Expanding the Antibody-Oligo Conjugate (AOC) Characterization Toolbox: Part 3- Partial and Complete AOC Conjugation Site Determination

Importance of the Topic

AOCs combine the high specificity of monoclonal antibodies with the gene-modulating capacity of oligonucleotides, offering a modular platform for targeted immunotherapies and oncology interventions. Precise determination of conjugation sites is essential to control product homogeneity, optimize biological activity, and meet regulatory standards for emerging biotherapeutic modalities.

Objectives and Study Overview

This third installment builds upon intact-mass and siRNA component analyses to establish complementary strategies for mapping partial and complete conjugation sites in AOCs. Two model AOCs with oligonucleotide-to-antibody ratios (OAR) of 1 and 2 were evaluated to demonstrate site localization at the subunit and peptide levels.

Methodology and Instrumentation

Subunit Analysis:

• FabRICATOR™ digestion of IgG1 into Fc and (Fd’+LC)2 fragments
• SCX-MS for charge-variant separation and mass assignment of conjugated subunits
• SEC-MS for orthogonal size-based profiling of subunit species

Peptide Mapping Workflow:

• Nucleoside digestion to cleave oligonucleotide backbones, retaining linker-tail adducts
• Denaturation, reduction (DTT), alkylation (IAM), and dilution
• RapiZyme™ trypsin digestion under optimized miscleavage conditions
• RPLC-MS analysis with positive-mode ESI; MSE and DDA fragmentation for site confirmation

Used Instrumentation

• BioAccord™ LC-MS System
• Xevo™ G3 QToF Mass Spectrometer
• ACQUITY™ Premier UPLC System
• BioResolve™ SCX mAb Column and ACQUITY UPLC Protein BEH SEC Column
• ACQUITY Premier Peptide CSH C18 Column

Key Findings and Discussion

• SCX-MS and SEC-MS localized conjugation exclusively to the Fab region above the hinge, indicating disulfide disruption at hinge cysteines.
• Nucleoside digestion enabled conversion of full oligo to a residual linker moiety, compatible with RPLC-MS peptide mapping.
• CID fragmentation of HC T16–17 peptides pinpointed primary conjugation at Cys222 and additional labeling at hinge-region cysteines.
• Quantitative XIC integration revealed site-specific occupancy ranging 10–17% (OAR 1) and 18–33% (OAR 2).

Practical Benefits and Applications

• Leverages existing peptide mapping protocols and software for AOC site characterization without bespoke reagents.
• Provides both rapid subunit-level screening and detailed peptide-level confirmation in a unified workflow.
• Supports process optimization, batch release testing, and regulatory filings by delivering site-specific conjugation profiles.

Future Trends and Opportunities

• Integration of automated digest and LC-MS platforms for high-throughput AOC screening.
• Adaptation to diverse linker chemistries and nucleic acid payloads, including single-strand and modified oligonucleotides.
• Application of machine-learning tools for predictive conjugation site modeling and real-time process control.
• Expansion to multi-payload conjugates and bispecific formats for advanced therapeutic designs.

Conclusion

The combined use of subunit SCX-MS/SEC-MS and a novel nucleoside-based peptide mapping workflow represents the first comprehensive approach to localize and quantify oligonucleotide conjugation sites on antibodies. This methodology harmonizes with existing analytical platforms, accelerating AOC development and quality control while ensuring precise product characterization.

References

1. Dovgan I, Koniev O, Kolodych S, Wagner A. 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 (AOCs): Current status and future perspectives. Pharmacological Research. 209, 107469 (2024).
3. Ippoliti S et al. Online IEX-MS of mAb Charge Variants Using a BioResolve SCX mAb Column, IonHance CX-MS pH Concentrates, and BioAccord System. Waters Appl. Note 720006672EN (2019).
4. Brandenburg C, Liu H, Kenrick S. Determination of Multiple Quality Attributes of Antibody-Oligonucleotide Conjugate with SEC-MALS and AEX-MALS. Waters-Wyatt White Paper WP8010 (2024).