Data for risk assessment

Regulatory authorities are increasingly aware that protein impurities, even at low levels, can affect patient safety and product stability. Some residual Host Cell Proteins (HCPs) have been observed to cause immunogenicity, adjuvant effects, or direct biological activity in patients and degradation of products or excipients during storage. Therefore, it is not enough that an ELISA reports a low number – you also need to investigate the individual HCPs.

For this reason, legal authorities call for orthogonal methods,  such as mass spectrometry (LC-MS analysis), to identify and quantify individual HCP impurities and for a thorough risk assessment of the resulting data. LC-MS can identify process-related impurities down to 0.1 ppm and allows for data-driven process optimization and evaluation of purification steps.

Why use LC-MS/MS for HCP analysis?

Patient safety and risk assessment

Rule out the presence of HCPs causing immunogenicity, adjuvant effects, or biological activity

Product stability and efficacy

Check for enzymes causing product degradation, modification, loss of activity, or excipient degradation

Manufacturing consistency and product purity

Evaluate purification process clearance, drug substance purity, or batch comparability

HCP-ELISAs tend to be biased towards abundant HCPs and are inherently immunologically weighted, which means they may fail to detect trace or non-immunogenic impurities in the final product. In addition, the readout of immunoassays is an aggregate sum of present HCPs, which does not confirm or deny the presence of specific HCPs of concern. 

Using mass spectrometry (MS), you will know which specific HCPs your product contains – and can evaluate if any of them may be of concern. 

Active enzymes need only be present at shallow levels to cause product or excipient degradation, or modification. This can lead to loss of drug activity (efficacy) or drug product stability upon storage. Examples of enzymes with significant impact on product or excipient stability include proteases which cleaves the protein biopharmaceutical, and tween-degrading enzymes; lipases, and esterases, which degrade Polysorbate 20 and Polysorbate 80 used as solubilizing excipient in the formulation buffer of many mAbs and biopharmaceuticals.


ELISAs are in general not able to detect specific HCPs at low ppm levels. By relying on HCP-ELISA only, you run the risk that your product may contain one or more HCPs of concern, which may degrade your product or cause adverse reactions in patients.


Since biosimilar products can be produced in different expression systems and are likely purified using processes other than the originator, the HCP impurity profile in the two products may differ.

Authorities ask that biosimilars are subject to just as extensive HCP analysis as their originators to verify that they do not contain HCPs of concern. In some cases, we have found that a biosimilar product is even cleaner than the originator, which has served as a quality attribute of the product.

Typical project process

You typically work with
these experts:

Solveig Beck Nielsen, expert on residual protein analysis

Solveig Beck Nielsen

PhD in Molecular Biology
Sai Sindhu Thangaraj,, expert on HCP coverage analysis and ELISA characterization

Sai Sindhu Thangaraj

PhD in Cardiovascular Inflammation
Christina Erika Hagensen, PhD

Christina Erika Hagensen

PhD in Biochemistry and Molecular Biology
  • Facilitate meetings

    Project scope

    We will 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.

  • Protein sample


    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.

  • SWATH LC-MS analysis and protein quantification using amino acid analysis


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

    A typical analysis includes:

    • Spike-in of internal standard proteins, enabling quantification at low ppm levels.
    • Enzymatic digest and analysis of peptides using LC-MS.
    • ELISA-MS analyses with an in-well digest of immunocaptured HCPs.
    • Setup of a custom-specific database.
  • 5000 mass spectrometry reports


    You will receive the report by email. As standard, the LC-MS HCP analysis report includes:

    • Objectives, description of analytical procedure, results, and conclusions.
    • A list of the number and the total amount of HCPs in the sample.
    • A list of individual HCPs and their quantities, highlighting HCPs of potential concern, including the physiochemical properties of the HCPs.
    • The coverage percentage of HCPs in ELISA.
    • Match between the ELISA standard and the harvest or mock samples.
    • ELISA Coverage of HCPs found in DS, including coverage of potential problematic HCPs.
    • Selected raw data, e.g., excel sheets.
  • Follow up on reports

    Follow up

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

Curious to know more?

Leading edge technologies

Whatever 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

A photo of an Alphalyse employee filling an ELISA plate with sample material.

