Decoding Oligonucleotide Stereochemistry: Individual Phosphorothioate Configuration Ratios and Intact Sequence
Md Rabiul Islam1, Leonard Yoon1, Nadia Tasnim Ahmed1
1Division of Pharmaceutical Quality Research II, Office of Pharmaceutical Quality Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, St. Louis, Missouri 63110, United States.
Analytical Chemistry
|June 18, 2026
Summary
High-resolution ion mobility mass spectrometry (HRIM-MS) enables simultaneous stereochemical analysis of oligonucleotide therapeutics. This method reveals discrepancies between intact sequence diastereomers and individual linkage configurations, crucial for drug quality and comparability.
Area of Science:
- Analytical Chemistry
- Pharmaceutical Science
- Biotechnology
Background:
- Phosphorothioate (PS) modifications in oligonucleotide therapeutics create complex diastereomeric mixtures.
- Stereochemical characterization is vital for drug development, regulatory approval, and generic comparability.
- Current analytical methods struggle with simultaneous, multi-level stereochemical analysis.
Purpose of the Study:
- To develop and demonstrate a high-resolution ion mobility mass spectrometry (HRIM-MS) methodology for concurrent stereochemical analysis of oligonucleotide therapeutics.
- To enable simultaneous characterization at both the intact sequence and individual PS linkage levels.
- To investigate potential discrepancies between intact sequence diastereomeric composition and individual PS linkage configurations.
Main Methods:
- Utilized high-resolution ion mobility mass spectrometry (HRIM-MS).
- Employed a phosphorothioate (PS)-modified 3-mer oligonucleotide as a model system.
- Performed in-source fragmentation to analyze individual PS linkages.
Main Results:
- Achieved baseline separation of all four expected diastereomers of the 3-mer sequence.
- Enabled direct measurement of intact sequence diastereomeric composition.
- Directly quantified Rp/Sp configuration ratios at individual PS linkages.
- Observed discrepancies between intact sequence diastereomeric profiles and predicted compositions based on individual linkage ratios.
Conclusions:
- HRIM-MS provides unprecedented concurrent stereochemical analysis of oligonucleotide therapeutics at multiple levels.
- Intact diastereomeric composition may not be predictable from individual PS linkage configurations alone.
- Comprehensive, multi-level stereochemical characterization is essential for ensuring quality, consistency, and comparability of oligonucleotide therapeutics.
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