Process-related residual proteins

Residual proteins from process additives must be determined in biopharmaceuticals

Do you want to analyze the process-related residual proteins in your drug substance?

We quantify specific proteins that are utilized in the manufacturing process to produce the protein of interest. Down-stream purification aims to reduce these process additives in the drug substance, but standard analyses are not able to detect these trace amounts. For instance, the protein process residuals include growth factors, Benzonase nuclease, Protein A, enzymes for site-specific PEGylation, aminopeptidase, etc.

You can use the results for:

  • FDA/EMA documentation of process-related residuals in final drug substance.
  • Evaluation of clearance throughout purification steps.
  • Process validation and establishment of batch acceptance criteria.

Alphalyse offers analysis development for your residual of interest, and analysis of both final drug substance and process samples. Above all, we perform low-to-sub ppm detection along with identification and quantification of key residual proteins.

The 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 also quantification of residual proteins.  What is SWATH?  >>
  • Finally, you receive a report with the identified and quantified residual protein in each sample

Send us a description of your project and we will be glad to discuss it with you.

host cell protein analysis by mass spectrometry

Analysis of Process-Related Residual Proteins

  • Identification of host cell proteins
  • SWATH LC-MS/MS analysis on Sciex TrippleTOF 6600 mass spectrometer
  • Database search in custom HCP Ion library
  • HCP list with pI, Mw, aa sequence, accesion number

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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.
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.

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

Ship your protein samples by a courier company of your own choice

Place your samples into micro tubes and pack in a suitable transport box. Remember to print and include the sample submission form.

  • Contact a courier company of your choice (FedEx, UPS, DHL, TNT, World Courier etc.). Use the following sentence to describe the content: “Protein material – Non-toxic, non-hazardous, for research purpose only“.
  • Use cool packs or cooling units if your samples should be stored below 5°C/40°F
  • Ship the samples to the office closest to you (Canadian customers, please ship to Europe):
Customers in the USA
Alphalyse, Inc.
299 California Avenue, Ste 200
Palo Alto, CA 94306
Att: Thomas Kofoed
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220 Odense SO
Att: Anette Rasmussen
Tel: +45 6310 6500


NB: The US office is CLOSED 24 DEC – 1 JAN.
Make sure that your samples are delivered OUTSIDE of this period

Order a prepaid FedEx Air Waybill < 1 lb./ 500g

A prepaid FedEx Air Waybill can be used for fast and easy shipment of samples in packages up to 1 lb./ 500g.
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack in suitable transport box. Remember to print and include the sample submission form.
  • Print the PDF document and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to send your samples stored at room temperature, this option is usually the right choice. If you fill your box with cooling packs, the weight will most often exceed 1 lb./500g.

Order a prepaid FedEx Air Waybill < 10 lb./5kg (e.g. for packages with cooling packs)

This prepaid FedEx Air Waybill can be used for shipment of samples in packages up to 10 lb./ 5kg, with or without cooling packs (do not use dry ice!).
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack them in a suitable transport box. Remember to print and include the sample submission form.
  • Fill with cooling units/cold packs (do not use dry ice!).
  • Print the PDF and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to fill your transportation box with cooling packs, select this shipping option. If you plan to use dry ice, you cannot use this Waybill!

Get a quote for shipment by World Courier

The World Courier service can be used for shipment of samples of all types. If you select this option, we will contact you to provide a quotation based on location and shipping conditions. You will receive a prepaid Air Waybill which will be invoiced together with your analysis results. This is usually a good option if FedEx does not service your location.
With the prepaid World Courier service you get:

  • GxP compliant processes to ensure maximal security and traceability
  • Door-to-door solution on a dedicated vehicle
  • Possibility of providing qualified packaging, temperature monitoring and dry ice
  • Shortest possible transit-time through “next flight out”-procedure
  • Customs clearance and help with paperwork
Order analysis now
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