Cold-chain storage remains a major challenge for mRNA products. Lyophilization could improve stability and simplify distribution, but freezing and dehydration risk damaging the RNA. Measuring this damage by HPLC can be difficult because intact mRNA and closely eluting degradation products produce broad, partially overlapping peaks.
We investigated the effects of freeze-drying and cryoprotectant formulation on the integrity and stability of unencapsulated mRNA. mRNA was lyophilized with sucrose, trehalose or other excipients, then compared with matched solution controls before and after drying and during storage across a range of temperatures.
Integrity was assessed using ion-pair reversed-phase HPLC. To detect changes that may be obscured by broad peak profiles, we developed a difference chromatogram approach that compares the full chromatographic trace between conditions. This captures both loss of full-length mRNA and the appearance of shorter degradation products.
mRNA was relatively robust to freeze-drying, with limited loss of integrity after drying and reconstitution. In an initial accelerated degradation study at 50 °C, lyophilized mRNA showed an approximately threefold longer apparent half-life than the matched solution control. Cryoprotectant-containing formulations also showed improved preservation of integrity during storage at elevated temperatures.
Overall, freeze-drying preserved mRNA integrity and improved stability relative to solution storage. Difference chromatogram analysis of IP-RP HPLC data provided a practical tool for formulation screening and stability assessment. Ongoing studies are examining the effects of formulation composition, storage temperature and duration on dry-state mRNA stability.