Key takeaways from this article:
- AOCs should not be viewed simply as ADCs with an alternative payload; the physicochemical properties of oligonucleotides introduce distinct analytical challenges and require a differentiated characterization strategy.
- Although UV-Vis spectroscopy and SEC remain useful within the analytical workflow, IEX, CE, and HRMS form the core analytical platform required for robust AOC characterization.
- The quality of the starting oligonucleotide defines the baseline impurity burden of the final conjugate, making upstream characterization essential before conjugation begins.
- Conjugation efficiency alone is not a sufficient indicator of product suitability; linker stability and overall conjugate integrity should be assessed directly.
- Early definition of CQAs is essential to ensure that analytical data generated during development can support later-stage regulatory submissions.
Antibody-oligonucleotide conjugates (AOCs) combine antibody-mediated targeting with an oligonucleotide designed to modulate gene expression (1). At first glance, AOCs may look like antibody-drug conjugates (ADCs) but with a different type of payload, leading to the assumption that analytical strategies established for ADCs characterization would be directly applicable to AOCs. In contrast to small-molecule payloads though, the phosphate backbone of oligonucleotides carry a negative charge, and present synthesis and sequence-related impurities, secondary structure, and enzymatic vulnerability. As a result, the analytical strategy to characterize AOCs cannot be transferred directly from conventional ADC paradigms.
Why do AOCs need a dedicated analytical strategy?
The three components of AOCs – the antibody, oligonucleotide, and linker – contribute a distinct set of critical quality attributes (CQAs) that must be evaluated within an integrated framework. As such, AOCs require a dedicated analytical strategy.
The antibody contributes target-binding specificity, Fc-related considerations, and the manufacturing constraints associated with biologic modalities.
The oligonucleotide introduces additional complexity through sequence-dependent properties, backbone chemistry, charge, molecular mass, and susceptibility to degradation. The linker is likewise an active determinant of product quality, with the potential to influence target binding, payload integrity, molecular stability, and impurity generation.
For that reason, CQAs should be defined early and aligned with analytical methods capable of resolving them with sufficient specificity to inform construct selection, process development, and eventual regulatory justification. When method development is deferred until later stages, minor analytical uncertainties can escalate into significant development delays.
How does oligonucleotide chemistry increase analytical complexity?
AOC analytical complexity arises primarily from two features that are not encountered to the same extent in ADCs: the generation of chemistry-driven product heterogeneity as oligonucleotides typically come as mixtures of diastereomers, and can include truncated sequences and sequence variants, and the physicochemical incompatibility that can emerge between the oligonucleotide and the antibody scaffold. The high anionic charge density of the oligonucleotide, together with its potential to form non-covalent intra- and intermolecular assemblies, can perturb charge-variant profiles and complicate intact-mass analysis. Consequently, ion-exchange chromatography (IEX) and high-resolution mass spectrometry (HRMS) workflows frequently require AOC-specific optimization rather than direct transfer from established ADC methods.
What do phosphorothioate modifications add to the product profile?
Phosphorothioate (PS) bonds increase the resistance of oligonucleotides to nuclease cleavage and are widely used to improve oligonucleotide stability; however, each PS linkage creates a chiral center, generating complex mixtures of diastereomers. Diastereomers can differ in their binding properties, biological activity, pharmacokinetics, enzymatic susceptibility, and, potentially, toxicity profile (2, 3). Accordingly, sequence confirmation alone is insufficient to establish molecular comparability. Control of PS stereochemical composition, or at minimum an understanding of its contribution to product heterogeneity, is important to maintain consistent biological activity.
Why does secondary structure matter for analytics?
Oligonucleotides can adopt conformations that materially affect both analytical behaviour and biological activity. Non-covalent interactions including hydrogen bonding, base stacking, and electrostatic forces govern molecular folding and structural stability.
Secondary structures, such as hairpins and other intramolecular folds, may form depending on sequence composition, ionic environment, and buffer conditions. These conformations can attenuate biological activity, even when the primary sequence is correct.
