ELISA characterization and coverage analysis

When you need to evaluate the quality of antibodies in a generic or process-specific HCP ELISA, advanced ELISA-MS™ analysis identifies each Host Cell Protein the ELISA does and does not measure, using a combination of immunocapture and mass spectrometry.

For ELISA-MS™, an early process sample containing the drug substance will be used for analysis avoiding the development of a null cell lysate from a mock fermentation. The Host Cell Proteins in the sample are immunocaptured using the ELISA antibodies and identified by LC-MS/MS, providing a list of the exact HCPs recognized by the ELISA antibodies. This list will be compared with all proteins found in the early process sample to evaluate the coverage of your ELISA. Additional HCP analysis of your drug substance (DS) allows you to compare the list of HCPs covered by your ELISA to the HCPs found in your DS.

Which data can Alphalyse provide for regulatory documentation?

Compare ELISAs and document HCP coverage

Document coverage percentage, identify covered vs. uncovered HCPs, and get a list of high-risk HCPs – enabling you to select the best fit ELISA for your product

Bridging studies

Characterize ELISA reagents and HCP standards to assess why the ELISA shows dilutional nonlinearity or inconsistent results

Troubleshooting ELISAs

Investigate issues caused by antibody/ drug substance cross-reactivity, insufficient specificity, incorrect standards for the calibration curve, etc.

A challenge faced by many of our clients is that different ELISA kits, even from the same vendor, can show very different results — sometimes more than 10 to 100-fold in- or decreases in HCP amounts — when analyzing identical batches.

Using LC-MS, we can uncover if the inconsistent results may be due to an unintended change in the HCP standard. Such a change can make it a poor fit for your drug sample or antibodies, resulting in a different HCP concentration readout even with the same HCP antibodies. Other reasons for the inconsistent results, which ELISA-MS™ can uncover, might be that a new generation of antibodies binds better — or worse — to the proteins in your sample.  

Regulatory authorities are concerned that abundant HCPs in a drug product may go undetected by ELISA or that your drug substance may contain undetected HCPs of concern that impact patient safety or drug stability. 

ELISA-MS™ data provides not only the coverage percentage of your ELISA but also a list of the exact HCPs recognized by your ELISA antibodies. In addition, we provide access to our extensive database of HCPs of concern – based on current regulatory guidelines, literature, analyses of commercially released products, and our experience from more than 350 client projects – allowing data-driven risk assessment of the HCPs in your product. This is solid documentation for regulatory authorities.

Not all commercial HCP ELISAs may be equally suitable for detecting the specific HCPs in your biologic. Comparing the coverage of various ELISAs — commercial kits or process-specific assays — allows you to pick the best and most fitting ELISA for your drug and process-related impurities. Furthermore, you must ensure that the ELISA standard resembles the HCPs in your drug substance, or the readout may be off.

ELISA-MS™ combines ELISA-based immunocapture with LC-MS analysis to investigate which ELISA antibodies bind to your early process sample. The result is a very accurate and detailed list of individual HCPs in the sample recognized by the ELISA antibodies. You can then compare and base your choice of kit not only on the total coverage but also on each ELISA’s ability to detect individual HCPs, e.g. HCPs of concern. Furthermore, LC-MS can be used to analyze the fit of the ELISA standard to your drug substance.

Typical project process

You typically work with
these experts:


Sai Sindhu Thangaraj

PhD in Cardiovascular Inflammation

Anette Holck Draborg

PhD in Immunology

Melissa Fernard Pedersen

MSc in Medicine with Industrial Specialization
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    Project scope

    We like to start with an online meeting to learn more about your project. Based on your needs, you will receive a draft proposal outlining the suggested analyses and expected timeframe.

  • samples-blue


    After signing the final project proposal, we will contact you for details about your samples. An estimated report delivery date will be sent as soon as we receive your samples.

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    An Alphalyse project leader will oversee the project and send you status updates by email at regular intervals.

    A typical analysis includes:

    • ELISA-MS™ analysis of selected HCP antibodies using a harvest sample.
    • LC-MS-based HCP analysis of harvest sample, mock sample, and/or drug substance (DS) sample.
  • 5000-+-reports-delivered-blue


    You will receive the analysis report by email. Depending on the project, it includes:

    • Objectives, description of analytical procedure, results, and conclusions.
    • Reporting of HCPs covered by the ELISA antibodies in the sample and their distribution, total coverage percentage.
    • Evaluation and comparison of HCP-profiles of the samples and/or to the ELISA standard.
    • HCP ELISA potential high responder HCPs.
    • HCP antibody binding of HCPs of potential concern (CHO projects only).
    • Selected raw data.
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    Follow up

    Upon completion of the project, your team is invited to go over the results at an online meeting.

Curious to know more?

learn more

Whatever ELISA-related challenge or question you may have, we are here to help you solve it. One of our protein analysis experts will discuss the best analysis approach or method for your project by email or online meeting – without obligation.

