Linker and Conjugation Chemistry in ADCs: Strategies and Best Practices

Vascular Nano-Delivery Using Tropism and Targeting
Vladimir R. Muzykantov, MD, PhD | Founding Co-Director, CT3N at UPenn

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Antibody–drug conjugates (ADCs) have become one of the fastest-growing classes of drugs in modern precision oncology. From early products like Mylotarg to widely marketed ADCs today, such as Enhertu, Padcev, and Zynlonta, linkers and conjugation chemistry have been proven to be core factors that determine ADC efficacy, stability, safety, and commercial success.

What is Linker and Conjugation Chemistry in ADC?

In an ADC structure, the linker is responsible for securely and stably connecting the small-molecule drug to the monoclonal antibody, while conjugation chemistry involves the specific chemical methods and reaction strategies used to achieve effective and controlled drug loading. Linkers and conjugation chemistry together define the functional performance of ADCs and their potential for clinical applications.

Fig. 1. Linker and conjugation chemistry in ADCs (BOC Sciences Authorized).

The Role of Linkers in ADC Stability and Selectivity

The choice of linker directly affects ADC stability and selectivity in vivo. An ideal linker should remain stable in the bloodstream to prevent premature drug release while enabling efficient payload release within target cells for therapeutic effect. Cleavable linkers release the payload in response to specific enzymes, acidic conditions, or reducing environments, whereas non-cleavable linkers rely on antibody degradation pathways. The chemical properties, spatial structure, and hydrophobic/hydrophilic modifications of the linker all influence ADC circulation stability and target selectivity.

How Conjugation Chemistry Shapes Drug-Antibody Functionality

Conjugation chemistry determines the attachment sites, conjugation efficiency, and distribution uniformity of the payload. Different conjugation approaches can affect the DAR (drug-to-antibody ratio), antibody conformation, and drug release characteristics. For example, lysine conjugation often results in random labeling, whereas cysteine conjugation via partial reduction of antibody disulfides enables controlled labeling. Enzyme-mediated and bioorthogonal conjugation can achieve highly uniform, site-specific attachment, optimizing both drug activity and safety.

Impact of Linker and Conjugation Chemistry on ADC Performance

The design of linkers and conjugation chemistry directly impacts overall ADC performance, including stability, pharmacokinetics, and targeting efficiency. Proper design can enhance therapeutic efficacy while minimizing off-target toxicity.

Impact on ADC Stability and Selectivity: Plasma stability is a key measure of ADC quality. High-stability linkers combined with controlled conjugation chemistry reduce non-specific drug release in circulation, improving target selectivity and reducing side effects.
Effects on DAR, Homogeneity, and Molecular Architecture: Conjugation strategies directly affect DAR and molecular uniformity. Random conjugation may lead to broad DAR distributions and product heterogeneity, while site-specific conjugation allows precise control of DAR and preserves antibody structural integrity, improving batch-to-batch consistency.
Influence on Pharmacokinetics and Biodistribution: Linker chemistry, drug attachment sites, and conjugation density influence ADC pharmacokinetics and in vivo distribution. Excessive hydrophobicity may cause aggregation and rapid clearance, whereas hydrophilic modifications can improve circulation half-life and target accumulation.
Control of Payload Release and Intracellular Processing: Linker design determines how the drug is released within target cells. Cleavable linkers enable enzymatic, acidic, or reductive-triggered payload release, while non-cleavable linkers rely on antibody degradation to release the active drug, precisely controlling drug exposure and concentration.
Safety and Toxicity Considerations: Linker and conjugation chemistry choices are directly related to ADC safety. Optimized linkers reduce non-specific release in the bloodstream, lowering systemic toxicity, and proper DAR and molecular uniformity help reduce liver and kidney toxicity risks.

Key Types of ADC Linkers and Linker Mechanisms

The selection and mechanistic design of ADC linkers are central to ADC performance. Linkers influence not only ADC stability in the bloodstream but also how efficiently the drug is released within target cells, directly affecting efficacy, safety, and pharmacokinetic profiles. Based on the release mechanism, ADC linkers are generally classified as cleavable, non-cleavable, or self-immolative spacer systems.

Cleavable Linkers: Enzyme-, Acid-, and Redox-Responsive Designs

Cleavable linkers are the most commonly used type in ADC development. Their core advantage lies in triggering drug release under specific stimuli, enabling "on-demand" delivery. This allows precise payload release inside target cells, enhancing antitumor activity while reducing systemic toxicity. Cleavable linkers are mainly categorized into three types:

Enzyme-Cleavable Linkers

These linkers are cleaved by proteases highly expressed in the tumor microenvironment or lysosomes. Examples include Cathepsin B-sensitive linkers (Val-Cit, Phe-Lys), and linkers targeting Cathepsin L, D, or K. Mechanistic features:

Efficient cleavage in lysosome-rich tumor cells.
Low cleavage in healthy tissues, ensuring high safety.
Rapid and abundant drug release.

