Literature
Problematic HCPs in drug development
May 2 2025, by Cecilie Hovitz Lautrup-Larsen, Scientific Communication Specialist, Alphalyse
How specific host cell proteins derail safety, stability, and regulatory approval
In biologic drug development, host cell proteins (HCPs) are a known challenge – but it’s not the total HCP amount alone that causes the biggest problems. A small subset of individual HCPs can significantly compromise safety, stability, and regulatory progress. These so-called “problematic HCPs” or “HCPs of concern” are a considerable cause of adverse patient reactions, formulation issues, and prolonged discussions with regulatory authorities.
As agencies like the FDA and EMA raise the bar for analytical documentation, developers must move beyond traditional ELISA-based quantification. Regulators now expect a deeper understanding of which HCPs are present and how they could affect the final drug product. This shift requires a change in both strategy and technology – especially for early-stage characterization.
In this article, we focus on six HCPs that repeatedly complicate drug development and show why identifying and managing them early on is critical for success.
Why specific HCPs are a development bottleneck
While it is possible to clear most HCPs to acceptable levels, some are particularly persistent – or high-risk – even in trace amounts. These proteins resist purification, evade antibody detection, and can interact with the drug or patient.
Some concerns about high-risk HCPs:
- Immunogenicity: Some HCPs are recognized by the immune system and can trigger adverse responses.
- Enzymatic activity: Others degrade excipients or the drug substance, reducing shelf life or stability.
- Biological interference: Some mimic human proteins and can interfere with the therapeutic drug function or endogene protein function.
- Purification resistance: Certain HCPs are challenging to remove and co-purify with the final drug.
When these proteins are not detected – or their quantity underestimated due to the limitations of ELISA – they often become a source of project delays during regulatory review. In the worst cases, they have resulted in failed clinical trials, withdrawn applications, or forced development of additional assays.
Six HCPs that repeatedly challenge drug development
Based on insights from published drug development articles, here are six HCPs that commonly raise red flags with regulatory authorities and internal quality teams:
1. Flagellin
Flagellin is a bacterial protein that activates the TLR5 pathway, triggering a strong innate immune response. It is highly immunogenic, even in trace amounts, and has been implicated in excessive immune activation in preclinical and clinical settings.
Why it is problematic:
- Extremely low safety threshold
- Not always detected by standard ELISA kits
- Difficult to remove from certain expression systems
- Known to delay regulatory approvals
2. Lysosomal acid lipase
This enzyme degrades polysorbate, a widely used excipient in biologic formulations. If undetected, it can silently compromise the drug’s long-term stability – sometimes after the product has already reached the market.
Why it is problematic:
- Degrades key excipients like polysorbate 20 and 80
- Impacts drug shelf life and physical integrity
- Often missed without targeted LC-MS analysis
- Can cause harmful degradation months into storage
3. PLBL2 (phospholipase B-like 2)
PLBL2 is a well-known example of an HCP that confounds ELISA assays. It can display non-linear dilution behavior, resulting in unpredictable – and unreliable – quantification. In some cases, it has also been linked to adverse immune responses in patients.
Why it is problematic:
- Causes inconsistent ELISA data
- May be falsely underestimated in release assays
- Has demonstrated immunogenic potential in vivo
- Not always cleared by standard purification
4. Clusterin
Clusterin is a chaperone protein that has been associated with increased immunogenicity in some biologics.
Why it is problematic:
- Interacts with therapeutic proteins
- Is associated with higher immunogenicity risk
- May not be recognized by commercial antibodies
- Difficult to clear completely from some processes
5. Protein disulfide isomerase (PDI)
PDI plays a natural role in protein folding but becomes a problem when it co-purifies with the drug substance. It promotes aggregation and misfolding, which can lead to reduced efficacy, stability concerns, immunogenicity, and even blood clot formation in patients.
Why it is problematic:
- Triggers protein aggregation
- Affects the structural stability of the drug
- May be enriched by therapeutic protein purification
- A threat to patient safety
6. Heat shock proteins (HSPs)
HSPs are another class of biologically active HCPs that can mimic human proteins. Their presence in final formulations has been linked to immune modulation and, in some cases, coagulation pathway activation. This poses safety concerns during regulatory review.
Why they are problematic:
- Can stimulate harmful immune responses
- May trigger autoimmune disorders
- Impede drug tolerability
- Linked to thrombosis risk
Why ELISA is not enough
Most biopharmaceutical developers rely on ELISA kits for HCP quantification. While useful for overall surveillance, ELISA:
- Does not identify individual HCPs
- Relies on polyclonal antibody recognition, which varies by kit and by animal
- May miss low-abundance or structurally unique HCPs like flagellin or PLBL2
- Cannot differentiate high-risk HCPs from benign ones
As a result, a drug substance may appear “clean” by ELISA standards while still containing HCPs that compromise safety or stability.
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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 LC-MS solves the problem
Liquid chromatography-mass spectrometry (LC-MS) enables individual identification and quantification of HCPs. When applied early in development, it provides critical insights that help biopharmaceutical developers:
- Determine whether a known problematic HCP is present
- Quantify trace amounts of immunogenic or enzymatic proteins
- Track clearance of individual HCPs across purification steps
- Provide regulators with detailed impurity profiles
Unlike ELISA, mass spectrometry (LC-MS)-based analysis is not affected by on antibody coverage. It can detect proteins based on their mass and sequence – making LC-MS the method of choice for identifying specific protein impurity risk factors.
Moving forward with confidence
The reality is simple: a few HCPs cause the majority of trouble. By identifying them early – before they trigger regulatory delays or force method development under pressure – developers can stay in control of their timelines and documentation.
Mass spectrometry is not just a tool for final characterization. It is an enabler of smarter, data-driven development. And when it comes to problematic HCPs, it is often the only way to see the whole picture.
Key takeaways
- A small group of HCPs – among them flagellin, PLBL2, lipases, PDI, HSPs, and clusterin – account for a disproportionate number of impurity challenges in biologic drug development.
- These proteins are known to impact immunogenicity, formulation integrity, long-term stability, and patient safety, making them high-risk impurities.
- Traditional methods like ELISA often fail to detect these HCPs accurately due to low antibody coverage and specificity limitations.
- LC-MS enables early identification and quantification of individual HCPs, helping developers make informed decisions, reduce regulatory risk, and maintain control throughout the development lifecycle.
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