Optimising the Engineering Design of ADCs: Mechanisms of Action, Coupling Chemistry and Other Considerations

With the advancement of new drug R&D, conjugate drugs have become a rapidly evolving direction in therapeutic development. Leveraging rational molecular design strategies, they optimize therapeutic regimens to improve clinical outcomes for patients. Conjugate drugs are constructed by combining biological and/or chemically synthesized components to achieve predefined therapeutic goals. Among them, antibody-drug conjugates (ADCs) represent a highly promising class of cutting-edge biopharmaceuticals. ADCs deliver potent cytotoxic agents or immunomodulators precisely to target cells and have attracted extensive attention in oncology. By covalently linking a monoclonal antibody (mAb) to a synthetic payload via a specific linker, ADCs leverage targeted delivery to enhance therapeutic potency while reducing off-target adverse reactions. This article describes the fundamental principles of ADC drug delivery and outlines the critical quality considerations that must be addressed during ADC development.

Introduction

Progress in pharmaceutical R&D has driven the continuous advancement of conjugate drugs, opening new avenues for optimized disease treatment. These therapeutics integrate diverse biological or chemical moieties to attain intended therapeutic effects. Covalent molecular conjugation enables selective targeting of specific cells and tissues, boosting efficacy while minimizing adverse events. The concept of targeted drug delivery was first proposed by Paul Ehrlich in the early 20th century, known as the classic “magic bullet” hypothesis, which envisioned drugs capable of reaching predefined cellular targets. As modern pharmaceutical technologies mature, the portfolio of conjugate drugs continues to expand. Researchers combine antibodies, polymers, liposomes and various bioactive molecules with pharmacophores to continuously explore new approaches for targeted delivery.

Antibody-drug conjugates (ADCs) constitute an innovative therapeutic modality designed to direct potent cytotoxic or immunomodulatory agents toward target cells, predominantly for the treatment of malignant tumors. As of June 2023, the U.S. Food and Drug Administration (FDA) had approved 11 ADC products, offering new treatment options for hematologic and solid malignancies including acute myeloid leukemia, lymphomas, breast cancer, gastric/gastroesophageal junction cancer, lung cancer, urothelial carcinoma, cervical cancer and ovarian cancer. Compared with conventional monoclonal antibodies or small-molecule therapies, ADCs have two core advantages:

1. Precise targeting: ADCs specifically recognize cells, tissues or receptors bearing matching biomarkers, making them particularly suitable for tumors with well-defined biomarkers.

2. Improved safety profile: Active payloads are delivered selectively to lesion sites, limiting non-specific damage to normal tissues and lowering the risk of adverse events.

A robust, traceable bioprocess platform underpins early-stage process development and quality studies for ADC programs. From strain revival and expansion, recombinant antibody expression to drug substance formulation, benchtop microbial fermenters, glass and stainless-steel bioreactors, and single-use bag bioreactor systems support two mainstream antibody expression routes: prokaryotic microbial expression and mammalian cell culture. Complementary tangential flow filtration (TFF) systems enable concentration of protein intermediates, buffer exchange and impurity clearance. Lyophilization systems provide an optional formulation strategy for formulation stability studies. These tools consistently deliver antibody intermediates meeting quality specifications, supplying reliable starting materials for subsequent conjugation, polishing purification and systematic evaluation of ADC critical quality attributes.

Characterization is a core component of ADC development, requiring systematic analysis of antibodies, synthetic payloads and final conjugated products. The unique molecular architecture of an ADC — a monoclonal antibody covalently bound to a drug payload via a linker (Figure 34.1) — dictates that its quality control framework must incorporate quality principles for both biological macromolecules and small-molecule drugs. Characterization activities encompass purity control to monitor unconjugated species, partially conjugated species, degradation products, process-related aggregates and fragments. Analytical methods must be sensitive and specific, covering monoclonal antibody intermediates, payloads, ADC drug substances and final drug products to assess purity, impurities and biological activity. As these novel therapeutics generate encouraging clinical data, understanding and implementing ADC-specific quality controls is essential to guaranteeing safety, efficacy and batch-to-batch consistency.

Figure 1. Schematic of antibody-drug conjugate components.

Combining features of biological macromolecules and synthetic small molecules, ADCs possess high structural complexity. The global CMC regulatory framework for ADCs continues to evolve. Even so, the International Council for Harmonisation (ICH), major pharmacopoeias (United States Pharmacopeia, European Pharmacopoeia), the FDA and EMA evaluate ADCs using risk-based assessment approaches adapted from established biologic and small-molecule control principles. Regulatory reviews focus on conjugation processes, purification strategies and stability studies, with particular attention to byproducts formed during conjugation and impurity risks associated with ADC drug substances and drug products. Intrinsic properties of the antibody intermediate, such as monomer purity and charge heterogeneity, propagate along the process chain into ADC drug substances and drug products and impact quality throughout the shelf life. Risks associated with linkers and payloads, including residual solvents, nitrosamines and degradation products, also require ongoing monitoring. This article discusses key quality considerations in ADC development.

