Antibody–Oligonucleotide Conjugates, Part 3: Analytical Control

By Robert Dream, consultant to the industry

In the first and second articles of this four-part series on antibody–oligonucleotide conjugates, I covered defining the AOC development plan, including the molecular architecture and the CMC consequence of “hybrid” identity; CMC development: defining the target product profile and clinical quality attributes of AOCs; manufacturing strategy, conjugation chemistry, process development and scale-up; and purification: the central manufacturing challenge. In this third article, I cover analytical control strategy, OAR/DAR measurement, mass spectrometry, capillary electrophoresis, peptide mapping and conjugate site analysis, bioanalytical characterization, potency assays, and stability and formulation.

Analytical Control Strategy

AOC characterization requires an orthogonal analytical toolbox. No single assay is likely to establish identity, purity, OAR, site occupancy, structural integrity, and biological activity.

A practical analytical control strategy can be divided into five levels:

Level 1: Identity

• Protein identity
• Oligonucleotide identity
• Intact AOC identity

Level 2: Purity

• Aggregates
• Fragments
• Free antibody
• Free oligonucleotide
• Process impurities

Level 3: Structural characterization

• Molecular weight
• OAR distribution
• Conjugation site
• Linker integrity
• Oligonucleotide integrity

Level 4: Functional characterization

• Antigen binding
• Receptor binding
• Cellular uptake
• RNA knockdown
• Functional potency

Level 5: Stability and degradation

• Deamidation
• Oxidation
• Fragmentation
• Oligonucleotide degradation
• Linker cleavage
• Aggregation
• Loss of potency

This orthogonal approach is consistent with the general philosophy of ICH Q6B, which emphasizes characterization as the scientific basis for appropriate specifications and control strategies.¹

OAR/DAR Measurement

The OAR is one of the most important AOC attributes. For ADCs, hydrophobic-interaction chromatography is commonly used because increasing payload numbers often produce predictable changes in hydrophobicity. This principle does not necessarily hold for AOCs.

The oligonucleotide's strong negative charge can instead dominate chromatographic behavior. Consequently, methods based on charge differences can be more informative.

Potential approaches include:

• anion exchange chromatography
• cation exchange chromatography
• capillary electrophoresis
• native SEC-MS
• native mass spectrometry
• UV-based calculations
• orthogonal chromatographic methods
• subunit analysis.

Recent work has demonstrated the use of capillary zone electrophoresis–mass spectrometry for resolving AOCs with different OAR values.^2,3,4

Importantly, OAR should ideally be reported as both: average OAR + OAR distribution rather than as a single number.

Mass Spectrometry

Mass spectrometry (MS) is indispensable for AOC characterization but presents unusual challenges. The oligonucleotide introduces substantial negative charge and may alter ionization efficiency. Consequently, a highly conjugated AOC may show a disproportionately weak MS response compared with lower-OAR species.

This creates a major analytical risk: MS signal intensity does not necessarily correspond directly to species abundance.

One study using native SEC–Orbitrap FTMS demonstrated a proteoform integration strategy for estimating OAR distributions, with reported average DAR errors below 10% in the investigated model systems.^5,6,7

Another practical observation is that denaturing MS can be problematic for some antibody–siRNA constructs because disruption of the native complex can make the intact conjugate difficult to observe. Native SEC-MS approaches can therefore be advantageous.⁸

Mass spectrometry is particularly valuable for:

• molecular weight confirmation
• linker characterization
• conjugation site analysis
• oligonucleotide identity
• degradation product identification
• mechanistic characterization.

However, MS should generally be considered an orthogonal technique rather than the sole quantitative release method for OAR.

Capillary Electrophoresis

Capillary electrophoresis offers an attractive alternative because AOC species with different oligonucleotide loading can possess substantially different electrophoretic mobilities.

Potential applications include:

• OAR distribution
• purity
• charge heterogeneity
• fragment analysis
• reduced antibody characterization.

The combination of electrophoretic separation and MS detection is particularly promising because it can simultaneously provide separation and structural identification. Recent literature identifies capillary electrophoresis, ion exchange chromatography, SEC, and MS as complementary analytical approaches for AOCs.²

Peptide Mapping And Conjugation Site Analysis

Site-specific characterization is considerably more complicated for AOCs than for conventional monoclonal antibodies. The attached oligonucleotide is large, highly charged, and chemically heterogeneous. Conventional proteolytic digestion followed by LC-MS/MS may therefore produce incomplete sequence coverage or inefficient detection of modified peptides.

Site mapping remains important because the conjugation site can affect:

• antigen binding
• receptor recognition
• internalization
• PK
• stability
• aggregation
• biological activity.

Advanced workflows may combine:

• enzymatic digestion
• subunit analysis
• native MS
• top-down MS
• middle-down MS
• orthogonal chromatographic separation.

The long-term goal should be quantitative determination of site occupancy and site distribution, not merely confirmation that conjugation occurred.

Bioanalytical Characterization

Characterizing the AOC in biological matrices is a separate challenge from characterizing the purified drug substance.

