HCP analysis of monoclonal antibodies

  • Check for HCPs that are not detected by ELISA
  • Investigate if your mAb contains HCPs known to be problematic
  • Compare individual HCPs in a biosimilar against the originator

Do you want to check your mAb for problematic HCPs?

Monoclonal antibodies are generally very clean due to efficient protein A purifications. However, it is still necessary to document the HCP level to the authorities.

Typically, you also want to confirm the HCP ELISA results by an orthogonal LC-MS analysis and check for problematic HCPs. For instance, HCPs degrading the drug product, e.g., phospholipase an...

Do you want to check your mAb for problematic HCPs?

Monoclonal antibodies are generally very clean due to efficient protein A purifications. However, it is still necessary to document the HCP level to the authorities.

Typically, you also want to confirm the HCP ELISA results by an orthogonal LC-MS analysis and check for problematic HCPs. For instance, HCPs degrading the drug product, e.g., phospholipase and cathepsin, or HCPs that are immunogenic in patients, e.g., clusterin.

Lower your limit of detection and check for problematic HCPs

Often, HCP ELISAs have sufficient coverage of HCPs from the CHO expression system. However, ELISAs have poor specificity and provide only a number for the total HCP level. Thus, abundant HCPs and potentially problematic HCPs can go undetected by ELISA. Especially CHO Host Cell Proteins of low MW and highly conserved proteins, e.g., ubiquitin, are poorly immunogenic. As a result, they are easily missed by the ELISA.

However, there is a good solution to this problem: 

Using our best practice LC-MS method, you get the total HCP amount by an orthogonal method to ELISA. In addition, you get a list of the individual HCPs and the amount of each. Moreover, by combining with a native digest optimized for mAbs, we can even lower the limit of detection to sub-ppm levels.

Check for specific Host Cell Proteins

As a unique service, our specialists will check for specific HCPs known to be of concern in mAb products and compare them to HCPs found in our study of commercially available mAbs. You can then use this info to ensure the stability and efficacy of your drug. Similarly, you can make changes to lower the risk of immunogenic reactions in patients. 

What is included in an orthogonal LC-MS assay:

Our CHO Host Cell Protein Assay was developed and tested on hundreds of mAb samples, including 16 commercial drugs. It is also worth noting that an assay for your mAb can be set up in a matter of weeks.

Analysis of HCP in mAbs includes the following:

  • An optimized sample preparation, specifically developed for monoclonal antibody samples.
  • Analysis using a robust microflow HPLC and SWATH® MS/MS system to ensure high reproducibility.
  • Spike-in of internal standard proteins (0-2000 ppm).  This step is important since it enables quantification linearity even to low ppm levels.
  • A report with both the total HCP number and a list of individual HCPs with amounts.
  • A check for potentially problematic HCPs, and a check for HCPs found in commercial products.
  • And finally, a follow-up meeting to go over the results.

When you work with us, you gain access to our extensive knowledge from the analysis of 100s of mAb samples for Host Cell Protein by mass spectrometry.

Contact us for a discussion on how to analyze HCP impurities in your mAb.

MoreLess details

The process:

  • 1 Contact us to discuss your project and receive a project proposal
  • 2 Analysis phase lead by an Alphalyse principal investigator
  • 3 Reporting of HCPs found, with check for specific HCPs
  • 4 Follow-up by email and in a virtual meeting

Let's work together

We offer customized solutions, contact us to discuss your project.

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Send us a description of your project and we shall be happy to present you with a solution.

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Testimonials

  • "We were hoping our product was cleaner, more stable, and safer than the originator. However, the results were so favorable that we can use them in our marketing."

    Cleaner than the original: mAb biosimilar did not contain problematic protease.

    Explore customer case
  • "With the results from the extensive characterization, we identified a scale-up problem and could optimize the process to increase mAb stability."

    Combining mass spec analyses for mAb characterization and batch-to-batch comparison

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Knowledge center

More information

We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

Technical details

How does the native digest method work?

Our native digest method to lower the LOD of mAb HCPs is inspired by the novel sample preparation which was presented by Huang et al. in 2017.

In our standard orthogonal HCP analysis we reduce and alkylate prior to protein digestion. However, the native digest is performed in a way that leaves the antibody nearly intact. This thus lowers the dynamic range for HCP detection by 1 or 2 orders of magnitude.

Sample preparation

Sample requirements:

Native digest and mass spectrometry are orthogonal methods to ELISA.

In order to obtain good data of the HCP levels with native digest and mass spectrometry we need sample material in sufficient amounts.

It is important to prepare samples in a clean laboratory so you avoid contamination with human keratin.
Please submit samples in liquid or lyophilized.

  1. The analysis typically requires a 1-2 mg of drug protein material for each sample.
  2. Above all, the samples should be free of large amounts of detergents.
  3. Keep the concentration >0.5 µg/µl.
  4. Use an Eppendorf Safe Lock tube, or similar tubes from your lab.
  5. Freeze or refrigerate to +4 oC for cold shipment of the liquid sample.

Meet the experts

For this type of analysis our experts include:

Do you need help?
Rikke Raaen Lund

PhD in Biomedicine

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Do you need help?
Janne Skaarup Crawford

BSc in Cell Biology and Biochemistry

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Do you need help?
Rie Bak Jäpelt

MSc Pharmacy, PhD Analytical Chemistry

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