ADC Structure

An ADC comprises three core building blocks: an antibody, a cytotoxic or immunomodulatory payload, and a chemical linker (Figure 34.2). Ideally, an ADC remains intact during systemic circulation until it reaches its target and releases the payload inside tumor cells. Fu et al. reviewed ADC structural design and summarized selection criteria for candidate molecule development.

Figure 2. Schematic of antibody-drug conjugate structure.

Antibody Intermediate

IgG antibodies are the most commonly used scaffolds for ADC development, with IgG1, IgG2, IgG3 and IgG4 subtypes widely adopted. Binding affinity between the antibody and tumor-associated surface antigen directly affects ADC cellular internalization and is a critical parameter governing pharmacological activity. Nevertheless, as described by Singh et al., antibody-antigen affinity requires careful optimization: excessively high affinity can impede antibody penetration deep into solid tumors, requiring a balance between target cell uptake efficiency and anti-tumor potency.

Linkers

The linker acts as a covalent bridge between the antibody and payload and directly governs ADC stability in circulation and therapeutic performance. The ideal linker is stable in systemic circulation and releases payload only within target tumor cells. Developers select cleavable or non-cleavable linkers according to the intended mechanism of action. Hydrazones and disulfides are chemically cleavable linkers, while glucuronide and peptide linkers are enzymatically cleavable; payload release is triggered by hydrolysis or intracellular enzymatic cleavage, respectively. Thioether and maleimidocaproyl linkers represent non-cleavable varieties, which are relatively resistant to chemical and enzymatic degradation. Payload release occurs after proteolytic degradation of the antibody backbone inside cells, releasing payload-bearing amino acid fragments to exert activity.

Payload

The payload is the functional moiety responsible for ADC pharmacologic action. It may exert direct cytotoxicity against tumor cells or function as an immunomodulator to remodel the tumor microenvironment and induce anti-tumor immune responses and immune memory. Frequently used ADC payloads include tubulin inhibitors, DNA-damaging agents, and immunomodulators such as TLR and STING agonists. These cytotoxic compounds are highly potent, and regulators impose strict requirements for containment and safety controls throughout formulation, manufacturing and storage.

Overall Control Strategy for ADCs

ADC control strategies integrate critical quality attributes (CQAs) for parenteral monoclonal antibody products and small-molecule drugs. Physicochemical characteristics of active species, degradation products and impurity levels form the foundation of control strategy design. Quality risks originating from the antibody scaffold are controlled at the antibody intermediate stage, while risks associated with linkers and payloads are managed at the payload intermediate stage. Bechtold-Peters et al. reported an illustrative control strategy for ADC development. Beyond standard physicochemical and microbiological testing for injectable products, ADC drug substances and drug products require quantitative evaluation of the following attributes:

· Size variants and charge variants

· Antigen-specific binding activity

· Functional assays aligned with the mechanism of action

· Drug-to-antibody ratio (DAR)

· Post-translational modifications and drug-related variants such as oxidation

The final control strategy must be supported by process characterization data to verify whether manufacturing parameters (including lyophilization, dilution, filling and other unit operations) affect critical quality attributes. Where process parameters significantly impact CQAs, parallel testing is required for both ADC drug substances and drug products.

Key Aspects of ADC Characterization

ADC characterization follows a sequential workflow from raw materials to finished products: first, separate characterization of antibody and linker-payload intermediates; second, characterization of conjugated ADC drug substance; and finally, characterization of the finished drug product. As independent intermediates, antibodies and linker-payload constructs follow characterization workflows broadly similar to conventional monoclonal antibodies and synthetic small-molecule drugs. As proposed by Wagh et al., the core of ADC quality research lies in detailed characterization of the ADC drug substance:

· Confirmation of predefined payload conjugation sites on the antibody and measurement of the drug-to-antibody ratio (DAR)

· Minimization of unconjugated antibody, free payload and associated degradation products

· Profiling of size variant and charge variant distributions of conjugated species

· Establishment of correlations between molecular activity and mechanism of action

Drug-to-Antibody Ratio (DAR)

Drug-to-antibody ratio (DAR) is defined as the average number of payload molecules covalently attached to a single antibody molecule. Technologies for covalent attachment of payloads to antibodies continue to evolve. Early non-specific conjugation targeting lysine or cysteine residues generates heterogeneous product mixtures, creating challenges for process reproducibility and quality control. Site-specific conjugation technologies use engineered amino acids, unnatural amino acids, glycans or peptide tags to enable site-directed attachment for precise control over DAR distribution.

