Literature
Flagellin – The hidden HCP that can jeopardize your drug development
by Thomas Kofoed, CEO, and Cecilie Hovitz Lautrup-Larsen, Scientific Communication Specialist, Alphalyse
Why this bacterial protein is considered a worst-case scenario for CMC processes
Host cell proteins (HCPs) are an unavoidable byproduct of biologic drug production. While many HCPs can be removed or reduced through purification, some are particularly problematic – even at trace levels – due to their strong immunogenicity, structural stability, or resistance to clearance. Flagellin is one such HCP that presents a worst-case scenario in chemistry, manufacturing, and controls (CMC) processes.
Flagellin, a bacterial protein commonly found in Escherichia coli expression systems, is a potent immune stimulator that interacts with toll-like receptor 5 (TLR5). Its presence, even in trace amounts, can compromise drug safety, trigger immune responses, and cause regulatory setbacks.
This article explores why flagellin is a high-risk contaminant, how it impacts biopharmaceutical development, and the most effective methods for detecting and eliminating it.
The risks of flagellin contamination
High immunogenicity – strong immune activation risk
Flagellin is one of the most immunostimulatory HCPs due to its interaction with the TLR5 receptor on immune cells. This makes it particularly concerning in biologic drug formulations where immune tolerance is critical.
Flagellin:
- Activates innate immune responses, leading to inflammation
- Increases the risk of adverse immunogenicity in patients
- Complicates regulatory approval due to its potential safety concerns
Example: Biologic drugs contaminated with flagellin have been shown to elicit excessive immune responses, even at very low concentrations, making regulatory approval challenging.
- Tends to co-purify with drug substances, leading to contamination
- May require additional purification steps, increasing the production costs
Example: Some downstream purification strategies fail to completely remove flagellin, requiring additional process modifications to meet regulatory requirements.
Regulatory challenges – heightened scrutiny from health authorities
Regulatory agencies are increasingly focused on individual HCPs, especially those with immunogenic potential.
- The FDA and EMA increasingly request individual identification and quantification of critical HCPs
- Failing to detect or remove flagellin can lead to regulatory delays or rejections
- Recent guidance, including ICH Q2(R2) and USP <1132.1>, emphasizes the need for more precise HCP analysis methods
Example: One of Alphalyse’s clients, a biopharma company, had to delay its biologics license application (BLA) submission after the FDA requested additional data on flagellin clearance, delaying market entry.
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Ebook: 6 host cell proteins that challenge drug development
Health authorities now emphasize the identification and quantification of individual HCPs. This shift reflects an increased understanding of how specific HCPs can influence product quality and patient safety, necessitating more advanced analytical approaches.
This ebook explores six HCPs of concern from the scientific literature, their harmful potential, and the lessons they offer for biopharmaceutical development.
How to detect and eliminate flagellin
Traditional ELISA-based detection – limited reliability
Enzyme-linked immunosorbent assay (ELISA) is a common method for HCP detection, but it has major limitations when dealing with flagellin.
- ELISA relies on polyclonal antibodies, which may not fully recognize flagellin
- Low abundance or structural differences may cause false negatives
- Commercial ELISA kits often lack specificity for problematic HCPs like flagellin
Takeaway: Relying on ELISA alone may result in undetected flagellin contamination, leading to regulatory and clinical risks.
Liquid chromatography-mass spectrometry (LC-MS) – the superior approach
Mass spectrometry has emerged as the new gold standard for detecting and quantifying individual HCPs, including flagellin.
- Provides high specificity, identifying individual HCPs rather than total protein levels
- Detects low-abundance flagellin even when it is missed by ELISA
- Supports regulatory compliance, aligning with USP <1132.1> and FDA expectations
Takeaway: Incorporating LC-MS into HCP analysis ensures precise flagellin detection, reducing regulatory risks.
Process optimization – improving purification strategies
To mitigate flagellin contamination, manufacturers must optimize downstream purification.
- Affinity chromatography: enhances removal of bacterial contaminants
- Multi-step purification: combining different methods increases clearance efficiency
- Process validation: continuous monitoring ensures consistency in flagellin removal
Takeaway: Refining purification strategies reduces flagellin-related risks, ensuring product safety and regulatory success.
Conclusion
Flagellin is one of the most problematic HCPs in biologic drug development due to its high immunogenicity and regulatory impact. Not detecting and removing it adequately can lead to clinical risks and delayed regulatory approvals.
Key takeaways
- Flagellin is a worst-case scenario HCP due to its strong immunogenicity
- Traditional ELISA methods may fail to detect flagellin, increasing the of clinical holds and regulatory delays
- LC-MS provides superior HCP analysis, ensuring compliance with regulatory expectations
- Optimizing purification processes is essential for effective flagellin removal
Next steps
- Evaluate your HCP detection strategy - does it reliably identify flagellin?
- Consider integrating LC-MS for more precise flagellin analysis.
- Refine your purification processes to enhance flagellin clearance.
For more information on advanced HCP detection, reach out to our team of experts.
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