Ion-Pair Reversed-Phase LC/MS Analysis of GalNAc-siRNA Conjugates under Denaturing and Non-Denaturing Conditions

Ion-Pair Reversed-Phase LC/MS Analysis of GalNAc-siRNA Conjugates under Denaturing and Non-Denaturing Conditions

Significance of the Topic:
Ion-pair reversed-phase LC/MS is a practical and widely used approach for characterizing therapeutic oligonucleotides, including siRNA conjugates bearing N-acetylgalactosamine (GalNAc) ligands. GalNAc conjugation is a leading strategy to direct oligonucleotides to hepatocytes via the asialoglycoprotein receptor (ASGPR). Reliable analytical methods that distinguish intact duplexes from dissociated strands and confirm molecular weight are critical for development, quality control, and regulatory documentation of GalNAc-siRNA therapeutics.

Objectives and Study Overview:
The study demonstrates ion-pair reversed-phase LC/MS workflows to (1) separate and identify GalNAc-conjugated siRNA in denaturing and non-denaturing elution conditions, and (2) confirm molecular masses of sense and antisense strands using a single-quadrupole mass spectrometer combined with sequence-aware data analysis software. The example uses a double-stranded siRNA sequence derived from Givosiran with a tri-antennary GalNAc modification at the 3′ terminus of the sense strand.

Methodology:

• Sample: Double-stranded GalNAc-siRNA conjugate (sense strand modified at 3′ with tri-GalNAc; nucleoside modifications include 2′-methoxy, 2′-deoxy-2′-fluoro, and phosphorothioate linkages).
• LC separation: Ion-pair reversed-phase UHPLC with 100 mM HFIP / 10 mM TEA in water (mobile phase A) and methanol (mobile phase B); Shim-pack Scepter Claris C18-300 column (100 × 2.1 mm, 1.9 µm); gradient 10%→50% B over 10 min, fast wash to 90% and return to 10%; flow 0.3 mL/min; injection 1 µL; column temperatures tested at 25 °C and 60 °C to probe non-denaturing vs denaturing behavior.
• MS detection: LCMS-2050 single-quadrupole mass spectrometer with heated DUIS ion source enabling combined ESI/APCI operation; negative ion mode; m/z range 550–2000; nebulizing gas 2.0 L/min, drying gas 5.0 L/min, heating gas 7.0 L/min; desolvation 450 °C, DL 200 °C.
• Data analysis: LabSolutions Insight Biologics software used to define oligonucleotide sequences (nucleobases, ribose types, linkers, and base/terminal modifications including Tri-GalNAc), compute theoretical masses and formulas, and assign component chromatograms by matching MS1 spectra to expected sequences.

Instrumentation Used:

• Nexera XS inert UHPLC (Shimadzu)
• LCMS-2050 single quadrupole mass spectrometer with DUIS heated ion source (Shimadzu)
• Shim-pack Scepter Claris C18-300 column (100 × 2.1 mm, 1.9 µm)
• LabSolutions Insight Biologics analysis software

Main Results and Discussion:

• Chromatographic behavior depended strongly on column temperature. At 25 °C (non-denaturing conditions) the GalNAc-siRNA eluted as a single main UV peak consistent with a duplex (sense/antisense co-elution).
• At 60 °C (denaturing conditions) the UV trace resolved into two peaks corresponding to separated sense and antisense single strands; LC/MS component chromatograms confirmed the identity and order of elution of each strand.
• Mass assignments for both sense and antisense strands matched theoretical molecular weights within ~1 Da at both temperatures, demonstrating the single-quadrupole system’s suitability for accurate molecular-weight confirmation in QC contexts.
• The LabSolutions Insight Biologics software effectively handled multiple sequence entries and modifications (including the tri-GalNAc 3′ modification), produced deconvoluted spectra, and generated component chromatograms based on MS1 matching.
• Limitations and complementarity: while single-quadrupole instruments provide reliable intact-mass confirmation and ease of use for routine QC, they do not support MS/MS sequence confirmation. For detailed sequence-level characterization and MS/MS fragmentation, higher-resolution instruments such as Q-TOF are recommended.

Benefits and Practical Applications:

• Rapid confirmation of molecular weight for GalNAc-siRNA conjugates to support manufacturing QC and release testing.
• Ability to discriminate duplex versus single-stranded states by simple temperature control of the LC column, facilitating assessment of duplex stability and dissociation behavior.
• Sequence-aware software streamlines identification when multiple oligonucleotide sequences or modified residues are present, reducing manual interpretation time.
• Single-quadrupole LC/MS provides a user-friendly platform with routine operational simplicity suitable for many analytical laboratories that do not require MS/MS.

Future Trends and Potential Applications:

• Greater adoption of high-resolution MS and MS/MS (Q-TOF/Orbitrap) for fragment-based sequence confirmation and impurity profiling, complementing single-quadrupole intact-mass workflows.
• Improved native and semi-native LC/MS conditions enabling preservation of higher-order structures and noncovalent assemblies for more comprehensive structural characterization.
• Optimized ion-pairing and volatile buffering strategies (or ion-pair alternatives) to balance chromatographic performance with MS sensitivity and instrument cleanliness.
• Automation of sequence library management and batch processing in analysis software to increase throughput for large oligonucleotide portfolios.
• Regulatory harmonization of mass-based QC approaches for oligonucleotide therapeutics and broader use in release testing and comparability studies.

Conclusion:
The reported ion-pair reversed-phase LC/MS workflow using the LCMS-2050 single-quadrupole system and LabSolutions Insight Biologics software provides an effective approach for confirming molecular weights and assessing duplex versus single-strand states of GalNAc-siRNA conjugates. Temperature-controlled chromatography enables straightforward discrimination between non-denatured duplexes and denatured single strands. Single-quadrupole systems offer practical advantages for routine QC, while higher-resolution MS remains necessary for in-depth sequence and fragmentation analysis.

References:

1. Nakazono J. Ion-Pair Reversed-Phase LC/MS Analysis of GalNAc-siRNA Conjugates under Denaturing and Non-Denaturing Conditions. Shimadzu Application News 01-01177-EN, Apr 2026.
2. Characterization of GalNAc-siRNA Conjugates Using a Quadrupole Time-of-Flight Mass Spectrometer. Shimadzu Application News No. 01-01176-EN.
3. Reversed-Phase Ion-Pair LC/MS Analysis of siRNA under Denaturing and Non-Denaturing Conditions. Shimadzu Application News No. 01-00915-EN.
4. Efficient Method Development for Separation of Capped mRNA Fragments. Shimadzu Application News No. 01-00898-EN.
5. Acknowledgment: Research supported by AMED grants JP21ae0121022, JP21ae0121023, and JP21ae0121024 (Project leader: Satoshi Obika).