Characterization of GalNAc-siRNA Conjugates Using a Quadrupole Time-of-Flight Mass Spectrometer

Significance of the topic

The development and quality control of oligonucleotide therapeutics—particularly chemically modified small interfering RNAs (siRNAs)—require robust analytical workflows for confirming identity, integrity and conjugation chemistry. GalNAc (N-acetylgalactosamine) conjugation is widely used to target hepatocytes via the asialoglycoprotein receptor, so reliable characterization of GalNAc-siRNA conjugates (molecular weight, sequence and modification mapping) is essential for research, development and release testing in biopharma.

Objectives and study overview

This application study demonstrates how ion-pair reversed-phase liquid chromatography coupled with a quadrupole time-of-flight (QTOF) mass spectrometer, combined with dedicated oligonucleotide analysis software, can be applied to (1) confirm molecular weight of GalNAc-conjugated siRNA strands and (2) obtain sequence confirmation by MS/MS. The work illustrates sample preparation and software configuration for automated multi-sequence analysis and visualization of sequence coverage, using a model siRNA derived from the givosiran sequence with common chemical modifications and a tri-antennary GalNAc moiety on the sense strand.

Methodology

Key methodological features:

• Chromatography: Ion-pair reversed-phase UHPLC under denaturing conditions (elevated column temperature) to separate denatured single strands derived from double-stranded siRNA.
• Mass spectrometry: High-resolution QTOF MS in negative electrospray ionization mode for MS1-based mass confirmation and data-dependent acquisition (DDA) MS/MS for fragmentation-based sequence analysis.
• Software: LabSolutions Insight Biologics used to define oligonucleotide sequences (bases, ribose modifications, backbone linkages, and terminal conjugates), to predict monoisotopic masses and structures, and to perform automated component extraction, deconvolution and fragment assignment for multiple sequences concurrently.

Instrumentation used

Instruments and principal settings used in this study:

• UHPLC: Nexera XS inert system with a Shim-pack Scepter Claris C18-300 column (100 mm × 2.1 mm, 1.9 µm).
• Mobile phases: A — 50 mM HFIP with 5 mM triethylamine in water; B — methanol. Gradient: ca. 10% B to 50% B over 10 min, ramp to high organic briefly, then re-equilibration.
• Chromatography conditions: 0.3 mL/min flow, column temperature ~60 °C, injection volume 2 µL.
• Mass spectrometer: LCMS-9050 QTOF operated in negative ESI; MS scan range m/z 550–2500; DDA MS/MS range m/z 100–2500. Typical source and gas parameters adjusted to support desolvation and stable ion currents for large oligonucleotide ions.
• Analysis software: LabSolutions Insight Biologics for sequence configuration, MS1 component reconstruction (charge-state summing and deconvolution), fragment assignment and graphical sequence coverage display.

Samples

The analyte was a double-stranded siRNA based on a therapeutic (givosiran) sequence. Both strands contain chemical modifications commonly used to improve stability and pharmacology: 2'-O-methyl and 2'-deoxy-2'-fluoro ribose substitutions, phosphorothioate backbone linkages at selected positions, and a tri-antennary GalNAc cluster appended to the 3'-terminus of the sense strand. The experimental chromatographic conditions were set to denaturing conditions so that the duplex dissociates and individual strands are detected separately.

Main results and discussion

Key outcomes from the LC/MS and MS/MS workflow:

• Chromatography: At the elevated column temperature used, the double-stranded siRNA dissociated and produced distinct UV-detectable peaks for the sense and antisense strands.
• Molecular weight confirmation: The LCMS-9050 provided high-quality multiply charged mass spectra for each strand. Deconvolution of the charge envelopes produced accurate intact molecular masses that matched the sequences and modifications defined in the software.
• Component extraction: The Insight Biologics software automatically generated component chromatograms by summing relevant charge states and isotopes, facilitating rapid assignment of chromatographic peaks to specified sequences.
• Sequence confirmation by MS/MS: DDA MS/MS fragmentation produced fragment ions corresponding to inter-nucleotide cleavages across both strands. The software listed top fragment ions and visualized sequence coverage using two complementary displays: a branch-mode showing fragment series types and a fill-mode showing intensity distribution and overall coverage.
• Coverage and modification mapping: Fragment ions spanned every inter-nucleotide bond, enabling 100% sequence coverage for both strands under the experimental conditions. Importantly, sequence confirmation extended to regions containing the GalNAc modification, demonstrating that the workflow can localize and confirm terminal conjugates.

Discussion points:

• The combination of ion-pair reversed-phase LC and high-resolution QTOF MS yields both separation and the spectral quality needed for intact mass confirmation of large, multiply charged oligonucleotides.
• MS/MS fragmentation under the applied DDA settings produced informative fragment ions for sequence assignment; visualization tools accelerate interpretation and QC decision-making.
• Software-driven multi-sequence handling and modification libraries reduce manual input and support routine analysis of panels of oligonucleotide sequences (useful in screening, process development and batch release).

Benefits and practical applications

This analytical configuration offers several practical advantages for oligonucleotide R&D and QC:

• Reliable intact mass verification of modified oligonucleotides and conjugates (e.g., GalNAc-siRNA).
• Comprehensive sequence confirmation using MS/MS with graphical coverage maps that simplify review and reporting.
• Support for multiple concurrent sequence analyses and configurable modification libraries, speeding workflows when many sequence variants must be characterized.
• Applicability to formulation, process development, impurity profiling and identity testing in preclinical and development-stage programs.

Future trends and potential applications

Anticipated developments and broader uses of this approach include:

• Higher throughput and automation: Integration with automated sample preparation and scheduled MS/MS acquisition to increase daily sample capacity for development and QC labs.
• Enhanced fragmentation methods: Optimization and adoption of alternative dissociation methods (including ion mobility–assisted separation) to improve localization of labile modifications and complex conjugates.
• Native and non-denaturing analyses: Complementary workflows under native conditions to preserve higher-order structure and ligand interactions where relevant.
• Regulatory and GMP implementation: Standardization of methods, software audit trails and validation procedures to support method transfer into regulated environments.
• Software advances: Machine learning–assisted spectral interpretation and expanded modification libraries to speed identification of unexpected variants and degradants.

Conclusion

The presented ion-pair reversed-phase UHPLC–QTOF MS workflow, combined with purpose-built oligonucleotide analysis software, delivers robust intact mass confirmation and comprehensive MS/MS-based sequence verification for GalNAc-conjugated siRNA. The approach handles chemically modified nucleotides and terminal conjugates, supports simultaneous analysis of multiple sequences, and provides clear visual outputs for sequence coverage—making it a practical solution for characterization tasks in oligonucleotide therapeutic development.

Reference

Shimadzu Corporation. Characterization of GalNAc-siRNA Conjugates Using a Quadrupole Time-of-Flight Mass Spectrometer. Application News, First Edition Apr. 2026, Application No. 01-01176-EN.

Related application notes cited in the original material:

1. Ion-Pair Reversed-Phase LC/MS Analysis of GalNAc-siRNA Conjugates under Denaturing and Non-Denaturing Conditions. Application News No. 01-01177-EN.
2. Ion-Pair Reversed-Phase LC/MS Analysis of siRNA under Denaturing and Non-Denaturing Conditions. Application News No. 01-00915-EN.
3. Efficient Method Development for Separation of Capped mRNA Fragments. Application News No. 01-00898-EN.

Acknowledgments: The original study acknowledged support from AMED (grant numbers JP21ae0121022, JP21ae0121023, JP21ae0121024).