Following administration, several species may coexist:

• Intact AOC
• Free antibody
• Free oligonucleotide
• Partially degraded AOC
• Linker-containing fragments
• Oligonucleotide metabolites/catabolites
• Antibody catabolites

Measuring only the free oligonucleotide may therefore provide an incomplete understanding of exposure. A recent bioanalytical framework recommends integrated analysis of the antibody, linker, and siRNA components using high-resolution MS together with chromatographic, affinity-based, and enzymatic sample preparation.^9,10 A mature AOC bioanalytical program should ideally distinguish:

Total antibody exposure ≠ intact AOC exposure ≠ total oligonucleotide exposure ≠ pharmacologically active oligonucleotide exposure.

This distinction is essential for establishing meaningful PK/PD relationships.

Potency Assays

An AOC can be chemically intact yet biologically inactive. Therefore, potency testing should reflect the complete mechanism of action. A useful potency cascade may include:

Target binding → receptor-mediated uptake → intracellular release/trafficking → RNA engagement → target knockdown → downstream functional response

A binding assay alone is unlikely to capture all relevant aspects of AOC activity. For an siRNA AOC, for example, a suitable potency assay might combine receptor-dependent cellular uptake with target mRNA or protein knockdown. For an ASO-based AOC, splice correction, target RNA reduction, or another mechanism-specific endpoint may be appropriate. The potency assay should therefore be linked explicitly to the proposed mechanism of action.

Stability And Formulation

AOC formulation introduces another layer of complexity. Potential degradation mechanisms include:

Protein-related

• Aggregation
• Fragmentation
• Oxidation
• Deamidation
• Disulfide scrambling

Oligonucleotide-related

• Backbone degradation
• Nuclease-mediated degradation
• Chemical hydrolysis
• Oxidative damage
• Loss or modification of chemical substituents

Conjugation-related

• Linker hydrolysis
• Deconjugation
• Site instability
• Payload loss

Product-level

• Intermolecular aggregation
• Adsorption
• Concentration-dependent instability
• Container interaction

Formulation development therefore needs to evaluate the entire conjugate rather than optimizing antibody stability and oligonucleotide stability independently.

Important formulation variables may include:

• pH
• ionic strength
• buffer composition
• excipient concentration
• protein concentration
• freeze–thaw exposure
• agitation
• light exposure
• temperature.

Stability-indicating methods should monitor both molecular integrity and biological potency.

Comparability And Process Changes

AOCs are likely to undergo substantial process evolution during development.

Examples include:

• new conjugation chemistry
• different antibody source
• scale-up
• new purification resin
• modified oligonucleotide synthesis
• change in OAR
• site-specific conjugation
• formulation change
• manufacturing site transfer.

Comparability should therefore focus on the attributes most closely associated with biological function.

For AOCs, a useful comparability package may include:

• intact molecular weight
• OAR distribution
• site occupancy
• SEC profile
• charge profile
• free oligonucleotide
• free antibody
• aggregation
• antigen binding
• receptor binding
• cellular uptake
• RNA knockdown
• potency
• stability.

A numerical similarity in average OAR is insufficient if the OAR distribution or conjugation site distribution changes.

In part 4 of this series, I’ll discuss regulatory considerations and future directions.

References:

1. ICH Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. FDA/ICH. Provides principles for characterization, specifications, identity, purity, quantity, and biological activity of biological products and conjugates.
2. Junutula JR, et al. Antibody-Oligonucleotide Conjugates: A Twist to Antibody-Drug Conjugates. Journal of Clinical Medicine. 2021;10(4):838. This review discusses AOC architectures, conjugation approaches, purification, and analytical characterization.
3. Advances in the pharmaceutical development of antibody oligonucleotide conjugates. European Journal of Pharmaceutical Sciences. 2025;215:107292.
4. Antibody-oligonucleotide conjugates: an emerging modality for precision RNA therapeutics. 2026 review discussing AOC development, site-specific conjugation, OAR, bioanalysis, manufacturing, and future challenges.
5. Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody-Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry. Analytical Chemistry. 2021. This work describes native SEC-Orbitrap FTMS for estimating AOC DAR/OAR distributions.
6. Bioanalytical approaches to support the development of antibody-oligonucleotide conjugate (AOC) therapeutic proteins. Xenobiotica. 2024;54(8). The paper describes integrated HRMS, chromatographic, affinity, and enzymatic strategies for AOC ADME characterization.
7. Bioanalytical Methods and Strategic Perspectives Addressing the Rising Complexity of Novel Bioconjugates and Delivery Routes for Biotherapeutics. 2022. Provides broader analytical and bioanalytical considerations for AOCs and other complex bioconjugates.
8. Protocol to generate, purify, and analyze antibody-oligonucleotide conjugates from off-the-shelf antibodies. 2024. Provides practical experimental approaches for AOC preparation and native SEC-MS characterization.
9. Structure–Activity Relationship of Antibody–Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody–siRNA Conjugates for Drug Development. Journal of Medicinal Chemistry. 2024.
10. Structure–Activity Relationship of Antibody–Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody–Phosphorodiamidate Morpholino Oligomer Conjugates for Drug Development. 2024.

About The Author:

Robert Dream is a recognized industry leader with over 35 years of experience in the life sciences sector, including executive leadership roles. He has successfully led projects, optimized processes, and scaled products by leveraging operational excellence and deep technological expertise. Business-minded and strategically focused, Dream brings functional knowledge across manufacturing, supply chain, and regulatory domains. His background includes extensive hands-on and senior executive experience in therapeutic biotechnology and biological product manufacturing at world-leading organizations. A prolific contributor to the industry, Dream has authored numerous articles, industry guidances, and delivered many presentations.