A Hybrid Fragmentation Strategy for Modified Oligos
Why a single fragmentation mode leaves gaps, and how a hybrid strategy closes them for confident, end-to-end characterization.
About this webinar
Mass-spectrometric sequencing of therapeutic oligonucleotides is constrained by the choice of fragmentation method. Electron-transfer dissociation (ETD) struggles to fully fragment modified backbones — particularly locked nucleic acids (LNA) — and higher-energy collisional dissociation (HCD) produces dense spectra that are slow to interpret and prone to coverage gaps. For antisense oligos that combine LNA modifications with phosphorothioate (PS) backbones, neither approach on its own delivers reliable end-to-end coverage.
This webinar presents a hybrid fragmentation approach that combines ETD and HCD in a single Orbitrap scan (EThcD), applied across a panel of 16-mer modified oligonucleotides — LNA-containing antisense oligos, full PS and PO backbones, and PS/PO mixtures. The method gives full sequence coverage from w-ions alone, with cleaner spectra than HCD and a controlled, a/w- and d/z-dominant fragment pattern suitable for automated assignment in Byos. Kevin will also walk through the Byos processing recipe used to interpret the data and the optimal EThcD energy identified for the 16-mer panel.
Key Takeaways
A single Orbitrap scan that covers modified oligos end-to-end. EThcD on highly-charged anionic precursors yields full w-ion coverage on LNA-containing antisense oligonucleotides and PS-backbone samples where ETD and HCD alone leave gaps.
A counter-intuitive PS-versus-PO finding. Phosphorothioate backbones fragment more efficiently under EThcD than phosphodiester backbones — with direct implications for impurity screening in PS therapeutics.
A practical EThcD processing recipe in Byos. Recommended energy settings, fragment-type configuration, and a strict-then-lenient pass strategy for confident sequence coverage and impurity detection.
Speaker
Kevin Hes — PhD candidate, Division of BioAnalytical Chemistry & MS-LaserLab, Vrije Universiteit Amsterdam. PhD jointly sponsored by AstraZeneca, GSK, and Novartis, with funding from the Dutch Research Council (NWO); co-supervised by Anouk Rijs and Govert Somsen. Part of the InnovATOR consortium InnovATOR consortium.
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