Early Spectroscopic Insights into RNA Modifications in LNP Formulations with Microfluidic Modulation Spectroscopy

Abstract

This study highlights the critical need for advanced analytical techniques in understanding RNA modifications within lipid nanoparticle (LNP) formulations, which are crucial for therapeutic RNA delivery. Ionizable lipids like SM-102, while enabling packaging within LNPs, can degrade and covalently modify mRNA, leading to potential loss of mRNA activity. Traditional methods for detecting these modifications often have time-consuming sample workup and do not report directly on structural integrity and covalent modifications.

Microfluidic Modulation Spectroscopy (MMS) offers a direct, label-free approach to monitor RNA chemical and structural integrity under formulation conditions. Our findings demonstrate MMS's ability to detect degradation-induced spectral changes in both polyA and polyC RNA samples. For polyA, MMS revealed subtle spectral perturbations consistent with ring-nitrogen modification, and UV-Vis showed a near-total loss of aromaticity, emphasizing the complementary insights provided by the techniques in combination. In polyC, MMS identified a new IR band at 1735 cm−1, suggesting abasic site formation, alongside addition changes indicative of cytosine perturbation. This research underscores MMS as a rapid, first-pass assay for assessing RNA degradation, guiding further characterization with orthogonal techniques to further explore degradation pathways.

MMS is a patented technology found in AuroraTX, Apollo and AQS instruments.