Glycosylation analysis

  • Exact molecular weight of glycoproteins
  • Peptide mapping of glycopeptides
  • Analysis of released N-glycans

Determine the glycan structure and heterogeneity

Glycosylation of proteins is one of the most common post translation modifications (PTMs) and also a critical quality attribute of biopharmaceutical products. Glyco-proteins contain ...

Determine the glycan structure and heterogeneity

Glycosylation of proteins is one of the most common post translation modifications (PTMs) and also a critical quality attribute of biopharmaceutical products. Glyco-proteins contain complex sugar moieties whose presence, absence, sites of attachment, and relative abundance can have a major impact on the efficacy, stability and activity of proteins and monoclonal antibodies (mAbs). Hence, it is essential to determine the glycan structure and heterogeneity, through glycosylation analysis.

A full glycosylation analysis includes the number, position and type of glycosylation in your protein or mAb. The analysis is especially useful for comparing different batches, originator versus biosimilar and process development samples.

Select from the following glycosylation analysis services:

Glycosylation analysis can be done at different levels. It can also be combined to achieve a full glycan characterization.

Service 1: Separation and exact molecular weight of protein/mAb glycoforms

  • Firstly, determination of exact molecular weight of glycoprotein by LC-MS (HILIC separation and online LC-MS).
  • Then, determination of exact molecular weight of deglycosylated protein by LC-MS (HILIC separation and online LC-MS).

Service 2: Separation and exact molecular weight of mAb sub-units

  • IdeS digestion and reduction to sub-units.
  • HILIC separation of both LC, Fd’ and single chain Fc (scFc) glycoforms.
  • Online LC-MS to determine the mass of each sub-unit.

Service 3: Glyco-peptide mapping 

  • Glycoprotein digest with one of more proteases (trypsin or Lys-C) and analyzed by LC-MS/MS.
  • Optional: Collection of glycopeptide fractions and offline MALDI TOF/TOF analysis.
  • Identification of glycopeptides by searching against drug substance sequence and glycostructure database.

Service 4: Released N-glycan analysis

  • PNGase F treatment to release N-linked glycans.
  • Fluorescent labelling using Waters RapiFluor Glycan labelling kit.
  • Separation of glycans by HILIC chromatography, detection by fluorescence HPLC and mass spectrometry.
  • Optional: Collection of the glycan fractions for offline MALDI TOF/TOF analysis.
  • Identification of both glycan profile and type, by FLD-trace and glycan mass.

Please send us a request so we can adjust the glycosylation analysis to meet your specific needs.

You can also take a look at our other protein characterization services.

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The process:

  • 1 Contact us to discuss your project and receive a project proposal
  • 2 Analysis phase lead by Alphalyse appointed principal investigator
  • 3 You receive a report with a clear presentation of the findings
  • 4 Follow-up by phone or email

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We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

Technical details

Introduction to glycoylation analysis

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).

HILIC glycosylation analysis

Separation of mAb subunits


HILIC separation of labelled mAb N-glycans followed by MS/MS

HILIC separation of labelled mAb N-glycans followed by MS/MS

Sample preparation

Sample requirements for glycosylation analysis

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 works on samples containing small amounts of salts and urea, but you get the best result 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 Eppendorf Safe Lock tubes, or similar high-quality 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
Related services

You can combine this analysis with other characterization analyses such as:

Meet the experts

For this type of analysis our experts include:

Do you need help?
Li Peng Lundgren

PhD in Protein Chemistry

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Do you need help?
Fen Yang Reske-Nielsen

PhD in Protein Chemistry

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Do you need help?
Stine Thyssen

PhD in Health Science

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