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Peptide mapping of mAb

Sequence confirmation of monoclonal antibody by LC MS

Do you need a sequence confirmation by high-coverage peptide mapping of mAb?

We offer to make a carefully designed peptide mapping of your mAb. Peptide mapping is used to confirm the amino acid sequence of monoclonal antibodies. Based on the aa sequence of your specific antibody we will select 2-5 digest enzymes for the experiment. This will ensure that we get almost 100% sequence coverage.

You can use the analysis for:

  • Process optimization: Use it to perform batch-to-batch comparison of sequence and modifications.
  • Biosimilars: Use  it to compare the sequence and modifications against originator mAb.

This is what our service includes:

  • We digest your sample with 2-5 enzymes.
  • We perform peptide mapping of mAb. We do this by nano LC MS MS or UV LC MS MS.
  • We compare to expected amino acid sequence. This includes comparison with a number of modifications.
  • You will receive a total sequence coverage map. Both for heavy chain and light chain.
  • The report includes a calculation of sequence coverage in percent.

Perhaps you want to combine with other mAb characterization services? We also offer: Molecular weight of mAbDisulfide analysis of mAb. • De-novo sequencing of mAb.

You can also send us a request if you need a custom service.

Services/Products
Protein analysis by Alphalyse

Antibody peptide mapping

  • Digestion with 1-3 enzymes, e.g. trypsin, Lys-C
  • UV LC-MS/MS, 90 min gradient
  • Common modifications included, e.g. deamidation, oxidation, truncations
  • Sequence coverage generally 75-100%

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Quality analysis, presented in easy-to-understand reports
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Technical details

Introduction
In Mass spectrometric Peptide Mapping the antibody is cleaved into smaller peptides using a specific protease. The peptides are analyzed by mass spectrometry and the observed peptides correlated to the antibody amino acid sequence.

The identified peptides in the peptide map thus confirms the specific amino acid sequences covered by the peptide map, as well as the identity of the antibody. Peptide mapping using a single protease will typically give a sequence coverage of 30-90%. By using two or more proteases this coverage can be increased to 95-100%.

Detailed characterization of an antibody requires careful investigation of the antibody sequence to select the best proteases, and choice of nano-flow LC-MS/MS or standard flow UV LC MS/MS to observe the peptides. The difference between the two techniques is related to the amount of material needed for the analysis – nano-flow can be conducted on low microgram antibody whereas the high-flow analysis requires at least 20 microgram per analysis. The high-flow analysis include a UV-trace of the chromatogram.

nano-flow LC-MS/MS
The peptide mapping using nanoLC-MS/MS is performed on a Dionex 3000 system running at 300 nl/min equipped with C18 capilary column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.

The analysis provide a Base peak chromatogram, and all MS/MS data is collected for peptides with mass/charge ratio from 200-1600 dalton.

high-flow LC-MS/MS with UV
The peptide mapping using high-flow LC-MS/MS is performed on a Agilent 1200 system running at 0.2 ml/min equipped with C18 column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.

The analysis provides both a UV- and a Base peak chromatogram, and all MS/MS data is collected for peptides with mass/charge ratio from 200-1600 dalton.

Sample requirements

Introduction
Antibody analysis by mass spectrometry requires a pure antibody in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin.

Antibody samples can be submitted in liquid or lyophilized.

  1. Purify the antibody

    1. The chromatographic antibody purity should be >90%
    2. Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS can be done on samples containing small amounts of salts, urea or detergent, but the best result are obtained with low buffer strength in volatile solvents without detergents
    3. Minimum amount ~ 0.5 mg
  2. Put the antibody sample into microcentrifuge tube

    1. Use a Eppendorf Safe Lock tube supplied, or similar tubes from your lab
    2. Lyophilize or speed vac the antibody to a solid sample to ensure stability during shipment
    3. Alternatively, freeze or refrigerate to +4 oC for cold shipment of the liquid sample

Sample shipment

shipping option
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
shipping option
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.

shipping option
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.
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.
  • 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.

shipping option
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

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