HCP levels in clinical batches

  • Compare the HCP levels between pre-clinical and clinical batches
  • Orthogonal method to ELISA to document HCP content in clinical batches
  • Regulatory documentation for FDA and EMA

Do you want to quantify hcp levels in pre- or clinical batches?

Before entering clinical trials and submitting NDA or BLA, it is highly critical to determine the level of process-related impurities, like Host Cell Proteins (HCPs). 

By applying mass spectrometry, you get the complete overview of the HCP content beforehand, to potentially avoid costly delays at later-stage trial approval. Also, you can ...

Do you want to quantify hcp levels in pre- or clinical batches?

Before entering clinical trials and submitting NDA or BLA, it is highly critical to determine the level of process-related impurities, like Host Cell Proteins (HCPs). 

By applying mass spectrometry, you get the complete overview of the HCP content beforehand, to potentially avoid costly delays at later-stage trial approval. Also, you can compare different clinical batches to check for manufacturing consistency. 

The method identifies and quantifies the host cell proteins in purified drug substance batches by SWATH LC-MS/MS. The analysis thus provides you with a more detailed and accurate HCP documentation than HCP ELISA. The total HCP content is measured in ng HCP/mg drug substance (ppm), and each host cell protein is identified and quantified individually.

For pure drug substance batches with low HCP levels (1-500 ppm) you receive a highly sensitive and reproducible analysis. It both delivers identification and quantification of host cell proteins down to low ppm concentrations.

An analysis of HCP levels includes:

  • Optimized sample preparation for pure drug substance samples
  • Spike-in of internal standard proteins (0-2000 ppm) for quantification linearity and LOD
  • LC-MS peptide analysis on a robust microflow HPLC and mass spectrometer, using SWATH LC-MS/MS for reproducible HCP identification and quantification
  • Report with table of total HCP and individual HCPs amounts, as well as comparison between batches

Send us a description of your project and we will contact you to discuss how you can benefit from an analysis of HCP levels.

MoreLess details

The process:

  • 1 Discussion of your project and preparation of a project proposal
  • 2 Analysis phase lead by Alphalyse appointed principal investigator
  • 3 Report w. list of individual HCPs and total HCP content, compared between batches
  • 4 Follow-up by phone or email

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Testimonials

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

    Analyses of key protein features, stability and impurity levels

    Explore customer case
  • "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

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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®?

The SWATH mass spec technology is a data independent acquisition strategy used to measure hcp levels. This means that we collect all MS/MS fragmentation data for every peptide in your sample.

This 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. We can thus perform MS/MS fragmentation covering the full mass range (m/z 350-1700) within a few seconds. We set up the individual windows based on analysis of your samples, and typically get 50-100 windows.

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

HCP analysis of clinical batch by using ion library

HCP analysis of Batch 1 with comparison of sample to ion library

The MS/MS data set links to the retention time and contains MS/MS data from multiple peptides. The extraction of these MS/MS data are done using a spectra library linking to peptide sequences in a library.

The spectra library can be prepared in many ways. The most simple 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. The library can be extended with new MS/MS data at any given time, and used for a research of old SWATH data.

HCP analysis of 3 clinical batches

HCP analysis of 3 clinical batches

Sample preparation

Sample requirements for analysis of HCP levels

Analysis of HCP levels using SWATH technology requires sample material in sufficient amounts to obtain good data. It is important to prepare samples in a clean laboratory to avoid contamination with human keratin.

Submit samples in liquid or lyophilized.

Samples for Library generation and SWATH analysis

  1. The analysis requires typically 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 supplied 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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