Author ORCID Identifier
https://orcid.org/0009-0009-1201-1149
Defense Date
2026
Document Type
Thesis
Degree Name
Master of Science
Department
Pharmaceutical Sciences
First Advisor
Matthew S. Halquist
Second Advisor
Sandro R. P. da Rocha
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.
Rights
© Jason C. Funderburk
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-6-2026
Included in
Analytical Chemistry Commons, Biochemistry Commons, Biological and Chemical Physics Commons, Investigative Techniques Commons, Lipids Commons, Medicinal Chemistry and Pharmaceutics Commons, Nanomedicine Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Other Pharmacy and Pharmaceutical Sciences Commons, Pharmaceutical Preparations Commons