Comprehensive LC-MS Analytical Assays for Antibody-Oligonucleotide Conjugate (AOC) and siRNA Linker-Payload Characterization

Significance of the topic

Analytical characterization of antibody-oligonucleotide conjugates (AOCs) is critical for the development of targeted biotherapeutics that combine the specificity of monoclonal antibodies with the functional diversity of oligonucleotide payloads. Rigorous assessment of conjugation efficiency, payload integrity, and antibody quality underpins safety, efficacy, and regulatory compliance in research and manufacturing.

Study objectives and overview

This study presents a comprehensive LC-MS workflow designed to characterize AOCs and siRNA linkers using orthogonal techniques. Key goals include mass confirmation of free antibody and siRNA starting materials, verification of linker incorporation on the sense strand, and precise determination of oligo-to-antibody ratio (OAR) for AOC samples targeting OAR values of 1 and 2.

Methodology and instrumentation

Five distinct LC-MS methods were applied on the BioAccord System (m/z 400–9000) to address diverse analytical needs:

• RPLC-MS (denaturing) using ACQUITY Premier BEH C4 Column for free mAb mass confirmation.
• Native SEC-MS on ACQUITY Protein BEH SEC 200 Å for intact mAb and AOC analysis.
• Native SCX-MS with BioResolve SCX mAb Column for charge-variant profiling and OAR assessment.
• HILIC-MS (denaturing and non-denaturing) on ACQUITY Glycoprotein BEH Amide for siRNA and siRNA-MCC confirmation.
• Ion-Pair Reversed-Phase (IPRP) MS on ACQUITY Premier Oligonucleotide BEH C18 for complementary confirmation of siRNA payloads.

Data acquisition was performed in UNIFI App, with processing in UNIFI or INTACT Mass App v1.9 enabling automated OAR calculation and compliance-ready reporting.

Main results and discussion

All five LC-MS assays delivered high-quality chromatographic separation and mass accuracy within 15 ppm of expected values. RPLC-MS and SEC-MS provided orthogonal confirmation of free mAb mass. HILIC-MS and IPRP-MS demonstrated consistent retention time shifts and mass evidence of MCC linker incorporation on the siRNA sense strand. SEC-MS and SCX-MS OAR values aligned closely with SEC-MALS data, confirming the robustness of OAR determination across platforms.

Benefits and practical applications

The integrated workflow offers laboratories:

• Rapid and reliable OAR measurements using multiple orthogonal LC-MS techniques.
• Automated data processing and reporting through user-friendly informatics tools.
• Flexible implementation from early discovery through process development and QC.

Future trends and opportunities

Advances in high-resolution native MS, real-time monitoring, and expanded multi-attribute methods will further streamline bioconjugate characterization. Integration with online analytics and machine-learning–driven data interpretation promises accelerated process optimization and enhanced product quality assurance.

Conclusion

This work establishes a versatile and comprehensive LC-MS platform for AOC and siRNA linker-payload characterization, delivering consistent mass confirmation, precise OAR determination, and robust analytical confidence across orthogonal techniques.

References

1. Dovgan I et al. Antibody-Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents. Bioconjugate Chem. 30, 2483–2501 (2019).
2. Jiao J et al. Overcoming limitations and advancing the therapeutic potential of antibody-oligonucleotide conjugates. Pharmacol Res. 209, 107469 (2024).
3. Shion H et al. Analysis of Antibody siRNA Conjugate Using BioAccord System. Waters Application Note 720007212 (2021).
4. Ippoliti S et al. Online IEX-MS of mAb Charge Variants Using BioResolve SCX mAb Column. Waters Application Note 720006672EN (2019).
5. Yogendrarajah P et al. Analysis of siRNA with Denaturing and Non-Denaturing Ion-Pair Reversed-Phase Methods. LCGC North America. 41:2 (2023).
6. Finny A et al. Reproducible Hydrophilic Interaction Chromatography for Denaturing and Non-Denaturing Analyses of Oligonucleotides. J Chrom A. 1733, 465285 (2024).
7. Doneanu C et al. LC-MS Analysis of siRNA, Single Guide RNA and Impurities Using BioAccord System. Waters Application Note 720007546 (2022).
8. Ippoliti S et al. BioAccord LC-MS System Enhancements for Improved Native MS Analysis. Waters Application Note 720008813EN (2025).
9. Ippoliti S et al. The INTACT Mass Application in waters_connect Platform Streamlines ADC DAR and Drug Distribution Analysis. Waters Application Note 720008818EN (2025).
10. Brandenburg C et al. Determination of Multiple Quality Attributes of Antibody-Oligonucleotide Conjugate with SEC-MALS and AEX-MALS. Waters-Wyatt White Paper (2024).