Absolute Quantification and Identification of RNA from RNA-Lipid Nanoparticles using High Resolution Mass Spectrometry

Abstract

RNA therapeutics are a rising drug category with potential use for a range of conditions encompassing infectious diseases to therapies for cancer, diseases, and genetic disorders. RNA-lipid nanoparticles (RNA-LNPs) are the prominent delivery method for these therapeutics approved products include mRNA vaccines and polyneuropathy treatments. The emergency use authorizations and orphan drug status of current RNA-LNP drugs has allowed approval without finalization of the regulatory analytical procedures for quality monitoring. The objective of the study was to develop an LC-MS assay to simultaneously measure identity and concentration of two therapeutically relevant intact RNA constructs extracted from RNA-LNPs to enhance quality monitoring techniques for potentially 20 approved RNA-based drugs. FLuc-S1 dsiRNA (57 bp) and custom single-stranded RNA (60 bp) constructs were formulated in LNPs at an N/P ratio of 6. RNA-LNPs were assessed for RNA concentration and encapsulation efficiency (EE%) using the Quant-iT™ RiboGreen™ assay and LNP size and polydispersity (PDI) with dynamic light scattering. RNA was extracted with isopropanol precipitation and diluted 100x for LC-MS characterization. Absolute quantification and identification of RNA was performed with a SCIEX X500R. The RNA-LNPs had a hydrodynamic diameter, PDI, and EE% within the targeted therapeutically relevant profile. The 60-mer had a measured concentration of 92.10 ± 6.90 ng/mL by fluorescence and 92.46 ± 7.77 ng/mL by LC-MS. The measured FLuc-S1 dsiRNA concentration was 77.75 ± 14.90 ng/mL, both RNA constructs were identified by LC-MS. Absolute quantification and identification of both RNA constructs was successfully performed by LC-MS and validated using ICH M10 bioanalytical guidelines.