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Pre-clinical and clinical batches

HCP analysis for documentation of pre-clinical and clinical batches

Do you want to identify host cell proteins in your pre-clinical tox batch or GMP phase I/II batches?

We have developed sensitive mass spectrometry and gel-based methods for detection, vizualization and relative quantification of host cell proteins down to low ppm concentrations. FDA and EMA are increasingly aware of mass spectrometry (MS) as an orthogonal method to ELISA.

Currently, we offer three orthogonal methods for comparison of batch-to-batch HCP profiles between pre-clinical and clinical batches:

  • HCP analysis by in-solution digestion and SWATH LC MS analysis
  • GeLC-MS/MS by 1D SDS PAGE, in-gel digestion and nanoLC MS protein identification
  • 2D PAGE MALDI MS by 2D electrophoresis, in-gel digestion and MALDI MS protein identification

The SWATH LC-MS method provides identification and relative quantification of HCPs down to low ppm concentrations. We digest the drug substance in-solution and analyse the complex peptide mixture by LC-MS/MS. Our scientists perform this analysis on a Sciex TrippleTOF 6600 instrument using data-independent SWATH data acquisition and ion library searching. The analysis is under development, promissing to deliver low-to-sub-ppm detection limits and highly reproducible quantification of each HCP.

The GeLC-MS/MS method is for identification of host cell proteins down to ~50 ppm. We separate the sample by 1D SDS PAGE; each lane is completely excised into 8 gel consecutive pieces and in-gel digested with trypsin. Afterwards, we analyze the peptides by high sensitivity nanoLC-MS/MS and search the data against a customized database containing host cell proteins from your specific cell line and the drug substance sequence. The MS-data is searched against this specifically designed database for HCP identification.

The GeLC-MS/MS analysis provides you with a comprehensive list of observed HCPs, amino acids sequences, theoretical pI/MW, database accession number, etc. The sensitivity of the analysis is in the low-to-sub nanogram range demonstrated on protein standards.

Read more about HCP analysis using GeLC-MS/MS in our poster Combining platform ELISA and orthogonal GeLC-MS/MS for HCP detection, and in the application note Detection of small HCPs by GeLC-MS/MS.

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(422 KB)

Download application note in high resolution
(4.6 MB)

The 2D PAGE with MALDI MS method is for visualization and identification of proteins separated by 2D PAGE according to pI and Mw. We stain the proteins by coomassie blue or fluorescense, then cut out the visible spots for in-gel trypsin digestion and MALDI MS/MS peptide analysis of each spot. The HCP protein identification is obtained by a MASCOT search in customized HCP protein database. Typically it identifies >95% of visible protein spots. The sensitivity of the analysis is demonstrated on known protein standards.

We also develop host cell protein analysis for up- and downstream bioprocess optimization

 

Services/Products
Protein analysis by Alphalyse

Identification of host cell proteins by GeLC MS/MS

  • Identification of host cell proteins
  • 1D PAGE nanoLC-MS/MS
  • Sensitivity ~50 ppm
  • HCP list with pI, Mw, aa sequence, acc number

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Quality analysis, presented in easy-to-understand reports
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Protein analysis by Alphalyse

ID of host cell proteins by in-solution digest & SWATH LC MS/MS

  • 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

$12,000.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote
Protein analysis by Alphalyse

Identification of host cell proteins by 2D PAGE & MALDI MS/MS

  • Identification of host cell proteins
  • 2D PAGE MALDI-MS/MS
  • Sensitivity ~all visible spots
  • HCP list with pI, Mw, aa sequence, acc number

$12,000.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote

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 400-1600) 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.

Introduction
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.

  1. Samples for Library generation

    1. The library generation requires typically a minimum of 100 ug of protein material.
    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
  2. Samples for SWATH analysis

    1. The SWATH analysis requires typically a minimum of 20 µg of protein material
    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

If the same sample is to be used for both library and SWATH analysis, just one vial will be needed (>120 µg).

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.

  • 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.
200 Page Mill Road, Suite 100
Palo Alto, CA, 94306
USA
Att: Jette Williams
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220, Odense
DENMARK
Att: Anette Rasmussen
Tel: +45 6310 6500
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.
  • 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. 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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