Guest Column | October 16, 2026

Antibody–Oligonucleotide Conjugates, Part 2: Manufacturing

By Robert Dream, consultant to the industry

In the first article of this four-part article series on antibody–oligonucleotide conjugates, I covered defining the AOC development plan, including the molecular architecture and the CMC consequence of “hybrid” identity, and CMC development: defining the target product profile and clinical quality attributes of AOCs. In part 2 of this four-part series, I cover manufacturing strategy, conjugation chemistry, process development and scale-up, and purification: the central manufacturing challenge.

Manufacturing Strategy

AOC manufacturing can be divided into three major manufacturing streams:

Antibody production → oligonucleotide production → conjugation and purification

These streams may be developed independently initially, but the final process must be designed as an integrated system.

Antibody manufacture

The antibody is typically produced using established recombinant expression platforms followed by conventional downstream purification. The antibody process must deliver an intermediate with appropriate:

purity
monomer content
charge profile
glycosylation
concentration
low levels of host-cell proteins and DNA
low bioburden/endotoxin
defined reactive site availability.

The conjugation process may impose additional requirements on the antibody. For example, reduction-based cysteine conjugation requires control of the disulfide state, whereas glycan-mediated conjugation requires control of glycan processing. Thus, an antibody that is acceptable as a conventional monoclonal antibody is not necessarily the optimal starting material for an AOC.

Oligonucleotide manufacture

Synthetic oligonucleotides are generally produced through iterative chemical synthesis, followed by cleavage/deprotection, purification, concentration, and isolation or formulation.

Important process variables include:

coupling efficiency
capping efficiency
oxidation or sulfurization
protecting-group removal
cleavage conditions
purification conditions
pooling strategy
concentration
storage conditions.

The EMA's current draft guideline specifically recognizes the distinctive manufacturing characteristics of synthetic oligonucleotides and provides recommendations on synthesis, impurities, purification, analytical characterization, specifications, and conjugation.^1,2,3

For AOCs, the oligonucleotide process must additionally deliver a defined conjugation handle or chemically compatible terminus. The design of this handle is itself a CMC consideration because it can affect synthesis yield, purification, conjugation efficiency, stability, and, ultimately, product heterogeneity.

Manufacturing Control Strategy

An integrated manufacturing control strategy for AOCs should encompass control of raw materials, antibody and oligonucleotide starting materials, conjugation, purification, drug substance, and drug product. The control strategy should be designed to ensure consistent product quality, control critical process parameters (CPPs) and critical quality attributes (CQAs), and maintain the intended structure, purity, potency, and stability of the AOC throughout manufacturing.

Raw Materials

Materials required for the manufacturing process
Identity and quality of starting materials
Appropriate specifications and supplier controls
Control of materials that may affect AOC quality or process performance

Antibody Manufacture

Antibody identity
Purity
Aggregation
Charge heterogeneity
Glycosylation
Other product- and process-related attributes relevant to AOC manufacture

Oligonucleotide Manufacture

Sequence identity
Chemical modifications
Purity
Process- and product-related impurities
Reactive group/handle identity and availability
Residual reagents and other relevant impurities

Conjugation

Antibody-to-oligonucleotide stoichiometry
pH
Temperature
Reaction time
Degree of conjugation / oligonucleotide-to-antibody ratio (OAR)
Control of conjugation-related impurities and side reactions

Purification

Removal of free oligonucleotide
Removal of free antibody
Removal or control of aggregates and other high-molecular-weight species
Control of OAR distribution
Removal of process-related impurities
Recovery and consistency of the desired AOC species

Drug Substance

Identity
Purity
OAR
Site occupancy/site-specific conjugation, as applicable
Aggregation and other relevant molecular attributes
Potency
Stability
Residual process-related impurities

Drug Product

Formulation composition
AOC concentration
Particulate control
Sterility and endotoxin control
Container-closure integrity
Stability
Other product-specific quality attributes relevant to the intended route of administration

This integrated control strategy provides a framework for manufacturing AOCs by linking controls across the antibody, oligonucleotide, conjugation, purification, drug substance, and drug product stages. Rather than treating each component as an independent manufacturing program, the strategy establishes interconnected controls to ensure that variability introduced at each stage is appropriately controlled and that the final AOC consistently meets predefined quality requirements.

Conjugation Chemistry

Conjugation is arguably the defining unit operation in AOC manufacturing. Several strategies have been explored.

Lysine conjugation

Lysine residues offer a relatively simple route because antibodies contain multiple accessible amines. The principal drawback is heterogeneity: multiple lysines may react, creating a broad population of conjugation sites and OAR species.

