Expanding the Antibody-Oligo Conjugate (AOC) Characterization Toolbox: Part 1- OAR Analysis and Conjugation Process Monitoring

Significance of the Topic

Antibody-oligonucleotide conjugates (AOCs) represent a cutting-edge class of biotherapeutics that leverage the targeting specificity of monoclonal antibodies and the gene-silencing potential of oligonucleotides. Accurate determination of the oligonucleotide-to-antibody ratio (OAR) is critical to ensure batch-to-batch consistency, therapeutic efficacy, and stability during development and commercialization.

Objective and Study Overview

This study aims to expand the analytical toolbox for AOC characterization by implementing non-denaturing liquid chromatography–mass spectrometry (LC-MS) techniques. Specifically, size-exclusion chromatography–mass spectrometry (SEC-MS) and strong cation exchange–mass spectrometry (SCX-MS) methods were evaluated on the BioAccord LC-MS System to monitor conjugation progress and accurately calculate OAR values.

Methodology and Used Instrumentation

Sample Preparation and Conjugates

• Free IgG1 antibody and two AOC samples (OAR 1.0 and OAR 2.0 by prior SEC-MALS/AEX-MALS) were prepared in ammonium acetate buffers for non-denaturing analysis.
• Denaturing reversed-phase LC-MS was used to validate free antibody mass.

Chromatography and Mass Spectrometry

• RPLC-MS: ACQUITY Premier BEH C4 column, 0.1% formic acid mobile phases.
• SEC-MS: ACQUITY Protein BEH SEC column, 50 mM ammonium acetate isocratic flow.
• SCX-MS: BioResolve SCX mAb column, pH and salt gradient with IonHance CX-MS concentrates.
• Detection: ACQUITY RDa detector in positive ESI, full scan 400–7000 m/z.
• Data Processing: UNIFI Application with INTACT Mass App for deconvolution and automated OAR calculation.

Main Results and Discussion

Free antibody mass determined by RPLC-MS and SEC-MS matched the theoretical value and revealed distinct charge envelopes under denaturing versus non-denaturing conditions. SEC-MS of AOCs showed clear mass shifts of ~14.8 kDa and ~29.7 kDa for single and double conjugation, respectively. Automated deconvolution yielded OAR values of 0.93 and 1.94 for Sample 1 and Sample 2, in close agreement with SEC-MALS/AEX-MALS data. SCX-MS provided equivalent mass characterization and allowed OAR determination through UV or extracted ion chromatogram integration. Orthogonal SEC-MALS analysis further demonstrated improved resolution for high-molecular-weight species and aggregated variants.

Benefits and Practical Applications

• Orthogonal non-denaturing workflows on a benchtop time-of-flight LC-MS platform.
• High-resolution MS detection offers precise OAR calculation and glycoform resolution.
• Robust, MS-compatible mobile phases simplify method transfer between SEC and SCX assays.
• Automated data processing accelerates conjugation process optimization and quality control.

Future Trends and Applications

Advancements may include integration of site-specific conjugation mapping, real-time process monitoring, coupling with ion mobility for structural insights, and expansion to alternative payloads such as peptides or small molecules. Enhanced informatics and machine learning could further streamline attribute monitoring and predict conjugation outcomes.

Conclusion

Non-denaturing SEC-MS and SCX-MS methods on the BioAccord LC-MS System provide reliable, high-resolution characterization of AOCs, enabling accurate OAR determination and process monitoring. These approaches complement light scattering techniques and support robust development of next-generation biotherapeutics.

Reference

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