Antibody-drug conjugates (ADCs) represent a highly complex class of biopharmaceuticals, combining the specificity of monoclonal antibodies with the potent cytotoxicity of small-molecule drugs. Chapter 40 of the 2018 reference work Biopharmaceutical Processing offers a concise yet insightful overview of the critical considerations in the development and manufacturing of ADCs. Drawing on experiences with lysine-based, interchain cysteine-based, and THIOMAB platforms, the chapter discusses the process control challenges and quality-critical attributes that shape successful ADC development.

Figure 1. Schematic of the three fundamental components of an ADC: the antibody, linker, and drug.

Quality Attributes in ADC Conjugation

ADC process development often revolves around the control of key quality attributes directly influenced by the conjugation process. These attributes, summarized in Table 1 of the original text, are particularly challenging to manage due to the inherent heterogeneity introduced during conjugation. Ensuring consistency in drug-to-antibody ratio (DAR), site-specific conjugation, and purity is vital, as these properties directly impact the pharmacokinetics, efficacy, and safety of the final product.

Table 1. Summary of Key ADC Product Quality Attributes

Segmented Manufacturing and the Strategic Role of Intermediates

ADC production typically follows a modular or segmented approach: antibody intermediates and cytotoxic drug-linker intermediates are manufactured separately on dedicated production lines and released after rigorous quality testing. This strategy offers significant advantages, including cost reduction by leveraging existing monoclonal antibody infrastructure and risk mitigation by introducing quality checkpoints upstream of the conjugation step.

High-quality intermediates help prevent failures due to raw material defects, minimize material loss, and reduce production delays. This modular model also simplifies quality control during conjugation and allows for flexible batch sizing, improving supply chain resilience.

Figure 2. A typical ADC product supply chain illustrating the separate production and release of antibody and small molecule drug intermediates, which are subsequently conjugated during ADC drug substance manufacturing.

Antibody Intermediates

As the structural scaffold of ADCs, the antibody's quality attributes profoundly impact the final product. While most release criteria align with therapeutic antibodies (e.g., residual HCPs, DNA), conjugation introduces additional requirements. Aggregation control becomes more stringent due to chemical modifications and solvent exposure during conjugation. Attributes such as antibody concentration and pH can be more relaxed, as they are adjustable later.

However, features directly affecting conjugation, such as trisulfide content in cysteine-based platforms or the modification state of C-terminal lysines in lysine-based platforms, must be tightly controlled. Free thiol content, for instance, determines DAR consistency batch-to-batch. Formulation excipients also need compatibility: in lysine-based conjugation, primary amine-containing buffers like Tris should be avoided due to competitive reactions with NHS esters.

Cytotoxic Drug-Linker Intermediates

Drug-linker intermediates, the cytotoxic payloads of ADCs, are typically produced via complex synthetic or semi-synthetic routes. Given their role in efficacy and toxicity, these intermediates are regulated similarly to small-molecule APIs, with strict specifications on purity and impurity profiling.

Particular attention is paid to reactive impurities that may undergo unintended conjugation with the antibody. Such conjugation variants are difficult to detect or remove post-reaction, while non-reactive impurities may be tolerable if proven to be removed consistently during purification.

Unlike traditional APIs, physical properties such as polymorphism or particle size are not major concerns since the drug-linkers are dissolved in organic solvents during conjugation. Residual solvent limits can also be higher due to efficient downstream removal, such as via tangential flow filtration (TFF).

Conjugation Process

Despite the diversity in conjugation chemistries across platforms, ADC manufacturing can generally be divided into three distinct stages: antibody functionalization, drug conjugation, and purification/formulation. The functionalization step is platform-dependent and critically determines the number and position of attachment sites, key to controlling average DAR and product heterogeneity.

Functionalization Chemistry Across Platforms

In interchain cysteine platforms, selective reduction of hinge-region disulfides exposes reactive thiols. THIOMAB technology introduces engineered cysteines at specific sites, typically requiring full reduction and reoxidation. Lysine-based conjugation involves modifying surface lysine residues with bifunctional linkers like SMCC.

Each method varies in sensitivity. Lysine conjugation is highly sensitive to pH, temperature, and concentration; slight deviations can drastically affect NHS ester hydrolysis and conjugation yield. Cysteine-based platforms are more tolerant, primarily influenced by reductant dosage. THIOMAB systems offer higher robustness via engineered site control and redox cycling.

