Considerations for chromatographic and mass spectrometry analytical methods for complex oligonucleotide therapeutic analysis
Nucleic Acid Insights 2026; 3(5), 351–360
DOI: 10.18609/nai.2026.042
The expanding complexity of oligonucleotide therapeutics creates challenges and opportunities within analytical characterization strategies. Centred on liquid chromatography with UV and mass spectrometry detection, this expert insight highlights ion‑pair reversed‑phase as a workhorse and the need for orthogonal separation modes to obtain conjugate‑driven selectivity. It details various mass spectrometry considerations for sequence and impurity analysis, dual‑polarity workflows and emerging ion mobility techniques for isomer or impurity resolution. Software limitations are discussed, as well as practical considerations of bioanalytical extraction bottlenecks, sensitivity trade‑offs, and the impact of the conjugate on resolution and robustness. The outlook emphasizes integrated approaches, DoE and automated method development, maturation of AI‑driven retention prediction and degradation pathway modelling and QC upskilling with fit‑for‑purpose instrumentation. Advancing highly specific, sensitive, and high‑resolution methods – supported by improved data handling and harmonized regulatory guidance on AI – will be critical to reliably characterize an evolving set of quality attributes for conjugate oligonucleotide therapeutics.