Lipid-conjugated antisense oligonucleotides (ASOs) improve pharmacokinetics and tissue uptake but present complex stability challenges. Understanding thermally driven degradation pathways is critical for shelf-life prediction and formulation design. This study evaluated accelerated stability of a gapmer ASO bearing a 5′-palmitate conjugate and 2′-O-methoxyethyl (MOE) termini. Heat-stress data were generated and modeled using Accelerated Stability Assessment Program (ASAP®) software, with degradation pathways and kinetic markers characterized by high-resolution LC-MS/MS. The objective was to identify shelf-life-limiting degradation mechanisms and assess heat-only accelerated stability modeling for lipid-modified ASOs.
A lipid-conjugated 16-mer phosphorothioate gapmer ASO standard (Waters, PN 186010747) was heat-stressed at 37, 50, 60, and 70°C for up to 168 hours. Samples were analyzed on a Vanquish Flex UHPLC system coupled to an Orbitrap Exploris 240 mass spectrometer operated in negative ESI mode. Separation was achieved on a Waters Premier BEH C18 Oligonucleotide column (2.1 × 50 mm, 1.7µm 130Å) using ion-pair reversed-phase liquid chromatography (IP-RPLC). High-resolution full MS and data-dependent MS/MS spectra were acquired. Data processing, quantitation, and Arrhenius-based shelf-life modeling were performed using BioPharma Finder and ASAP®prime software.
Heat stress produced a simple, ordered degradation profile well suited for Arrhenius modeling. The dominant early degradation pathway was hydrolytic loss of the 5′-palmitate conjugate. This transformation increased with temperature and time and was the primary kinetic driver in ASAPprime modeling. With extended heat exposure, a shortmer ladder emerged, initiated predominantly from the 3′-terminus, sequentially losing MOE-modified nucleotides before entering the DNA gap. Early shortmers (n−1 and n−2) retained the lipid conjugate, whereas longer stress durations produced delipidated shortmers. Minor levels of phosphorothioate desulfurization (−16 Da variants) were detected at higher temperatures but remained too low to impact kinetic modeling. Notably, lipid chain oxidation or nucleobase oxidation were not observed under heat-only conditions, confirming that degradation chemistry remained hydrolytic and Arrhenius-consistent. ASAPprime modeling predicted a refrigerated shelf-life of more than 1 year and 6-12 months at room temperature, with palmitate linker hydrolysis identified as the shelf-life-limiting pathway. These results demonstrate HR-MS and ASAP modelling as powerful tools for shelf-life prediction of lipid-conjugated gapmer ASOs.