Process-related residual proteins

  • 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?

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.

Mass spectrometry can quantify specific proteins that are utilized in the manufacturing process.

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.

Analysis includes:

  • Assay setup and sample analysis for specific residual protein in your pharmaceutical
  • LC-MS analysis in a robust microflow HPLC coupled online to the Sciex TrippleTOF 6600 mass spectrometer
  • SWATH LC-MS for reproducible identification and quantification of residual proteins
  • Report with the identified and quantified residual protein in each sample
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 w. quantity of specific residual proteins in each sample or purification step
  • 4 Follow-up by phone or email

Start the collaboration

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

Ask a question or get a quote

Send us a description of your project and we shall be happy to present you with a solution.

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We encourage you to call us or schedule a meeting to discuss your project in detail.

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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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  • "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. 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.

Quantification of specific HCPs

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  still being able 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 can 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. The library can be extended with new MS/MS data at any given time, and used for a research of old SWATH data.

MRM areas of the corresponding non-isotope peptides

MRM areas of the corresponding non-isotope peptides

Sample preparation

Sample requirements for SWATH LC-MS/MS

HCP analysis using SWATH technology requires sample material in sufficient amounts to obtain good data. It is important that samples are prepared 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
Related services

Meet the experts

For this type of analysis our experts include:

Do you need help?
Janne Skaarup Crawford

BSc in Cell Biology and Biochemistry

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Do you need help?
Rikke Raaen Lund

PhD in Biomedicine

Read more
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