Representative product: Brentuximab vedotin (Adcetris) uses a Val-Cit linker to release MMAE via Cathepsin B cleavage.

Acid-Cleavable Linkers

These linkers exploit the low-pH environment in tumors or intracellular vesicles to release the payload. Common structures include Hydrazone and Cis-aconityl groups. Mechanistic features:

Accelerated cleavage at pH 6–4.
Suitable for antibody targets that undergo rapid endocytosis and lysosomal trafficking.
May prematurely release in inflamed tissue or acidic plasma regions, requiring careful stability evaluation.

Representative product: Gemtuzumab ozogamicin (Mylotarg) uses an acid-sensitive hydrazone linker to release calicheamicin.

Redox-Responsive Linkers

These linkers release the payload in response to high intracellular glutathione (GSH) or other reducing conditions in tumor cells, commonly featuring disulfide bonds or electronically tuned disulfides. Mechanistic features:

Intracellular GSH in tumor cells is 2–10 times higher than in normal cells, providing selectivity.
Release rate can be tuned through molecular design.
Highly controllable drug release pathways, widely used for potent cytotoxic small molecules.

Non-Cleavable Linkers and Their Advantages in Payload Retention

Non-cleavable linkers are structurally stable and do not break under external stimuli in circulation or within target cells. Payload release typically relies on antibody degradation in lysosomes, known as a "complete degradation release" mechanism. Typical types include:

Thioether linkers (e.g., SMCC).
Highly stable carbon-chain or amide structures.

Advantages and applications:

Extremely high plasma stability: prevents premature drug release and reduces systemic toxicity, ideal for ADCs with long half-lives and strong antibody stability.
Reduced off-target toxicity: payload is only released after complete antibody degradation, enhancing safety.
Defined, controllable release mechanisms: suitable for drugs requiring precise release kinetics, e.g., amine-modified toxins or unstable small molecules.

Representative product: T-DM1 (Kadcyla) uses a non-cleavable SMCC linker to release the Lys-MCC-DM1 metabolite via antibody degradation.

Non-cleavable linkers are suitable for:

Projects requiring maximum stability and minimized off-target effects.
Payloads that do not need rapid burst release.
Treatments with moderate molecular toxicity but requiring high cumulative concentration.

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ADC Linker Functional Groups and Reactivity

The functional groups of a linker directly determine the selectivity, stability, and drug-release behavior of ADC conjugation reactions, making them core elements for constructing high-quality ADC molecules. By carefully selecting and designing typical functional groups such as maleimides, NHS esters, hydrazones, disulfides, and carbamates, ADC structural uniformity, plasma stability, and release kinetics can be significantly optimized.

Common Functional Moieties (Maleimide, NHS Ester, Hydrazone, Disulfide, Carbamate, etc.)

Maleimide-Based Functional Groups – Maleimides are widely used for cysteine conjugation due to their high thiol selectivity, forming thioether linkages via Michael addition with antibody thiols. Advantages include mild reaction conditions, high selectivity, and reliable conjugation efficiency. Attention must be paid to possible "retro-Michael" reactions leading to deconjugation, which can be mitigated by stabilizing modifications such as pyridyl-maleimide.
NHS Esters – NHS esters react with lysine residues on antibodies to form stable amide bonds and are commonly used in lysine conjugation. The reaction is mild and occurs under neutral or slightly basic conditions. Due to the wide distribution of lysine residues, heterogeneity may be high, necessitating reaction optimization during formulation development.
Hydrazide and Aldehyde Functional Groups – Used to form controllable chemical bonds such as hydrazones or acylhydrazones for acid-sensitive release or site-specific conjugation. Hydrazides react with aldehydes to form hydrazone linkages, suitable for acid-sensitive linkers, and can be combined with oxidized glycosylated antibodies for more precise site-selective attachment.
Click Chemistry Functional Groups – Includes azide, alkyne, tetrazine, etc., with high specificity, efficiency, and biocompatibility. CuAAC (copper-catalyzed azide–alkyne cycloaddition) is effective for in vitro conjugation but requires metal residue control. SPAAC (strain-promoted azide–alkyne cycloaddition) is suitable for biological systems, enabling mild and selective conjugation.
Disulfide-Based Functional Groups – Disulfide linkages are commonly used in reduction-sensitive linkers, cleaving rapidly in the high-GSH environment of tumor cells. They can be used to design reductively triggered payload release mechanisms. Stability can be tuned to prevent premature degradation in plasma.