Multiple novel conjugation platforms have emerged. The microbial transglutaminase-mediated site-specific conjugation reported by Anami et al. uses branched linkers to reproducibly achieve a DAR of 4. Chen and Levengood et al. described designs with dual pharmacophores per linker, compatible with cysteine-based conjugation workflows. Kumar et al. presented a branched linker platform capable of site-specifically generating ADCs with DAR values of 4, 5 and 8. These technological advances expand ADC design space and enable payloads with moderate potency to be considered for ADC applications.

High molecular heterogeneity creates analytical challenges for accurate DAR quantification. A 2021 review by Matsuda summarized three primary DAR measurement modalities: HPLC/UV, LC-QTOF/MS and UV-Vis spectrophotometry. Hydrophobic interaction chromatography (HIC) and reversed-phase HPLC are routine chromatographic approaches, using 280 nm UV peak areas to calculate DAR distributions. LC-QTOF/MS and UV-Vis spectrophotometry serve as orthogonal methods to confirm molecular weights of individual DAR species and complete qualitative and quantitative evidence packages.

Impurities Generated during ADC Manufacturing

Conjugation reactions are designed to generate ADC species with the target DAR, yet reaction mixtures are inherently multi-component systems containing unconjugated antibody, free payload and conjugated byproducts differing in size and charge. Unconjugated antibody competes for antigen binding and reduces ADC potency. Free unconjugated payload cannot undergo targeted internalization; it not only diminishes efficacy but also elevates off-target toxicity risk. Wagh et al. defined key impurity categories requiring quantitative monitoring during ADC development:

· Unconjugated antibody: Equivalent to the DAR = 0 fraction and quantifiable using the same DAR analytical workflows;

· Charge variants: Arise from post-translational modifications and manufacturing conditions including deamidation, oxidation, sialylation and incomplete N/Cterminal processing. They may compromise ADC stability and biological activity and are typically monitored via ion-exchange chromatography (IEX) or capillary isoelectric focusing (cIEF), classified as acidic or basic variants relative to the main peak;

· Size variants: Include ADC aggregates, particles and degradative fragments, as well as antibody fragments conjugated to payloads or antibodies carrying degraded payloads. Owing to the hydrophobicity introduced by linkers and payloads, ADCs have a greater tendency to aggregate than naked antibodies. Fragmentation arises from peptide bond cleavage and carries risks of immunogenicity and altered in vivo pharmacokinetics. Common analytical tools include size-exclusion chromatography (SEC), CE-SDS, multi-angle light scattering coupled with SEC, dynamic light scattering and light obscuration particle counting;

· Free drug-related impurities: Even after purification steps, incomplete conjugation generates free payloads, drug-linker complexes and associated degradants. Additional degradation products may form during storage or systemic circulation, raising safety risks and reducing efficacy. Trace free payloads are commonly quantified by ELISA, reversed-phase HPLC and solid-phase extraction (SPE) coupled detection.

Continuous monitoring of unconjugated antibody, charge variants, size variants and related attributes is required to assess shelf-life purity and ensure safety and efficacy of ADC drug substances and drug products. A thorough understanding of how unit operations affect these quality attributes is a prerequisite for advancing ADC candidates into clinical development and commercial manufacturing.

ADC Potency

ADC potency assessment addresses two core dimensions: the specific binding capacity of the antibody domain to the target antigen, and the ability of the molecule to mediate the intended biological effect. Bechtold-Peters et al. outlined evolving potency testing strategies across development stages. In early research, antibody ELISA binding assays are suitable for candidate screening. At later development stages, cell-based functional cytotoxicity assays must be incorporated into specifications for ADC drug substances and drug products. Cytotoxicity assays represent the gold standard for evaluating in vitro anti-tumor activity, quantifying viable or dead tumor cells post-treatment. Readouts are derived from tetrazole reduction, protease labelling or ATP quantification, reporting cellular metabolic or enzymatic activity. Reduced cell viability correlates inversely with ADC cytotoxic potency.

Conclusion

ADCs represent a promising class of anti-tumor therapeutics, combining the targeting capability of monoclonal antibodies with the activity of cytotoxic or immunomodulatory moieties. They selectively recognize target cells and deliver potent payloads, yet their development pathway presents multiple challenges requiring iterative optimization of antibody scaffold, payload, linker and conjugation process. ADC development is a multidisciplinary effort spanning biology, chemistry, pharmacology and immunology. Despite technical hurdles, multiple approved products and abundant clinical-stage candidates validate the clinical value of this modality. As new technologies and design strategies emerge, ADC molecular design, delivery efficiency and developability will continue to improve. Supported by benchtop microbial fermenters, diversified bioreactor platforms, tangential flow filtration systems and lyophilization infrastructure, research teams can establish a full CMC workflow covering antibody expression, intermediate purification and formulation to efficiently support process development and quality studies for ADC candidates.

Source: S. Agarwal, E. Kikovska-Stojanovska, Chapter 34 - Antibody-drug conjugates. Specification of Drug Substances and Products (Third Edition), 2025.