Heightened interest in messenger RNA (mRNA) therapeutics has accelerated the need for analytical methodologies that facilitate the production of supplies for clinical trials. Forced degradation studies are routinely conducted to provide an understanding of potential weak spots in the molecule that are exploited by stresses encountered during bulk purification, production, shipment, and storage. Consequently, temperature fluctuations and excursions are often experienced during these unit operations and may accelerate mRNA degradation. Here, we present a concise panel of chromatography-based stability-indicating assays for evaluating thermally stressed in vitro transcribed (IVT) mRNA as part of a forced degradation study. We found that addition of EDTA to the mRNAs prior to heat exposure reduced the extent of degradation, suggesting that transcripts may be fragmenting via a divalent metal-ion mediated pathway. Trace divalent metal contamination that can accelerate RNA instability is likely carried over from upstream steps. We demonstrate the application of these methods to evaluate the critical quality attributes (CQAs) of mRNAs as well as to detect intrinsic process- and product-related impurities.
Keywords: Forced degradation; Liquid chromatography; Multi-angle light scattering; Stability-indicating assay; messenger RNA.
Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.