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Abstract
Background
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.
Methods
FLuc dsiRNA (57 bp) and custom ssRNA (60bp) 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 an X500R LC-QToF.
Results
The RNA-LNPs had a hydrodynamic diameter, PDI, and %EE within the target profile. The ssRNA 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 dsiRNA concentration was 77.75 ± 14.90 ng/mL, both RNA constructs were identified by LC-MS.
Conclusions
Absolute quantification and identification of both RNA constructs was successfully performed by LC-MS and validated using ICH M10 bioanalytical guidelines.
Publication Date
2026
Keywords
RNA, RNA-LNP, LC-MS, LC-MS/MS, Critical Quality Attributes (CQAs), Top-Down MS, MS Method Development, RNA Quantification, RNA Identification
Disciplines
Analytical, Diagnostic and Therapeutic Techniques and Equipment | Biochemistry | Biological and Chemical Physics | Lipids | Medicinal Chemistry and Pharmaceutics | Nanomedicine | Nanotechnology | Nucleic Acids, Nucleotides, and Nucleosides | Pharmaceutical Preparations | Pharmacy and Pharmaceutical Sciences
Faculty Advisor/Mentor
Dr. Matthew S. Halquist
Is Part Of
VCU Graduate Research Posters
Included in
Analytical, Diagnostic and Therapeutic Techniques and Equipment Commons, Biochemistry Commons, Biological and Chemical Physics Commons, Lipids Commons, Medicinal Chemistry and Pharmaceutics Commons, Nanomedicine Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Pharmaceutical Preparations Commons, Pharmacy and Pharmaceutical Sciences Commons