Accordingly, sequence design, formulation development, and analytical characterization should be approached in an integrated, rather than sequential, manner. A formulation optimized for antibody stability, but permissive of unfavorable oligonucleotide self-association, may result in a conjugate with compromised functional performance.
Are standard ADC analytical methods enough for AOCs?
Traditional ADC analytical methods are necessary but not sufficient for the characterization of AOCs. While UV–Vis spectroscopy and size‑exclusion chromatography (SEC) are valuable for assessing concentration, aggregation, and overall profile, they do not address the oligonucleotide‑specific attributes that ultimately determine whether an AOC is fully characterized.
Which advanced analytical technologies are needed?
Three techniques are particularly valuable in this context:
- AEX enables the resolution of charge-based heterogeneity
- Capillary electrophoresis (CE) provides high-resolution separation based on both size and charge
- High-resolution mass spectrometry (HRMS) particularly when coupled with IEX or SEC under native MS conditions, supports identity confirmation, impurity assignment, and assessment of the complete AOC assembly, including oligonucleotide-to-antibody ratios and non-covalent complexes
In combination, these approaches dissect charge variants, sequence-related impurities, conjugation heterogeneity, and product-related species that would be missed by any single technique. Although SEC remains useful, it should be considered part of a broader analytical panel rather than a standalone decision tool. A clean SEC profile does not provide insight into diastereomeric composition or oligonucleotide impurities, or whether the conjugate exhibits the intended payload distribution. Relying on a single method is often what leads to extensive re-characterization at the IND stage.
How does impurity profiling guide AOC development?
Impurity profiling determines whether the oligonucleotide input, conjugation reaction, and purification process can produce a controllable product, and where each one fails.
Solid-phase oligonucleotide synthesis can generate a range of impurities. These include truncated sequences, missed incorporations, incomplete deprotection products, diastereomers, and self-hybridized species. Some of these impurities can co-elute with the target oligonucleotide, thereby establishing the baseline impurity burden carried into downstream conjugation. If such species enter the conjugation reaction, they can propagate into the final conjugate and materially increase the burden placed on subsequent purification steps.
What do orthogonal methods reveal that single assays hide?
Orthogonal methods reveal the different attributes of the molecule while single assays often fail to detect heterogeneity. When IEX, CE, and HRMS provide consistent results, confidence in the characterization increases. Conversely, when their outputs diverge, this is equally informative – such discrepancies often reveal underlying heterogeneity that a single method would not detect. This has important regulatory implications as health authorities are placing greater emphasis on the characterization of oligonucleotide-related impurities, particularly those that may impact safety or biological function, such as diastereomeric variants and co-eluting species (4–6). As a result, analytical methods must be robust enough to support both internal development decisions and the regulatory submissions that depend on them.
What does conjugation analytics actually need to measure?
Conjugation analytics need to demonstrate that the oligonucleotide is attached at the intended conjugation sites, and that bioconjugation did not compromise antibody binding, oligonucleotide integrity, or stability.
Why isn’t conjugation efficiency enough on its own?
A high conjugation yield does not, in isolation, ensure a viable or high-quality candidate. Instability of the linker, increased aggregation, alterations in the antibody binding profile, or loss of functional integrity for the conjugated oligonucleotide can all compromise product performance despite an apparently favorable yield. Accordingly, yield should be viewed as a necessary but insufficient metric.
The analytical strategy must instead address a set of interrelated questions in parallel: whether the intended conjugate has formed as designed, whether the species distribution is consistent and controlled, whether aggregation has increased, whether antibody binding functionality is retained, and whether the oligonucleotide component remains structurally and functionally intact.
How does linker choice change the analytical risk profile?
