Development of combined bio-RP-HPLC-UV and UHPLC-ELSD methods for quantitation of mRNA-LNP content before and after size-exclusion chromatography.

Abstract

Following the COVID-19 pandemic, mRNA vaccines have been widely deployed, with lipid nanoparticles (LNPs) serving as the prevailing delivery vehicles for mRNA vaccines. Separation and quantitative determination of mRNA and LNP lipid components have therefore attracted considerable attention. Reported mRNA assays suffer from several limitations, including interferences originating from metallic instrument components and complex formulation matrices, insufficient resolution, and prolonged elution durations. Existing analytical approaches for LNP lipid quantification usually involve labor-intensive sample pretreatment and fail to eliminate matrix interferences. Even reported methods capable of simultaneous measurement of nucleic acids and lipids still exhibit deficiencies in resolution and sensitivity. Herein, an integrated analytical platform combining Bio-RP-HPLC-UV and UHPLC-ELSD was established. A biocompatible inert liquid-chromatography system was adopted for mRNA quantification to mitigate adsorption and carry-over effects commonly observed in conventional stainless-steel systems, while chromatographic conditions were optimized to shorten analysis time. A 2/6-port valve-switching splitting device was installed upstream of the UHPLC-ELSD detector to prevent rapid detector contamination induced by direct injection of complex mRNA-LNP formulation matrices and substantially reduce instrument-maintenance frequency. Mobile-phase compositions were further optimized to improve resolution and sensitivity for LNP lipid detection. The validated workflow exhibited favorable precision and excellent linearity (R > 0.9990). The recovery of the mRNA quantification method ranged from 87.89% to 100.31%, and the mean recoveries of the four LNP lipids at three concentration levels varied between 99.52% and 102.46%, guaranteeing assay accuracy. Furthermore, this integrated platform was applied to characterize mRNA-LNP samples before and after size-exclusion-chromatography (SEC) purification. Correlation analysis was performed among mRNA contents, drug-to-lipid ratios, and particle-size data acquired by high-throughput dynamic light scattering (DLS). Results from method-validation experiments and testing of three batches of samples demonstrated that the proposed platform enables simultaneous quantification of mRNA and lipids in mRNA-LNP formulations and multi-attribute parameter correlation verification throughout the SEC purification procedure. Satisfactory inter-batch consistency was achieved across three production batches. This work provides technical support for constructing a facile and practicable quality-evaluation system for mRNA-LNP vaccine formulations.

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