Blog: Why results obtained by new ELISA kit differ from the original

Bridging from one HCP-ELISA kit to another, even just a new version, is not as straightforward as it sounds
Characterization of HCP ELISA standard

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 on ELISA standard

Characterization of a mock sample before ELISA development

CDMO: "The data saved us from using an unsuitable mock sample in development of a process-specific ELISA"
Evaluating five commercial HCP-ELISAs kits

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"
Analyzing individual A549 HCPs by mass spectrometry

Analysis of individual human residual proteins

US-based C&GT developer: "We can follow the reduction of specific A549 HCP in our adenovirus-based product"
HCP coverage analysis on uncommon expression system

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!"
Investigating HCPs in mAbs using a native digest

Video: Orthogonal HCPs of mAb product

A native digest approach lowered LOD - to reveal potentially problematic HCPs
mab webinar

Webinar: Why do we need orthogonal HCP analysis for mAbs?

How to investigate your mAb for potentially problematic HCPs < 1ppm
case study native digest lod mab

Revealing HCPs not detected by ELISA in antibody-drug product

mAb developer: "We combine ELISA and LC-MS to avoid having any HCP impurity surprises in Phase 2 or 3"
purified mab hcp

Purified mAb with difficult-to-remove HCP impurity (ubiquitin)

CMC Director: "The analysis' level of detail enables risk assessment of Host Cell Proteins in our mAb DS."
documenting hcps mAb biosimilar

Video: Documenting HCPs in mAb biosimilar vs. originator

Investigation of why one mAb biosimilar was more stable than the originator
Comparison of biosimilars to originator mAbs

Cleaner than the original: mAb biosimilar vs. originator

Biosimilar manufacturer: "We can use the HCP impurity data for both regulatory filing and marketing material"
mab study webinar

Webinar: Comparison of impurity profiles – and problematic HCPs – in mAbs

Study of 58 mAbs including 16 commercial originator and biosimilars
top 20 hcps in mabs

PDF: Top 20 Host Cell Proteins found in monoclonal antibodies

Our HCP profile study of 58 mAbs showed somewhat surprising results
HCP MS analysis for gene therapy products

Analyzing heterogeneous HCP mix from multiple species

Gene therapy developer: "We went from inconsistent, low-coverage ELISAs to reproducible and detailed HCP analysis"

Benefits of LC-MS HCP analysis for risk assessment

  • Identify specific HCPs of concern found in you drug samples (e.g. lipases, heat-shock proteins)

  • Quantify each of the HCPs of concern

  • Use LC-MS data (pI and MW for each protein) for data -driven process optimization to remove specific unwanted HCPs

  • Evaluate if your ELISA is capable of detecting HCPs of concern found in your process

  • Set up an MRM-LC-MS assay to check for specific proteins of concern

  • Follow up with validation of the assay under GMP

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
“Extremely professional service, high-level quality of the results, and excellent communication”

Program Manager
European Biotech
Testimonial Alphalyse
"Honestly, we were astonished to see the mass spec results: The detailed report enables us to optimize our CMC process and remove specific HCPs if we find it relevant."

Director, Regulatory CMC
Oncology-focused biopharmaceutical company
Testimonial Alphalyse
"We didn't want surprises in Phase 2 or 3, so we supplemented the HCP ELISA data with an orthogonal method. We got the results within a few weeks and could quickly implement them in our development process."

VP, Process Development and Manufacturing
Drug discovery company
Testimonial Alphalyse
"We hoped our biosimilar product was cleaner, more stable, and safer than the originator. However, the results were so favorable that not only can we use them as documentation for regulatory filing, but also as part of our marketing"

Head of Analytical Development
Developer of a mAb-biosimilar
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 knew 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
Aicuris logo
"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

Knowledge center

Why use native digestion instead of classic digestion with reduction and alkylation?

Unlike classic reduction and alkylation, native digestion largely keeps the antibody drug substance (DS) intact while Host Cell Protein (HCP) impurities digest into peptides. This procedure allows precipitation of the antibody DS, so the remaining supernatant with HCP peptides can be run through the mass spectrometer at a higher concentration.

This method increases peptide identification and lowers the LLOD to around 0.1 ppm, enabling a deeper look into potentially problematic HCPs and a more comprehensive risk assessment.

Can the identity of a specific HCP be coupled with its potential immunogenicity?

We have constructed an extensive database of HCPs of concern – based on the Biphorum database, 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.

As part of our analytical services, we check the list of all identified proteins in your project against our database and report, which might be of concern, providing you with links to the corresponding reference sources.

What sensitivity can you achieve for HCP characterization and quantification?

Our quantitative mass spectrometry-based assay is suitable for all protein systems. It typically achieves a lower limit of detection (LLOD) of 1-10 ppm, whereas the lower limit of quantification (LLOQ) is around 10-25 ppm.

For mAbs, we can increase the sensitivity to 0.1 ppm using a native digestion assay. On request, we can also set up an MRM-based assay for one or more individual protein(s) of interest with an LLOQ of 0.1 ppm.

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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