HCP profiling for bioprocess optimization

  • Evaluate clearance of individual HCPs between purification steps
  • Compare HCP profiles for tech-transfer batches between CMOs
  • Monitor HCP content during process changes and scale-up

Do you want to improve and speed up your process development?

The best way to identify Host Cell Proteins (HCPs) in your process samples is by LC-MS analysis. For bioprocess optimization, you will benefit from an optimized workflow providing reproducible HCP profiling through identification and quantification of individual HCPs.

The HCP analysis provides the identity and quantity of each HCP, in addition t...

Do you want to improve and speed up your process development?

The best way to identify Host Cell Proteins (HCPs) in your process samples is by LC-MS analysis. For bioprocess optimization, you will benefit from an optimized workflow providing reproducible HCP profiling through identification and quantification of individual HCPs.

The HCP analysis provides the identity and quantity of each HCP, in addition to the total HCP content (ng HCP/mg drug substance). Thus, the results can be used for data-driven optimization of downstream purification to remove process-related impurities and bring down HCP levels.

HCP profiling for all expressions systems and types of samples

One of the biggest advantages of LC-MS-based HCP analysis is that the same assay can be applied to all expression systems and all types of samples – from early harvest to pure drug substance. Thus, HCPs can be determined in, e.g., the four main cell-based expression systems: bacterial, yeast, insect, and mammalian. The only requirement is that the organism’s proteome needs to be sequenced.

Common yeast hosts include Pichia pastorisSaccharomyces cerevisiaeSchizosaccharomyces pombe, and Hansanuela polymorpha. Besides Escherichia coli, which we often analyze, common bacterial hosts include Lactococcus lactis, Bacillus subtilis, Pseudomonas fluorescens, Staphylococcus aureus, and Brevibacillus choshinensis. Insect cell lines, such as SF21, Autographa californica, Lepidoptera, and Spodoptera frugiperda, are also worth mentioning. Finally, we see many cell lines of mammalian origin, including CHO, HEK 293, HeLa, L929, BHK, Y79, U2OS, HepG2, NIH3T3, NS0, P19, CAD, MCF-7, and J558L.

The standard HCP profiling service includes

  • Generic sample preparation for process samples in different matrices
  • LC-MS peptide analysis on a robust microflow HPLC and Sciex TripleTOF 6600 mass spectrometer using SWATH mass spectrometry (LC-MS/MS) for reproducible HCP identification and quantification
  • Report with a list of HCPs and their physiochemical properties (MW, pI, AA sequence, database accession number) for each process-sample – for use in your bioprocess optimization.

Send us a description of your project, and we will be glad to discuss HCP profiling analysis with you.

MoreLess details

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 listing individual HCPs with physio-chemical properties - for each process step
  • 4 Follow up by phone or email

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

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Testimonials

  • "Alphalyse´s HCP analysis saved us the development of an ELISA assay that may not have worked anyway."

    Innovative development of an orthogonal method for Host Cell Protein analysis

    Explore customer case
  • "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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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

What is SWATH LC-MS/MS-based HCP analysis?

The SWATH mass spec technology is a data-independent acquisition strategy. It means that all MS/MS fragmentation data for every peptide in your sample is collected. However, it is only possible due to the high speed of modern mass spectrometers.

The Sciex 6600 TripleTof instrument makes it possible to divide the whole mass range into sequential windows of size from 5-50 daltons and still perform MS/MS fragmentation covering the entire mass range (m/z 350-1700) within a few seconds. We set up the separate windows based on analysis of your samples and typically get 50-100 windows.

In this way, we can obtain MS/MS data for all peptides without having the instrument to select specific peptides for MS/MS fragmentation.

Table of HCPs in different process step samples

Table of HCPs in different process step samples

The MS/MS data set is linked to the retention time and can contain MS/MS data from multiple peptides. These MS/MS data are extracted using a spectra library linking to peptide sequences in a library.

The spectra library can be prepared in many ways. The simplest way is to run a few samples in data-dependent acquisition mode to collect good MS/MS data. Or it can be more advanced based on off-line fragmentation followed by MS/MS analysis of each fraction. Finally, the library can be extended with new MS/MS data at any given time and used to research old SWATH data.

8 selected HCPs through the 6 process steps

Eight selected HCPs through the six process steps

Sample preparation

Sample requirements for SWATH LC-MS/MS

HCP analysis using SWATH technology requires sample material in sufficient amounts to obtain good data. Also, samples must be prepared in a clean laboratory to avoid contamination with human keratin.

You may submit samples in liquid or lyophilized.

Samples for Library generation and SWATH HCP analysis

  1. The analysis requires a minimum of 500-1000 μg of protein material for each sample
  2. The samples should not contain large amounts of detergents
  3. The concentration should be >0.5 µg/µl
  4. Use an Eppendorf Safe Lock tube or similar tubes
  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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