Figure 3. Comparison of antibody functionalization steps across three major ADC conjugation platforms.

Precision in Reagent Addition

Reagent dosing precision is a major concern during scale-up. For interchain cysteine systems, free thiol generation is linearly proportional to the reductant amount. A 5% dosing error can shift DAR by a similar margin—unacceptable in commercial production, where DAR specs are often as narrow as ±10%.

To mitigate cumulative variance from antibody concentration errors (±5%), volume transfers (±1%), and solid weighing (±1%), engineering strategies include using gravimetric methods instead of volumetric pipetting, correcting for purity, transferring by weight when possible, and diluting reagents to increase addition volumes. Implementing such controls can reduce reagent variability to within a few percent of the target molar ratio.

Controlling Heterogeneity in the Final Product

Heterogeneity in DAR distribution and conjugation sites is an inherent trait of ADCs, especially for lysine and cysteine-based platforms. For commercial products like Adcetris® and Kadcyla®, a range of DAR species is both expected and acceptable, provided inter-batch consistency is maintained.

However, since DAR affects PK/PD and toxicity, ensuring a reproducible distribution is a regulatory and therapeutic necessity. Even when heterogeneity is allowed, rigorous control over the functionalization and conjugation steps is essential to ensure that the composition profile stays within validated limits throughout scale-up and commercial production.

Learn more about Creative Biogene's ADC Pharmacodynamic Evaluation Services.

Conclusion

The process development and commercial-scale production of ADCs represent a sophisticated integration of deep scientific insight and meticulous engineering control. Success in this endeavor rests on three fundamental pillars: a comprehensive understanding of the physicochemical properties of the antibody, linker, and cytotoxic payload; stringent and targeted quality control of both antibody and small-molecule intermediates; and precise optimization and robust control of the conjugation process, especially during antibody functionalization and conjugation reaction phases.

The three mainstream conjugation platforms—lysine-based, interchain cysteine-based, and THIOMAB—exhibit significant differences in process sensitivity, product heterogeneity, and technical challenges. This necessitates the development of platform-specific strategies. Core principles throughout include: reinforcing control over intermediates, particularly critical sources of variability such as trisulfide bonds and conjugatable impurities, to prevent process failures; pursuing extreme accuracy in reagent addition during the functionalization step while applying DAR-based heterogeneity control strategies; and delicately balancing reaction efficiency with suppression of side reactions during conjugation (notably off-target conjugation and aggregation). Additionally, leveraging the design advantages of site-specific conjugation technologies can significantly enhance process robustness.

Only through such a systematic, science- and risk-based approach can ADCs transition efficiently, stably, and at scale from laboratory to commercial manufacturing, ultimately ensuring the reliable delivery of safe and effective targeted cancer therapies to patients. If you have specific enzyme types or conjugation workflows in mind, Creative Biogene's experts are available to help tailor separation strategies and scale-up solutions accordingly.

FAQ

Q: Does ADC multi-step conjugation require continuous real-time monitoring during scale-up?

A: Yes. Multi-step conjugation reactions—such as two-step, three-step, or dual-payload approaches (e.g., lysine + cysteine or glycan + cysteine combinations)—require careful real-time control during each step, as conjugation efficiency directly impacts final product quality. Real-time monitoring is essential during both process development and scale-up.

Summary: In multi-step ADC conjugation and scale-up, real-time monitoring should be flexibly applied based on process complexity and maturity. Continuous monitoring is advisable during early development and should only be reduced after sufficient process validation.

Q: How can enzymes used in site-specific conjugation be removed post-reaction?

A: In enzyme-mediated site-specific conjugation (e.g., using transaminases or sortase), it is crucial to remove catalytic enzymes post-reaction to prevent interference with product purity and activity. Depending on enzyme characteristics (e.g., presence of affinity tags) and reaction composition, several strategies are available:

Recommended Approach:

• If the enzyme carries an affinity tag, use affinity chromatography for precise and gentle removal.
• If the enzyme differs significantly in size or charge from the product, physical separation methods (e.g., SEC or IEX) can be used, though optimization is critical.
• Avoid harsh chemical removal methods unless the product is proven stable under such conditions.