Glycosylation analysis

Determination of number, position and type of glycosylation in proteins

The glycan structure is a critical attribute of your protein!

We offer determination of the number, position and type of glycosylation in your drug substance protein. The analysis can be used for comparing different batches or originator versus biosimilar.

Glycosylation of proteins is one of the most common post translation modifications in biopharmaceuticals. Glyco-proteins contain complex sugar moieties whose presence, absence, sites of attachment, and relative abundance can have a major impact on the efficacy and stability of the drug substance.


Glycosylation analysis can be done at three different levels and you can combine one or more to get the full glycan characterization:

Part 1: Analysis on intact protein

  • Exact molecular weight of glycoprotein determined by UV-LC ESI-MS
  • Exact molecular weight of deglycosylated protein determined by UV-LC ESI-MS

Part 2: Glyco-peptide mapping 

  • Glycoprotein is digested with one of more proteases and analyzed by nano LC-MS/MS
  • Searched against drug substance sequence and glycostructure database

Part 3: Released N-glycan analysis and quantitation

  • PNGase F treatment to release N-linked glycans
  • Fluorescent labelling using RapiFluor-MS labelling kit
  • Separation of glycans by HILIC chromatography, detection by fluorescence HPLC and mass spectrometry
  • Glycan profile and type by FLD-trace and glycan mass

You can also send us a request so we can adjust the analysis to meet your specific needs

Protein analysis by Alphalyse

Glycosylation analysis of drug substance protein

  • Number, position and type of glycosylation
  • Exact MW of intact and deglycosylated protein
  • Glycopeptide mapping by LC-MS and MALDI
  • Released N-glycan analysis by fluorescence and LC-MS

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The identification and characterization of glycosylated proteins is not straightforward, and typically requires a range of different analysis to solve the task. Alphalyse provides a range of different analysis that can provide detailed information about glycosylations. The specifc analysis will depend on the protein and the complexity of the glycosylation (O- and N-linked, number of glycosylations etc.).

Intact protein analysis
The intact mass is determined very accurate by ESI-MS analysis on a high resolution Q-Tof instrument (Bruker Impact). The analysis of N-glycans is performed before and after treatment PNGase F, an amidase that cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides.

Glycopeptide analysis
The glycoprotein is digested with a set of proteases cutting the protein at frequent locations in the peptide backbone and the resulting small glycopeptide fragment are analyzed by LC-ESI-MS/MS analysis. The analysis can be combined with specific fraction collection of glycopeptides and/or glyco-enrichment by HILIC chromatography followed by MALDI-TOF MS/MS analysis. All data is compared with expected sequence of the drug substance (protein).

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

Protein samples can be submitted in liquid or lyophilized.

  1. Purify the protein/peptide

    1. The chromatographic protein 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 protein sample into microcentrifuge tube

    1. Use a Eppendorf Safe Lock tube supplied, or similar tubes from your lab
    2. Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment
    3. Alternatively, 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.

  • 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
Att: Jette Williams
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220, Odense
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
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