Expanding the Antibody-Oligo Conjugate (AOC) Characterization Toolbox: Part 2- Denaturing and Non-denaturing Analysis of siRNA Payload

Importance of the Topic

siRNA conjugated to antibodies improves targeted delivery of gene silencing therapeutics. Reliable characterization of both free and linker-functionalized siRNA building blocks is crucial for quality control and drug development. Robust analytical workflows enable impurity tracking and ensure precise stoichiometry, reducing risk in AOC manufacturing.

Objectives and Study Overview

This study evaluates denaturing and non-denaturing HILIC-MS alongside denaturing ion-pairing RP-MS (IPRP-MS) for the analysis of a model siRNA (siCOL1a1) and its linker-conjugated derivative (siCOL1a1-MCC) using the BioAccord LC-MS platform. The goal is to compare chromatographic modes and validate a streamlined workflow for comprehensive payload characterization.

Methodology and Sample Preparation

• Sample Types: Free siCOL1a1 and MCC-linker-functionalized siCOL1a1 at 5 µM.
• Chromatographic Modes:
  • HILIC-MS: Denaturing at 90 °C; non-denaturing at 35 °C; mobile phases based on ammonium acetate buffer and acetonitrile.
  • IPRP-MS: Denaturing at 80 °C; HFIP/TEA-based aqueous and methanolic mobile phases.
• Data Acquisition: Electrospray negative mode (m/z 400–7000), with intelligent data capture for MS/MS fragmentation.

Used Instrumentation

• LC System: ACQUITY Premier UPLC with TUV detector.
• Mass Detector: ACQUITY RDa TOF.
• Columns: BEH Amide HILIC and BEH C18 IPRP oligonucleotide columns.
• Informatics: waters_connect UNIFI with INTACT Mass Application and CONFIRM Sequence App.

Main Results and Discussion

• HILIC-MS Non-denaturing vs Denaturing: The siRNA duplex remained intact at 35 °C and dissociated into sense/antisense strands at 90 °C, matching expected masses within 2–15 ppm.
• Linker Conjugation: HILIC-MS and IPRP-MS confirmed selective attachment of the MCC linker to the 3′ end of the sense strand, revealing a +219 Da mass shift and detecting a hydrolysis impurity (+18 Da).
• IPRP-MS Orthogonality: Denaturing IPRP-MS separated strands in reverse elution order, providing complementary confirmation of species and retention behaviors.
• Data Processing: INTACT Mass deconvolution and CONFIRM Sequence mapping achieved >78% sequence coverage and accurate modification assignment.

Benefits and Practical Applications

• Single-platform analysis of both free siRNA building blocks and intact AOC molecules enhances lab efficiency.
• Orthogonal chromatographic techniques support impurity profiling and confirmation of conjugation sites.
• Use of less toxic HILIC reagents simplifies instrument maintenance and reduces environmental impact.
• Integrated informatics accelerates workflow, supporting compliance and regulatory readiness.

Future Trends and Potential Applications

Antibody-oligonucleotide conjugates will continue to evolve, requiring advanced MS-based assays and informatics. Future developments may include real-time monitoring of conjugation reactions, higher-throughput HILIC-MS protocols, and deeper integration with AI-driven data analysis to further streamline therapeutic development.

Conclusion

Denaturing and non-denaturing HILIC-MS combined with IPRP-MS on the BioAccord system delivers a comprehensive, orthogonal strategy for siRNA payload characterization, supporting robust AOC development and quality control.

References

1. Finny A, Johnston T, Sauner B et al. Reproducible HILIC for Denaturing and Non-Denaturing Analyses of Oligonucleotides. Waters Application Note, 2024.
2. Yogendrarajah P et al. Analysis of siRNA with Denaturing and Non-Denaturing IPRP Methods. LCGC North America, 2023.
3. Doneanu C et al. LC-MS Analysis of siRNA and Impurities Using BioAccord. Waters Application Note, 2022.
4. Shion H et al. Analysis of Antibody siRNA Conjugate Using BioAccord System. Waters Application Note, 2021.
5. Brandenburg C, Liu H, Kenrick S. Determination of Quality Attributes of AOC with SEC-MALS and AEX-MALS. Wyatt White Paper, 2024.
6. Gilar M et al. Impact of Mobile and Stationary Phases on siRNA Duplex Stability. J Chrom A, 2024.