Monitoring Cathepsins in Antibody-Drug Conjugate (ADC) Development

Emerging Risks, Linker Stability, and the Need for Protein-Specific HCP Monitoring

Antibody-drug conjugates (ADCs) continue to reshape oncology therapeutics by combining the targeting specificity of monoclonal antibodies with the potency of highly cytotoxic payloads. As ADC pipelines rapidly expand, manufacturers and assay developers such as ICL are also discovering that ADCs introduce unique bioprocessing and stability challenges that are not typically encountered with traditional monoclonal antibodies.

A recent article in Genetic Engineering & Biotechnology News titled “Adopting Creative Chemistry to Optimize Bioprocessing Workflow” highlighted growing industry concerns surrounding ADC stability, linker integrity, and the role of host cell proteins (HCPs) during manufacturing and storage.
According to Sunny Zhou, ADCs are particularly vulnerable because the same linker chemistry designed for payload release in patients may also become susceptible to enzymatic cleavage from residual HCP contaminants during production.

“With these ADC linkers, enzymes that didn’t create problems before might do so now.”

This growing awareness is placing increased focus on proteolytic host cell proteins especially cathepsins.

Why Cathepsins Matter in ADC Manufacturing

Many modern ADCs utilize protease-cleavable linker technologies such as Val-Cit and related peptide linker systems. These linkers are intentionally engineered to be cleaved intracellularly by lysosomal proteases, enabling controlled release of the cytotoxic payload once the ADC enters target cells.

Among the enzymes associated with this process are:

• Cathepsin B
• Cathepsin D
• Cathepsin L
• Cathepsin Z

The importance of Cathepsin B in ADC linker cleavage has been well established for more than two decades. In a landmark study published in Bioconjugate Chemistry, Dubowchik et al. demonstrated the successful use of cathepsin B-sensitive dipeptide linkers for intracellular payload release in immunoconjugates. This work helped establish protease-cleavable linker technology as a foundational component of many modern ADC platforms.
While lysosomal cleavage is essential for therapeutic efficacy, residual cathepsins present during manufacturing or storage may also contribute to:

• Premature linker cleavage
• Payload degradation
• Product instability
• Aggregation formation
• Reduced shelf-life
• Off-target toxicity
• Excipient degradation

As ADCs become increasingly complex, even trace levels of problematic proteases may create meaningful product risk.

Additional studies evaluating ADC linker-payload stability have shown that enzymatic degradation pathways can significantly impact ADC efficacy, pharmacokinetics, and long-term stability. Research published in Bioconjugate Chemistry examining linker degradation in plasma further reinforced how proteolytic activity can alter ADC performance and payload integrity.
Zhou also noted that ADC payloads themselves can introduce additional stability concerns. Because many payloads absorb strongly above 280 nm, ADCs may become more susceptible to light-induced damage, including crosslinking and aggregation. Some manufacturers are already adapting production workflows using dim, red, or yellow lighting conditions to minimize photochemical degradation.

Together, these findings reinforce an important industry trend:
ADC manufacturing requires a more targeted and risk-based approach to impurity monitoring.

The Limitation of Generic HCP Testing

Traditional generic HCP ELISAs remain valuable tools for measuring overall residual host cell protein burden. However, they do not identify which specific proteins remain in the product.

This distinction is especially important for enzymes such as cathepsins, where low concentrations may still have biological consequences.

A low total HCP result does not necessarily mean low risk.

For ADC developers, understanding the presence and concentration of individual proteases can provide deeper insight into:

• Linker stability risk
• Purification effectiveness
• Process robustness
• Root-cause investigations
• Comparability studies
• Long-term product stability

Increasingly, manufacturers are adopting orthogonal analytical strategies that combine:

• LC-MS for protein identification
• Generic HCP ELISAs for total HCP assessment
• Protein-specific ELISAs for targeted quantification of known high-risk proteins

This layered approach helps provide a clearer understanding of which problematic proteins may still remain after purification.

Protein-Specific Cathepsin ELISAs for ADC Workflows

To support targeted HCP monitoring strategies, Immunology Consultants Laboratory (ICL) offers a portfolio of protein-specific ELISA kits for sensitive detection of CHO-derived cathepsins:

These assays are designed to help ADC developers and biopharmaceutical manufacturers monitor specific cathepsins that may impact linker stability and overall product quality.

Protein-specific ELISAs can be particularly useful during:

• Cell line selection
• Process development
• Downstream purification optimization
• Resin evaluation
• Stability studies
• Final product investigations
• Risk assessment workflows

The Future of ADC Characterization

As ADC technologies evolve, manufacturers are recognizing that traditional analytical approaches may no longer be sufficient on their own. The industry is moving toward more sophisticated characterization strategies focused on identifying and mitigating specific high-risk impurities before they impact product performance.

The work highlighted by Zhou and others reflects a broader shift toward creative chemistry and smarter bioprocessing workflows, including targeted impurity removal, improved linker stability, and enhanced analytical monitoring.

For biopharmaceutical developers, protein-specific cathepsin monitoring represents an important opportunity to support the next generation of ADC manufacturing strategies.

Because in ADC development: Low total HCP does not always mean low risk.

Discuss your ADC & HCP strategy

If you would like to explore how cathepsin monitoring and HCP assays can support your ADC development, our specialists at Bio‑Connect are happy to help. Whether you need advice on your workflow design or support in selecting the right ELISAs and related products, you can always reach out via your Bio‑Connect account manager or use the contact form on our website. Together, we will identify the most suitable solutions for your specific project.

References

1. Raper V. Adopting Creative Chemistry to Optimize Bioprocessing Workflow. Genetic Engineering & Biotechnology News.
2. Dubowchik GM et al. Cathepsin B-sensitive dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjugate Chemistry.
3. Studies evaluating linker-payload stability and protease-mediated ADC degradation pathways in plasma and bioprocessing environments.
4. Industry publications on host cell protein risk assessment, orthogonal HCP characterization, and ADC stability strategies.