Hydrophilic vs. Hydrophobic Modifications

The hydrophilic or hydrophobic nature of the linker directly affects ADC physicochemical properties and in vivo behavior, including solubility, stability, aggregation tendency, and pharmacokinetics.

Hydrophilic Modifications

Significantly improve solubility and reduce aggregation from hydrophobic interactions.
Enhance plasma stability and circulation time, reducing non-specific tissue binding.
Commonly achieved via PEG, glycosylation, or polar side chains.

Hydrophobic Modifications

Improve payload–antibody binding or membrane penetration.
Excessive hydrophobicity may cause aggregation, faster clearance, or reduced plasma stability, requiring careful structural optimization.
Hydrophilic linker segments are often introduced to balance hydrophobic payloads.

Overall, balancing hydrophilic and hydrophobic properties is critical for ADC stability, reduced immunogenicity, and optimized tissue distribution.

Influence of Functional Groups on Cleavage Kinetics

The chemical mechanism of the functional group determines its cleavage rate in physiological and pathological environments, affecting drug release timing, location, and efficiency.

Acid-sensitive groups (e.g., Hydrazone): Cleave rapidly in acidic lysosomal environments (pH 4–6) for selective tumor-cell payload release.
Redox-sensitive groups (e.g., Disulfide): Cleave in high-GSH tumor cells while remaining stable in plasma, enabling precise release.
Enzyme-sensitive structures (e.g., specific peptides, Carbamate + Self-Immolative Spacer): Cleavage mediated by lysosomal enzymes such as Cathepsin B for high tissue selectivity.
Stable structures (e.g., Carbamate or partially modified Maleimide): Provide high equilibrium stability, releasing the drug mainly after antibody degradation, suitable for delayed-release ADC designs.

Fine-tuning functional groups allows precise control over release rates, tissue specificity, and cellular uptake, a key strategy for optimizing the ADC therapeutic window.

Conjugation Strategies for Precise Payload Attachment

Payload conjugation strategies are critical for ADC uniformity, stability, and efficacy. With the evolution of ADC technology, methods have progressed from early random conjugation to site-specific approaches with controlled DAR and high homogeneity. Different strategies affect not only ADC structural uniformity but also pharmacokinetics, intracellular processing, and overall therapeutic window.

Fig. 2. ADC conjugation strategies (BOC Sciences Authorized).

Lysine Conjugation (Random labeling, high DAR variability)

Lysine conjugation utilizes abundant lysine residues on the antibody surface via reactive groups such as NHS esters to form stable amide bonds. Key features:

Many potential reaction sites (~80–90 lysines), leading to broad DAR distribution.
Simple and cost-effective, used in early ADCs such as T-DM1.
High structural heterogeneity may affect pharmacokinetics and biodistribution.
Still applicable in certain therapeutic contexts, e.g., rapidly turned-over tissue targets.

Lysine conjugation is suitable for early screening or ADCs with low structural uniformity requirements but is gradually being replaced by more precise methods.

Cysteine Conjugation (Partial reduction of disulfide bonds)

Cysteine conjugation exposes thiols by reducing native antibody disulfides and reacts with maleimide or pyridyl-maleimide groups to form thioether bonds.

Advantages:

Fewer potential sites than lysine (typically 8), resulting in higher homogeneity.
DAR can be controlled (commonly 2, 4, or 8).
More controllable than lysine conjugation, with better pharmacokinetics, safety, and stability.

Notes:

Some maleimides may undergo retro-Michael reactions, requiring stabilization.
Reductive control is critical for correct structure and aggregation risk.

Cysteine conjugation is widely used in commercial ADCs and is the mainstream choice for highly stable, homogeneous ADCs.

Enzyme-Mediated Conjugation (e.g., Transglutaminase, Site-specific conjugation with improved homogeneity)

Enzyme-mediated strategies (e.g., Transglutaminase, Formylglycine-generating enzyme, Sortase A) enable stable bond formation at specific antibody sites and represent third-generation site-specific conjugation technologies.

Features:

Highly specific reaction sites, yielding nearly homogeneous ADCs.
Fixed DAR (e.g., 2 or 4), significantly enhancing stability and predictability.
Well-suited for late-stage development and commercialization due to better batch consistency.

Typical methods:

TGase conjugation – Forms amide bonds between specific glutamine residues and amines.
Glycan remodeling – Introduces aldehydes at Fc glycans for site-controlled conjugation.

