Quantify Host Cell Proteins of concern

  • Determine HCPs specific to your process
  • Obtain the exact amount in all purification steps
  • Results to be used for removal of immunogenic and proteolytic proteins

Do you want to monitor specific HCPs in your drug substance?

Individual HCPs can influence product safety even at very low level. The problematic HCPs include:

  • Immunogenicity: Induce immune response against drug substance or human protein
  • Enzymatic activity: Degrade the drug substance or co...

Do you want to monitor specific HCPs in your drug substance?

Individual HCPs can influence product safety even at very low level. The problematic HCPs include:

  • Immunogenicity: Induce immune response against drug substance or human protein
  • Enzymatic activity: Degrade the drug substance or constituents in the drug product
  • Biological activity or homology to the drug substance

Therefore, we offer to determine the quantity of specific HCPs in your sample – by a targeted and sensitive LC-MS/MS analysis based on Multiple Reaction Monitoring (MRM) methods.

The analysis include identification of unique signature peptides for the specific protein. For quantitation, we preferable use the intact reference protein if available, or alternatively, the synthetic isotope-labeled signature peptides. The reference protein or the labeled peptides are spiked in  known amounts into the samples to generate standard curves for linearity and calibration.

Our standard service includes

  • Identification of up to 3 unique signature peptides for quantitation
  • Standard curves and linearity assessment for dilution series of the HCP of concern and quantification standards
  • Absolute quantification of HCPs of concern by MRM LC-MS/MS
  • Finally, reporting with determined protein amounts and analysis details
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The process:

  • 1 Contact us to discuss your project and receive a project proposal
  • 2 Analysis phase lead by Alphalyse appointed principal investigator
  • 3 Report w. the absolute quantity of individual HCPs
  • 4 Follow-up by phone or email

Start the collaboration

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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  • "The most sensitive, yet robust, Host Cell Protein analysis we’ve seen"

    Analyses of key protein features, stability and impurity levels

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  • "We shortened the purification process with at least 1 year"

    Detailed host cell protein overview enables targeted elimination of impurities

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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 description


The absolute quantification of specific protein in complex matrices are based on the selection of specific peptides as signature peptides and used for assay development based on Multiple-Reaction-Monitoring (MRM). The project are typically divided into three parts.

Part 1: Feasibility study

The purpose of the feasibility study is to investigate the possibility to develop an assay that can be used for the quantification of the specific protein in the matrix. The feasibility study will include:

  1. Planning phase – evaluate potential sample preparation protocols and signature peptides, i.e. specific marker peptides, based on the protein sequences.
  2. Digest and LC-MS/MS analysis to ensure that specific signature peptides are formed and that this/these peptides do not appear as part of miss-cleaved peptides or modified peptides (oxidized, deamidation etc.). The digest will be performed with urea as denaturation buffer and using the proteases Lys-C and trypsin.
  3. Short report evaluating the possibility for Alphalyse being able to develop an assay that can be used for the purpose of the project including an estimation of LOQ and LOD.
Calibration curve for MRM quantification

Standard curve for MRM quantification of specific HCP

Part 2: Assay development

  1. Optimizing the sample preparation and digest of the proteins for mass spectrometric identification of released peptides.
  2. Ordering of isotope labeled peptide standards (heavy peptides).
  3. Sample preparation and digest of the protein with addition of heavy peptides for quantitative measurements. Selection of optimal product ions for each peptide. Evaluate quantitative results and select quantitative peptides for quantification assay.
  4. Integrate workflow; sample prep. incl., reduction/alkylation, protein digestion, peptide cleanup, LC-MS/MS analysis and MRM measurement. Quantitative measurements using heavy peptides and external calibration curves on the protein reference standard.
  5. Qualification of quantification assay; demonstration of measurement range, linearity, precision and accuracy.

Part 3: Sample analysis.

  1. Using the final assay to measure real samples.
SWATH MRM chromatogram

MRM areas of the corresponding non-isotope peptides

Sample preparation

Sample requirements for protein quantification

Protein quantification by mass spectrometry requires a protein in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin. To develop MRM quantification assays requires the protein in pure form and a matrix sample without the protein similar to the matrix of the real samples to be analyzed.

Protein samples can be submitted in liquid or lyophilized.

  1. Purify the protein

    1. The chromatographic protein/peptide purity should be >90%
    2. Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS can be done on samples containing small amounts of salts, urea or detergent, but the best result are obtained with low buffer strength in volatile solvents without detergents
    3. Minimum amount ~ 1 mg
  2. Put the protein sample into microcentrifuge tube

    1. Use a Eppendorf Safe Lock tube supplied, or similar tubes from your lab
    2. Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment
    3. Alternatively, 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?
Janne Skaarup Crawford

BSc in Cell Biology and Biochemistry

Read more
Do you need help?
Rikke Raaen Lund

PhD in Biomedicine

Read more
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