Process-related impurities

  • Document process-related residuals in final drug substance
  • Evaluate clearance throughout purification steps
  • Validate process and establish acceptance criteria

Do you want to monitor residual proteins added during manufacturing?

Mass spectrometry can quantify specific proteins that are utilized in the manufacturing process, with a customized process-related impurities analysis. This is especially important in the development of gene therapy products.

Common process residuals include added growth factors, Benzonase® Nu...

Do you want to monitor residual proteins added during manufacturing?

Mass spectrometry can quantify specific proteins that are utilized in the manufacturing process, with a customized process-related impurities analysis. This is especially important in the development of gene therapy products.

Common process residuals include added growth factors, Benzonase® Nuclease, Protein A, enzymes for site-specific PEGylation, aminopeptidase, etc. Downstream purification aims to reduce these process additives in the drug substance, but standard analyses cannot detect trace amounts.

Therefore, Alphalyse offers analysis development for your residual protein of interest, in both final drug substance and process samples with low-to-sub ppm detection limit.

A process related impurities analysis includes:

  • Assay setup and sample analysis for specific residuals in your pharmaceutical
  • LC-MS analysis in a robust microflow HPLC coupled online to the Sciex TripleTOF 6600 mass spectrometer
  • SWATH LC-MS/MS for reproducible identification and quantification of process related impurities
  • Report with the identified and quantified impurities in each sample
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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. quantity of specific process related impurities in each sample or step
  • 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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    Testimonials

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

      Detailed host cell protein overview enables targeted elimination of impurities

      See more
    • "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 spectrometry technology is a data independent acquisition strategy used for e.g. residual protein and process related impurities analysis. This means that all MS/MS fragmentation data for every peptide in your sample is collected.

    This is only possible due to the high speed of modern mass spectrometers.

    Calibration curve for MRM quantification

    Standard curve for MRM quantification of specific HCP

    The Sciex 6600 TripleTof instrument makes it possible to divide the whole mass range into sequential windows of size from 5-50 daltons. And it makes it easy to 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 therefore obtain MS/MS data for all peptides without having the instrument to select certain peptides for MS/MS fragmentation.

    The MS/MS data set is linked to the retention time and can contain 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. We can extend the library with new MS/MS data at any given time, and use it for a research of old SWATH data.

    SWATH MRM chromatogram

    MRM areas of the corresponding non-isotope peptides

    Sample preparation

    Sample requirements for process related impurities analysis with SWATH LC-MS/MS

    HCP analysis 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.

    Samples can be submitted 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 a 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?
    Janne Skaarup Crawford

    BSc in Cell Biology and Biochemistry

    Read more
    Do you need help?
    Rikke Raaen Lund

    PhD in Biomedicine

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