Enzyme-mediated conjugation is favored in advanced ADC platforms, improving tissue distribution and efficacy consistency.

Click Chemistry and Bioorthogonal Approaches (Azide–alkyne cycloaddition, Tetrazine–TCO pairs)

Click chemistry offers high selectivity, rapid reaction, and excellent biocompatibility, representing a key direction in future ADC development. Common bioorthogonal reactions include:

1. Azide–Alkyne Cycloaddition

CuAAC (copper-catalyzed) – highly efficient, metal residue must be controlled.
SPAAC (copper-free) – suitable for in vivo applications, safer and reliable.

2. Tetrazine–TCO Click

Extremely fast kinetics, efficient conjugation at very low concentrations.
Used for in vivo bioorthogonal labeling and prodrug release systems.

3. Strain-Promoted Reactions

Catalyst-free, easy to operate, compatible with biological systems.

Advantages:

High site specificity for homogeneous structures.
Mild reaction conditions, protecting sensitive payloads and antibody structure.
Can be combined with enzyme-mediated or genetic engineering strategies to create next-generation ADCs with controllable DAR.

Linker–Payload Design Considerations for Modern ADCs

In modern ADC development, the synergistic design of linkers and payloads is a core determinant of efficacy, stability, safety, and pharmacokinetic properties. An ideal linker–payload system should feature high stability, controllable release, strong tissue selectivity, manageable toxicity, and compatibility with antibody structures. Rational design not only expands the therapeutic window but also significantly enhances the commercial feasibility of ADCs.

Balancing Payload Reactivity With Linker Compatibility

Payloads are often highly reactive or hydrophobic, and their compatibility with the linker directly impacts conjugation efficiency, storage stability, and final product homogeneity. Key design principles include:

Protecting or selectively exposing reactive groups on the payload – for example, cytotoxic molecules with multiple reactive sites require protecting groups or derivatization to ensure correct conjugation.
Ensuring linker functional groups are chemically compatible with the payload – e.g., DNA topoisomerase I inhibitor SN-38 suits carbamate or self-immolative spacers, while Auristatin-class molecules prefer maleimide or peptide-cleavable linkers.
Avoiding excessive reactivity that may degrade or isomerize the payload – sensitive payloads require the linker to provide protection against light, heat, or pH.
Maintaining cytotoxic activity post-conjugation – certain payloads may lose activity if conjugation is improper, making structural optimization critical.

Spacer Engineering for Improved Solubility and Flexibility

Spacers between the linker and payload play a critical role in modulating spatial conformation, enhancing drug solubility, and reducing aggregation risks. Optimization strategies include:

Introducing hydrophilic spacer elements (e.g., PEG or polar amino acid fragments) to reduce ADC hydrophobicity, increase solubility, and improve plasma stability – particularly important for highly hydrophobic Auristatin payloads.
Using flexible or semi-rigid structures to modulate spatial conformation, enabling the payload to engage target enzymes or exert cytotoxic activity more effectively.
Enhancing accessibility for intracellular enzymatic cleavage – certain enzyme-sensitive linkers (e.g., Val-Cit) require appropriate spacers for Cathepsin B recognition.
Reducing antibody aggregation and non-specific binding – spacers minimize hydrophobic interactions with plasma proteins, improving pharmacokinetics.

Rational spacer design significantly enhances ADC pharmacokinetic performance and is a key tool in modern ADC optimization.

Strategies for Reducing Off-Target Toxicity

Off-target toxicity is a major limitation on ADC dosing and safety. Structural optimization at the linker–payload level can markedly reduce systemic toxicity and widen the therapeutic window. Key strategies include:

Enhancing linker stability to prevent premature release in plasma – use more stable maleimide derivatives or non-cleavable linkers; employ stabilized disulfides or enzymatic cleavage strategies.
Improving tissue-selective release to minimize healthy tissue exposure – employ acid-, enzyme-, or reduction-sensitive linkers that trigger payload release only in specific tumor microenvironments.
Controlling DAR to avoid toxicity accumulation from high DAR – optimize conjugation conditions to maintain DAR at 2–4, reducing rapid clearance, enhanced non-specific binding, and liver or bone marrow toxicity.
Optimizing payload structure for selectivity and safety – employ prodrug strategies or structural derivatization to reduce intrinsic payload toxicity; match linkers to appropriate release mechanisms to avoid non-specific activation.
Utilizing bioorthogonal responsive systems for site-specific activation – tetrazine–TCO or similar bioorthogonal systems allow selective payload release in specific lesion microenvironments or under external triggers, minimizing systemic exposure and off-target damage.