Linker design fundamentally determines both the nature and timing of the attributes that must be characterized (7). Cleavable linkers are engineered to release the oligonucleotide under defined intracellular conditions, typically through enzymatic processing following cellular uptake, whereas non‑cleavable linkers prioritize plasma stability and rely on downstream intracellular degradation pathways – such as endolysosomal processing of the antibody – to generate the active oligonucleotide species, often retaining residual linker and amino acid fragments. Consequently, linker structure, length, and chemistry directly influence both stability and biological activity. In the context of AOCs, the presence of a highly negatively charged oligonucleotide and a comparatively large protein scaffold introduces significant electrostatic and steric constraints, the impact of which must be empirically characterized rather than inferred, as they can materially affect conjugate behavior, stability, and performance.
What’s the issue with maleimide-thiol chemistry in AOCs?
Maleimide-thiol chemistry remains a practical and widely used conjugation approach, but its applicability in AOCs must be supported by construct-specific stability data. Thiosuccinimide linkages can undergo retro-Michael addition and related deconjugation pathways, depending on linker architecture and conjugation site (8). Conversely, succinimide-ring hydrolysis can stabilize certain maleimide-derived conjugates, and apparently similar chemistries may exhibit markedly different stability profiles depending on molecular context (9). For that reason, linker stability should be established experimentally rather than inferred from conjugation efficiency alone. Alternative disulfide-rebridging strategies, such as ThioBridge®, may offer advantages where tighter control of conjugation site and product distribution is required, but those benefits should be demonstrated for the specific AOC architecture under evaluation.
How should AOC purification and formulation decisions be made?
AOC purification and formulation decisions should be made against product quality, material recovery, stability, and function together.
What trade-offs come with additional purification steps?
Yield is typically a primary consideration in purification development; however, oligonucleotide-derived species exhibit behavior that is markedly different from small‑molecule payload impurities. As a result, additional purification steps – such as anion-exchange chromatography (AEX) – are often required, each contributing to cumulative material loss. The key question is whether the process can deliver the required product quality at a scale compatible with manufacturing.
A purification method that performs adequately at a small scale but results in significant material losses represents a substantial risk when translated to manufacturing. Conversely, approaches that maximize recovery while failing to adequately resolve impurity species may introduce regulatory concerns. Consequently, purification development must be evaluated holistically, with continuous monitoring of impurity clearance, aggregation, conjugate integrity, residual unconjugated components, and overall recovery at each stage. These interdependent parameters define a set of unavoidable trade-offs that must be carefully balanced.
What should AOC stability studies track?
Degradation, aggregation, conjugate integrity, oligonucleotide structure, and biological activity must all be monitored over time. Formulation development must simultaneously stabilize both the antibody and the oligonucleotide, two components with distinct and often competing liabilities. Oligonucleotides are susceptible to nuclease-mediated degradation and require preservation of structural integrity throughout storage and delivery, whereas antibodies must remain conformationally stable, non‑aggregated, and capable of target binding.
Accordingly, formulation development involves systematic evaluation of parameters such as pH, ionic strength, excipient composition, stabilizing agents, storage conditions, and degradation pathways. Typical formulations may incorporate surfactants, cryoprotectants, buffering systems, and tonicity modifiers. Long-term stability studies are essential to confirm retention of biological activity over the intended shelf life. When instability is observed – manifesting as degradation, aggregation, or loss of activity – the root cause is frequently upstream, arising from linker chemistry, conjugation conditions, purification processes, or oligonucleotide design. As such, formulation should not be relied upon to compensate for deficiencies in molecular design or process development, and analytical data should be used to trace instability back to its origin.
How do analytical datasets support regulatory readiness?
Analytical datasets support regulatory readiness by integrating purity, stability, safety, manufacturability, and functional performance into a single, internally consistent framework that reflects the behavior of the AOC. Regulatory expectations for AOCs remain in evolution, as these molecules combine attributes of biologics, oligonucleotide therapeutics, and conjugated products. While existing oligonucleotide guidance provides a useful foundation, AOC-specific requirements must be substantiated through comprehensive, product-specific data spanning analytical characterization, stability, safety, and manufacturing controls (4–6).