Client stories

Troubleshooting HCP ELISA results using LC-MS and ELISA-MS™

Biotech: "We found out that the increase in HCP amount in our DS was due to a shift in the ELISA calibration
case study

Characterization of an ELISA standard

CDMO: "The data saved us from using an unsuitable mock sample in development of a process-specific ELISA"

Selecting the best HCP-ELISA kit out of five

Antibody therapeutics developer: "We found the kit with best coverage of HCPs overall and individual HCPs of concern"

Video: Trouble-shooting ELISA results using mass spectrometry

A change in ELISA standard may have a dramatic effect on measured HCP levels

HCP-ELISA coverage analysis without a null cell line

Developer of immuno-oncology biologics: "Thanks to the data, we knew which HCP-ELISA kit to use!"

Video: Selecting the best suited HCP-ELISA

Comparing 5 CHO kits from 4 suppliers to find the one with the best coverage

Video: HCP coverage analysis by ELISA-MS

Measuring the coverage of your HCP ELISA without a null cell line
3 coverage analysis ella ms

Video: Problems with non-dilutional linearity of ELISA

This biotech company had issues with the validation of its ELISA

Ebook: Comparison of HCP ELISA coverage methods

Get an overview of the most common methods for measuring ELISA coverage
3 reasons to check the coverage of your HCP-ELISA

Webinar: 3 reasons to check the coverage of your HCP-ELISA

Using ELISA-MS™ for detecting which individual impurities are measured

ELISA characterization and coverage analysis

  • Fast assay setup and analysis – 8-12 weeks.

  • No null cell line is necessary.

  • Requires only a low amount of antibody.

  • Applicable for all types of biologics: Monoclonal antibodies, recombinant proteins, gene therapy, vaccines, etc.

  • Suitable for all commercial, custom, process- and platform-specific ELISAs.

  • Tested on commercial ELISAs from all major vendors.

What clients say

Testimonial Alphalyse
"The analysis showed that the new ELISA standard made the calibration slope shift, resulting in a misinterpretation of the total amount of HCPs in the drug sample"

Director QC
EU-based biotech
Testimonial Alphalyse
“I found the group at Alphalyse knowledgeable, easy to work with, and helpful in
planning the study”

VP CMC and Quality
US Biotech
Testimonial Alphalyse
“We found out why our PG13 ELISA kit had such low coverage of our gene therapy’s HCPs”

Chief Scientific Officer
Top10 Pharma Company
Testimonial Alphalyse
“Extremely professional service, high-level quality of the results, and excellent communication”

Program Manager
European Biotech
Testimonial Alphalyse
"Thanks to the ELISA-MS data, we knew which kit to use for the setup and validation of our HCP ELISA assay! Furthermore, we new exactly which individual HCPs were covered by
the ELISA antibodies"

Director CMC
US Biotech
Testimonial Alphalyse
"Based on the data provided by Alphalyse, we settled on a kit that rovided both excellent coverage of the HCPs overall and the individual HCPs of concern"

Senior CMC specialist

US-based Biotech
"Alphalyse provided a very well-designed and executed HCP analysis, fruitful technical discussions, and flexibility in terms of
writing the report."

Thore Schmedt, Associate Director
AiCuris Anti-infective Cures AG, Germany
"Using the Alphalyse LC-MS/MS coverage method in HCP ELISA selection, we estimate a savings of approximately $1M and, likely, one year of development time."

Lars Skriver Senior Science Officer
SAVARA Aps, Denmark

Knowledge center

How do the measured MS HCP levels compare to measured ELISA HCP levels?

The principles of ELISAs and LC-MS are inherently different. However, in our experience, a fit-for-purpose ELISA with good coverage of relevant HCPs performs comparably to LC-MS analysis. However, if the ELISA is unsuitable for the product, e.g., a generic ELISA with low coverage of HCPs in the specific process, the measured amounts of HCPs might be entirely off in the ELISA.

ELISA-MS™ analysis combines immunocapture with mass spectrometry and can determine if your HCP-ELISA is fit-for-purpose and capable of accurately detecting the HCPs of concern in your product.

How do you ensure digesting only the antigen and not the antibodies in the ELISA-MS analysis?

After adding the antigen, we perform a gentle protein/antigen digestion directly in the ELISA plate. This procedure leaves the ELISA antibodies largely intact as they are somewhat resistant to the proteases, unlike the HCPs. In addition, we check the antibody sequences against our extensive protein database to eliminate any antibody peptides generated by the sample preparation.

Why is label-free quantification of HCP preferable?

We prefer to use intact label-free protein standards for HCP quantification because they resemble the protein impurities in the sample and will be exposed to the same conditions as the HCPs during sample preparation. Isotope-labeled peptide standards would not be exposed to digestion and sample clean-up, which makes them unable to account for the effects of sample preparation on the sample proteins.

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.

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