Manufacturing and Analytical Challenges in Linker Chemistry

Linker chemistry not only defines ADC molecular structure and functional attributes but also profoundly affects manufacturability, batch consistency, and commercial viability. From lab development to industrial-scale production, linker–payload systems face multiple process and analytical challenges that require systematic optimization and stringent quality control to ensure safety, stability, and batch uniformity.

Process Control in Conjugation Reactions

Linker chemistry is highly sensitive to temperature, pH, solvent type, and reductant concentration.

Precise control of reaction kinetics is necessary to avoid over-reduction that damages antibody structure or skews DAR.
Reaction systems must be optimized to minimize side reactions, such as maleimide retro-Michael opening or NHS ester hydrolysis.
Scale-up requires ensuring uniform mixing and local concentration, preventing batch-to-batch variability.

Analytical Characterization of Linker–Payload Integrity

The complex structural composition of ADCs requires precise analytical tools for quality monitoring.

Multi-dimensional methods such as LC–MS, CE–SDS, and HIC are used to assess DAR distribution, conjugation sites, and linker stability.
Cleavable linkers must be evaluated for stability and trigger characteristics in plasma and lysosomal environments.
Stress tests and stability studies assess the risk of degradation, side reactions, or payload detachment.

Aggregation and Solubility Issues

Linkers and payloads are often highly hydrophobic, affecting antibody solubility and aggregation.

High DAR or hydrophobic payloads increase aggregation risks during production.
Hydrophilic spacers, optimized buffer systems, or lower reaction temperatures can mitigate aggregation.
Aggregation affects product stability and may trigger immunogenicity or rapid clearance.

Heterogeneity in Product Composition

Linker chemistry directly impacts ADC heterogeneity, a key factor in quality consistency.

Random lysine or cysteine conjugation can produce DAR variation, uneven conjugation sites, or structural diversity.
Uneven reduction during conjugation may lead to inconsistent antibody chain structures.
Process optimization, site-specific conjugation, or antibody engineering is required to reduce heterogeneity.

Scale-Up Considerations for Commercial Production

Transitioning from lab-scale to industrial-scale production must address process inconsistencies and quality fluctuations.

Mixing efficiency, mass transfer rates, and local reaction environments differ significantly in large reactors and require validation.
A robust QbD (Quality by Design) framework must ensure control over critical process parameters (CPPs).
Solvent handling, purification efficiency, and online monitoring must meet large-scale production demands.
Product stability must remain consistent over extended storage and transport conditions.

Linker and Conjugation Chemistry Platform at BOC Sciences

BOC Sciences has extensive experience in ADC linker and conjugation chemistry, integrating chemical design, payload synthesis, site-specific conjugation, and analytical characterization. With advanced technology platforms and expert teams, we provide high-homogeneity, high-stability, and highly controllable ADC solutions for clients, supporting multi-stage needs from early R&D to commercial production. Our services focus on structural optimization and controlled drug release while ensuring manufacturability and batch consistency, accelerating project timelines and enhancing ADC efficacy and safety.

Custom Linker Design and Optimization

Optimize linker stability to prevent premature plasma degradation.
Adjust spacer length and hydrophilic/hydrophobic properties to improve solubility and intracellular cleavability.
Align drug-release kinetics with antibody structure for high DAR control and homogeneity.

Payload Development and Synthesis

Design and derivatize Auristatin, MMAE/MMAF, PBD, topoisomerase inhibitors, and other payloads.
Optimize chemical modifications for conjugation efficiency, plasma stability, and targeted release.
Offer small-scale R&D or pilot-scale synthesis according to client needs.

Site-Specific Conjugation Technology Solutions

Cysteine, lysine, and glycan remodeling conjugation strategies.
Bioorthogonal click chemistry (Azide–Alkyne, Tetrazine–TCO, etc.).
Achieve high-selectivity conjugation, homogeneous DAR, and optimized pharmacokinetics.

Analytical Support and Payload–Linker Characterization

DAR measurement, conjugation site identification, and structural homogeneity analysis.
Stability assessment of cleavable and non-cleavable linkers.
Aggregation, solubility, and stress stability testing to support R&D and commercial quality control.

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References

Su Z, et al. Antibody-drug conjugates: Recent advances in linker chemistry. Acta Pharm Sin B. 2021; 11(12): 3889–3907. DOI: 10.1016/j.apsb.2021.03.042. PMID: 35024314.
Aoyama M, et al. Linker and Conjugation Site Synergy in Antibody-Drug Conjugates: Impacts on Biological Activity. Bioconjug Chem. 2024; 35(10): 1568–1576. DOI: 10.1021/acs.bioconjchem.4c00348. PMID: 39363433.

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