Why define critical quality attributes be defined early?
Early definition of critical quality attributes (CQAs) converts complex analytical observations into actionable development criteria. When established alongside the target product profile (TPP), CQAs provide a framework for linking purity, stability, efficacy, safety, and manufacturability to specific analytical readouts. In that context, the analytical strategy becomes a decision-enabling function rather than a retrospective characterization exercise.
How does Abzena support analytics-led AOC development?
Abzena support analytics-led AOC development by characterizing oligonucleotide inputs supplied by clients or specialist oligonucleotide manufacturers, then connecting those data to conjugation, purification, formulation, stability, and regulatory decisions. The aim is that analytical findings inform process changes and decisions.
Our AOC capabilities include the advanced analytics this work demands: IEX, CE, and HRMS for input characterization and impurity assignment. We support conjugation process development through linker selection, conjugation chemistry, reaction conditions, conjugation efficiency, stability, and antibody binding assessment. Where needed, analytical findings are interpreted alongside bioassay, binding, immunogenicity, and safety data so that impurity and stability profiles link to biological relevance. Regulatory support covers requirements, documentation, and responses to agency queries.
What that produces, across a program, is a dataset that can carry construct selection, impurity control, stability, manufacturability, and regulatory decision-making – and reads consistently across the antibody, the oligonucleotide, and the chemistry that joins them. AOC development is complex, and advanced analytics make it interpretable. That’s the difference between a program that moves forward and one that stalls.
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Frequently Asked Questions
How are AOCs analytically different from ADCs?
The oligonucleotide payload introduces charge-based heterogeneity, sequence-related impurities (truncations, deletions, diastereomers), secondary-structure behavior, and enzymatic-degradation vulnerability – none of which typically appear with small-molecule payloads. Methods routinely used for ADCs (UV-Vis, SEC) stay useful for AOCs but are not sufficient on their own.
Which analytical methods are most useful for AOC characterization?
For oligonucleotide-specific characterization, the working trio is IEX (charge variants), CE (size and charge species), and HRMS (identity and impurity assignment). SEC, UV-Vis, and bioassays support the overall picture but rarely answer the decisive questions in isolation.
What’s the role of CQAs in AOC development?
CQAs convert analytical observations into go/no-go decisions. Defined early, alongside a TPP, they align analytics with construct selection, impurity control, stability strategy, and regulatory submission, rather than treating each as a separate workstream.
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References
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- S. T. Crooke, T. A. Vickers, X. Liang, Phosphorothioate modified oligonucleotide–protein interactions. Nucleic Acids Res. 48, 5235–5253 (2020).
- S. T. Crooke, P. P. Seth, T. A. Vickers, X. Liang, The Interaction of Phosphorothioate-Containing RNA Targeted Drugs with Proteins Is a Critical Determinant of the Therapeutic Effects of These Agents. J. Am. Chem. Soc. 142, 14754–14771 (2020).
- Development and manufacture of oligonucleotides, European Medicines Agency (EMA). https://www.ema.europa.eu/en/development-manufacture-oligonucleotides-scientific-guideline.
- Clinical Pharmacology Considerations for the Development of Oligonucleotide Therapeutics, Food and Drug Administration (FDA). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/clinical-pharmacology-considerations-development-oligonucleotide-therapeutics.
- Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics, Food and Drug Administration (FDA). https://www.fda.gov/media/183496/download.
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- Y. Wang, F. Xie, L. Liu, X. Xu, S. Fan, W. Zhong, X. Zhou, Development of applicable thiol-linked antibody–drug conjugates with improved stability and therapeutic index. Drug Delivery 29, 754–766 (2022).
- M. Lahnsteiner, A. Kastner, J. Mayr, A. Roller, B. K. Keppler, C. R. Kowol, Improving the Stability of Maleimide–Thiol Conjugation for Drug Targeting. Chem. A Eur. J. 26, 15867–15870 (2020).
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