attributes (QAs) must be monitored to ensure product efficacy, stability, and safety. Encapsulation efficiency (EE) and mRNA integrity are particularly critical, yet current analytical approaches often require separate assays, challenging sample handling, and limited selectivity toward free versus encapsulated species. Therefore, new chromatographic methods capable of resolving these species and supporting multi-payload formulations remain a major unmet need.
RESULTS: To rapidly obtain multiple QAs for mRNA-LNP formulations, we present an optimized two-dimensional liquid chromatography (2D-LC) workflow combining anion-exchange chromatography (AEX) in the first dimension (D) and ion-pair reversed-phase liquid chromatography (IP-RPLC) in the second dimension (D). TheD-AEX evaluated encapsulation efficiency of mRNA within lipid nanoparticles based on the mRNA ratio of intact and disrupted drug products, whileD-IP-RPLC assessed integrity profiles and mRNA-lipid adducts. Attention was paid to theD optimization to eliminate solvent incompatibilities and ensure efficient transfer between dimensions. This approach enables simultaneous determination of EE, mRNA integrity, mRNA-lipid adducts, and transcript ratios in multi-payload mRNA-LNP formulations. It also provides chromatographic assessment of free mRNA integrity within formulations. This method furthermore enables the characterization of additional species and confirms the presence of surface-associated mRNA. The workflow demonstrates good selectivity and applicability to both mono- and multi-payload mRNA-LNP products. 1 2 1 2 2
SIGNIFICANCE: Overall, the developed 2D-LC platform is a powerful analytical tool for comprehensive mRNA-LNP characterization. By enabling simultaneous assessment of several critical QAs, including EE, integrity, transcript ratios, and mRNA-lipid adducts, it streamlines analytical workflows and reduces reliance on multiple independent assays. This approach provides mechanistic insight into LNP structure, including detection of surface-associated RNA species, and establishes an innovative tool to support formulation development, process optimization, drug products release, and stability studies for next-generation mRNA vaccines and therapeutics.
