Deciphering Phosphorothioate Isomerism in Synthetic Oligonucleotides Using the Unique Geometry of the Cyclic IMS P20 Mass Spectrometer

Deciphering Phosphorothioate Isomerism in Synthetic Oligonucleotides Using Cyclic IMS P20 Mass Spectrometry

Importance of the topic

Phosphorothioate (PS) modification of the nucleic acid backbone is a widespread chemical strategy to improve stability and pharmacokinetics of therapeutic oligonucleotides. Each PS linkage creates a chiral phosphorus center (Rp or Sp), and combinations of such linkages produce complex diastereomeric mixtures that can influence potency, durability and safety. Accurate, site-specific measurement of PS stereochemistry is therefore increasingly important for discovery, process development and regulatory compliance when developing antisense oligonucleotides (ASOs), siRNA and other RNA therapeutics.

Study objectives and overview

This application study demonstrates an analytical workflow that leverages the geometry and high-resolution multipass ion mobility capability of the Waters Cyclic IMS P20 Mass Spectrometer to (i) generate internal dimer fragment ions that contain a single PS linkage, (ii) separate Rp and Sp diastereomers by cyclic ion mobility, and (iii) quantify the relative isomer ratios for individual linker positions. The method is illustrated using a 16‑nucleotide lipid‑conjugated ASO standard and is presented as extensible to a range of therapeutic oligonucleotides.

Methodology and analytical strategy

The core strategy exploits pre‑ion mobility fragmentation (CID in the trap collision cell upstream of the IMS device) to produce internal fragment ions, specifically nucleotide dimers that include exactly one PS linker. Key procedural elements:

• Sample introduction: direct infusion after desalting or optional LC coupling.
• Pre‑IMS fragmentation: quadrupole isolation of a chosen precursor charge state followed by CID in the trap collision cell to generate a broad fragment population including internal dimers.
• Dimer selection and ion mobility: target dimer ions are subjected to high‑resolution multipass Cyclic ion mobility separation to resolve diastereomer arrival‑time distributions.
• Quantification: relative Rp/Sp ratios are obtained from integrated peak areas of mobility‑separated peaks using MassLynx software.
• Resolving isomeric dimers: when identical dimer sequences occur at multiple positions, two orthogonal approaches are used — in‑source CID to generate larger unique first‑generation fragments followed by MS/MS, or MS/MS followed by IMSn slicing and secondary dissociation (pseudo‑MS3) — to link a given dimer to its specific backbone location.

Used instrumentation

• Waters Cyclic IMS P20 Mass Spectrometer with multipass high‑resolution ion mobility.
• Quadrupole mass filter for precursor selection.
• Trap collision cell for CID fragmentation upstream of IMS.
• Capability for in‑source CID (cone voltage) and IMSn slicing for mobility selection.
• MassLynx software for data processing and peak integration.

Main results and discussion

The workflow successfully separated and quantified Rp/Sp diastereomers of multiple individual PS linkers within the LCASO 16‑mer standard. Dimeric internal fragments containing a single PS provided tractable species for site‑specific stereochemistry measurement. The Cyclic IMS geometry (pre‑IMS fragmentation) proved decisive when identical dimer compositions occurred at distinct backbone positions, because larger upstream fragments or IMSn slicing allowed unambiguous assignment of the dimer origin. Representative measured ratios from the study included isomer pairs resolved to near‑equimolar distributions (e.g., ~48:52 and ~40:60 for specific linkers), illustrating sensitivity to modest stereochemical imbalance. The authors note that absolute mobility assignment to Rp versus Sp requires stereopure reference materials or computational modelling; the presented method provides robust relative quantification at individual linkers.

Key advantages and practical applications

• Site‑specific stereochemistry measurement: by analysing dimeric internal fragments, the method enables characterization of single PS linkers rather than ensemble averages across the full oligonucleotide.
• High resolving power: multipass Cyclic IMS resolves diastereomer arrival times that are unresolved by many chromatographic approaches.
• Flexibility: compatible with direct infusion workflows or LC coupling and adaptable to ASOs, siRNA and sgRNA formats.
• Regulatory and process control value: provides data to support correlations between stereochemistry and biological performance and to monitor batch consistency during process development and scale‑up.

Limitations and considerations

• Absolute stereochemical assignment (Rp vs Sp) is not intrinsic to mobility separation and requires stereopure standards or complementary modelling/structural methods to definitively label peaks.
• Generation of suitable internal dimers depends on fragmentation efficiency and sequence context; some linkers may be less accessible to produce isolated dimers.
• Complex mixtures with many sequence repeats increase the need for MSn strategies to assign dimer origin, adding experimental complexity.

Future trends and potential applications

• Integration with stereopure standards and computational structural modelling to enable unambiguous Rp/Sp peak assignment and build libraries of mobility fingerprints.
• Automation of IMSn acquisition and data processing to streamline site‑specific stereochemistry mapping across larger panels of oligonucleotide candidates.
• Routine deployment in quality control for clinical‑stage oligonucleotide manufacturing as regulatory expectations for stereochemical characterization evolve.
• Extension to fully phosphorothioated therapeutics and chemically diversified backbones, including conjugated modalities, to study structure–activity relationships tied to stereochemistry.

Conclusion

The described approach combines pre‑IMS CID fragmentation with high‑resolution multipass ion mobility on the Cyclic IMS P20 platform to separate and quantify phosphorothioate diastereomers at single‑linker resolution using internal dimer fragments. This capability offers a practical and sensitive route to monitor PS stereochemistry during oligonucleotide discovery and development, supporting mechanistic studies, process control and regulatory submissions. While definitive Rp/Sp assignment requires additional stereopure standards or modelling, the method delivers reliable relative ratios and a pathway to more comprehensive stereochemical characterization across diverse oligonucleotide therapeutics.

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