Why Mass Spectrometry is becoming the gold standard of HCP analysis

by Thomas Kofoed, CEO, and Jette Friis Thirup, Head of Business Development, Alphalyse

A comparison of ELISA vs. LC-MS in host cell protein detection

Host Cell Proteins (HCPs) are critical process-related impurities in biopharmaceuticals, arising from the production process. If not adequately removed, they can compromise drug safety, stability, and efficacy. Historically, Enzyme-Linked Immunosorbent Assay (ELISA) has been the industry standard for HCP detection. However, Liquid Chromatography-Mass Spectrometry (LC-MS) is rapidly gaining recognition as the superior alternative due to its ability to provide a more detailed and accurate analysis.

With increasing regulatory emphasis on individual HCP identification, many biopharmaceutical companies are turning to LC-MS to enhance product safety and regulatory compliance. This article explores why mass spectrometry is the future of HCP analysis and how it compares to traditional ELISA methods.

Limitations of ELISA in HCP detection

ELISA has been widely used for decades due to its sensitivity, ease of use, and cost-effectiveness. However, it has significant limitations that restrict its ability to provide a complete and precise HCP profile.

Dependence on antibodies

ELISA relies on polyclonal antibodies that bind to HCPs in a sample.
Problem: The antibodies may not recognize all HCPs, leading to undetected impurities.
Different ELISA kits produce inconsistent results depending on antibody coverage.

Example: If an ELISA kit lacks antibodies recognizing specific HCPs, the assay will underestimate the amount of HCPs and not be able to monitor batch-to-batch variations.

Inability to identify individual HCPs

ELISA provides total HCP quantification but does not differentiate between individual proteins.
Some HCPs are more problematic than others, and regulatory agencies now require their identification.
Problem: A high total HCP level does not indicate whether critical HCPs (such as proteases or immunogenic proteins) are present.

Example: Two drugs may have identical total HCP levels, but one could contain a highly immunogenic HCP while the other does not. ELISA would fail to detect this crucial difference.

Risk of “Jackpot HCPs”

Some HCPs are highly immunogenic, meaning ELISA antibodies bind preferentially to them.
Problem: This can cause an overrepresentation of certain HCPs while underestimating others.

Example: An HCP with strong antibody recognition may appear more abundant than it actually is, skewing data and misleading developers.

Lack of standardization across processes

Each biopharmaceutical requires a specific ELISA kit tailored to its cell line.
Problem: Developers must create custom ELISA, which is costly and time-consuming.

Example: A commercial ELISA kit is optimized for the antigens used during immunization, and if the actual antigens (HCPs) in a specific project deviate from these the ELISA kit will most likely not be suitable for HCP characterization.

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Video: Are MS-based HCP data accepted in regulatory submissions?

Bryant McLaughlin on FDA and EMA views upon MS-based HCP data

Thomas Kofoed, CEO of Alphalyse, recently interviewed our long-term client Bryant McLaughlin, a Californian CMC executive who has used Alphalyse data in projects for several biotech companies.

Hear about Bryant's experience using mass spectrometry (MS) to document host cell protein (HCP) impurities for regulatory submissions in this 2-minute video.

Experience with FDA and EMA views on MS-based HCP data

Why LC-MS is the future of HCP detection

Mass spectrometry (LC-MS) overcomes the limitations of ELISA by offering unparalleled specificity, sensitivity, and regulatory alignment.

Identifies and quantifies individual HCPs

Unlike ELISA, LC-MS provides detailed identification and quantification of each HCP present in a sample.

Pinpoints specific proteins that may affect drug stability or immunogenicity.
Allows trend analysis of individual HCPs over time, improving process consistency.
Enhances risk assessment by determining the presence of critical HCPs.

Example: If a protease is present at low levels, this will not be reported by the ELISA, while LC-MS would detect it and allow early process adjustments.

Higher sensitivity and accuracy

Detects HCPs at trace levels (as low as parts per million).
Minimizes false negatives that occur in ELISA due to poor antibody recognition.
Provides batch-to-batch consistency, reducing variability in results.

Example: A study comparing ELISA and LC-MS showed that ELISA underestimated HCP content in some samples by 30-40%, whereas LC-MS provided a more accurate risk profile.

Eliminates the need for antibodies

No reliance on antibody specificity - LC-MS detects almost all proteins present (>95%).
No risk of "Jackpot HCPs" skewing results.
Can be used across multiple expression systems (CHO, E. coli, yeast, etc.).

Example: Unlike ELISA, an LC-MS method developed for one biologic can often be applied to multiple drug products with minimal modifications.

Regulatory endorsement and industry trends

Regulatory agencies increasingly recognize LC-MS as a preferred orthogonal method for HCP detection.

The FDA and EMA emphasize the need for individual HCP identification rather than total HCP levels.
USP General Chapter <1132.1> provides guidelines for LC-MS-based HCP measurement.
Pharmaceutical companies incorporating LC-MS face fewer regulatory questions and thus delays in approvals.

Conclusion

While ELISA remains a valuable tool, it is no longer sufficient as a standalone HCP analysis method. LC-MS provides superior accuracy, specificity, and regulatory alignment, making it the new gold standard for biopharmaceutical companies.

Key takeaways

ELISA is limited by antibody specificity, making it unreliable for complex biologics.
LC-MS offers precise identification and quantification of individual HCPs.
Regulatory bodies are increasingly requesting mass spectrometry-based HCP analysis.

Next steps

Evaluate whether your current HCP strategy meets the latest regulatory expectations.
Consider integrating LC-MS as an orthogonal method alongside ELISA.
Stay ahead of evolving compliance requirements, including USP <1132.1> and FDA guidance.

Would you like more information on implementing LC-MS in your HCP analysis workflow? Contact us today!

Talk to us

Whatever protein-related challenge or question you may have, we would love to help. Our experts can help you decide on the best analytical approach for your project by email or online meeting - providing advice without obligation.