Advantages include:

familiar chemistry
straightforward implementation
potentially high reaction efficiency.

Limitations include:

site heterogeneity
variable OAR
potential effects on antibody binding
more difficult structural characterization.

Cysteine conjugation

Native or engineered cysteine residues can provide greater control than nonspecific lysine chemistry. Controlled reduction of interchain disulfides followed by conjugation can generate defined species, although partial reduction and reoxidation must be carefully controlled.

Engineered cysteine strategies can further improve site specificity.

Glycan-based conjugation

The Fc glycan can be enzymatically or chemically modified to create a defined attachment site. Glycan-based strategies can provide improved site control while preserving antigen-binding regions.

The major CMC considerations include:

glycan heterogeneity
enzymatic reaction efficiency
residual enzymes
site occupancy
structural integrity
scale-up of enzymatic processing.

Site-specific enzymatic or engineered approaches

The direction of the field is increasingly toward site-specific conjugation. Enzymatic approaches, engineered amino acids, and antibody engineering can reduce product heterogeneity and simplify analytical characterization. Recent studies comparing different conjugation sites and chemistries indicate that conjugation location can substantially influence PK and siRNA delivery.^4,5 This creates a strong development argument for considering analytical tractability during molecular design, rather than treating analytics as a downstream problem.

Process Development And Scale-Up

AOC conjugation is often performed under conditions that differ substantially from either antibody purification or oligonucleotide synthesis.

Important process parameters include:

protein concentration
oligonucleotide concentration
molar equivalence
pH
ionic strength
temperature
reaction time
reducing-agent concentration, where applicable
linker concentration
mixing
order of addition
quench conditions.

A key challenge is achieving sufficient conjugation without inducing:

antibody aggregation
fragmentation
oligonucleotide degradation
over-conjugation
antibody binding loss
linker hydrolysis
nonproductive side reactions.

Process development should therefore employ a quality by design framework in which critical process parameters (CPPs) are linked experimentally to CQAs.

ICH Q11³ provides a useful general framework for understanding and developing drug substance manufacturing processes, including identification and control of process-related impurities and establishment of an appropriate process control strategy.^6,7,8 For AOCs, however, the traditional framework needs to be extended because the process simultaneously controls protein and nucleic acid attributes.

Purification: The Central Manufacturing Challenge

Purification may be the most difficult manufacturing step because the product contains two molecular domains with fundamentally different physicochemical properties.

Potential impurities include:

unconjugated antibody
free oligonucleotide
under-conjugated AOC
over-conjugated AOC
aggregated AOC
antibody fragments
modified/free linker
residual conjugation reagents
process enzymes
residual solvents
oligonucleotide truncations.

Traditional protein purification cannot necessarily resolve all these species.

Size-exclusion chromatography

SEC is valuable for:

aggregate measurement
monomer assessment
fragment detection.

However, SEC generally does not provide adequate resolution for all OAR species.

Ion exchange chromatography

Ion exchange approaches can be particularly valuable because the oligonucleotide introduces substantial negative charge. Anion exchange chromatography can therefore separate AOCs according to oligonucleotide loading and charge. Recent experimental studies have used strong anion exchange chromatography for both purification and OAR assessment.^4,9

Affinity chromatography

Protein A remains useful for antibody-based capture, although its ability to separate AOC species is limited. Depending on the conjugation architecture, affinity purification may be useful upstream or downstream of conjugation.

Mixed-mode approaches

Mixed-mode chromatography may ultimately become particularly useful because it can exploit combinations of:

charge
hydrophobicity
size
protein interactions.

The ideal purification train will depend strongly on the AOC architecture.

In part 3 of this series, I’ll delve into analytical control and in part 4 I’ll discuss regulatory considerations and future directions.

References:

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.
Guideline on the Development and Manufacture of Oligonucleotides. European Medicines Agency, draft guideline, 2024. Addresses synthetic oligonucleotide manufacturing, characterization, specifications, analytical control, impurities, and conjugation.
ICH Q11: Development and Manufacture of Drug Substances. International Council for Harmonisation. Provides principles for process understanding, impurity control, and drug-substance manufacturing development.
Structure–Activity Relationship of Antibody–Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody–siRNA Conjugates for Drug Development. Journal of Medicinal Chemistry. 2024.
Structure–Activity Relationship of Antibody–Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody–Phosphorodiamidate Morpholino Oligomer Conjugates for Drug Development. 2024.
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.
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.
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.
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.

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.