The Rise of Top‐Down Mass Spectrometry for the Characterization of Oligonucleotide Therapeutics

The therapeutic landscape has been rapidly expanding beyond traditional small molecules and biologics in recent years with the emergence of oligonucleotide therapeutics. As these oligonucleotide modalities grow in complexity and chemical modifications, conventional digestion-based mass spectrometry strategies start to yield redundant and ambiguous information. Alternatively, the top-down approach has the potential to provide a holistic view of therapeutic oligonucleotides. This review examines the rise of top-down mass spectrometry for the characterization of therapeutic oligonucleotides, with a focus on technological and methodological advances between 2020 and 2025. We cover sample preparation, sample introduction including separation, ionization, mass analysis, fragmentation approaches, data analysis strategies, and a discussion of long oligonucleotide and beyond for oligonucleotide top-down analysis. Finally, we provide a practical top-down workflow guidance on therapeutic oligonucleotides and conclude with a forward-looking perspective on the frontiers of the field.