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Glycosylation analysis

  • Exact molecular weight of glycoproteins
  • Peptide map of glycopeptides
  • Analysis of released N-glycans
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Technical details, sample info, experts etc.

Determine the glycan structure and heterogeneity of protein or antibody

Glycosylation of proteins is one of the most common post-translation modifications (PTMs) and also a critical quality attribute of biopharmaceutical products. Glycoproteins contain complex sugar moieties whose presence, absence, sites of attachment, and relative abundance can significan...

Determine the glycan structure and heterogeneity of protein or antibody

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

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

Which glycosylation analysis services should you choose?

Protein glycosylation analysis can be done at different levels. It can also be combined to achieve a complete glycan characterization. Below you can see examples of our services:

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

  • Firstly, determination of the exact molecular weight of glycoprotein by LC-MS (HILIC separation and online LC-MS).
  • Then, determination of the 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 LC, Fd, and single chain Fc (scFc) glycoforms.
  • Online LC-MS to determine the mass of each sub-unit.

Service 3: Glycopeptide map 

  • Glycoprotein digest with one of more proteases (trypsin or Lys-C) and analyzed by LC-MS/MS.
  • Identification of glycopeptides by searching against drug substance sequence and glycostructure database.

Service 4: Released N-glycan analysis

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 are very pleased Alphalyses high-quality antibody characterization services. We ship hundreds of samples and always receive the analytic results shortly after."

    High-throughput characterization analysis of antibodies for fast quality assessment

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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:

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Technical details

Introduction to glycosylation analysis

Identifying and characterizing glycosylated proteins are not straightforward and typically require various analyses to solve the task. The specific analysis will depend on the protein and the complexity of the glycosylation (O- and N-linked, number of glycosylation, etc.). Alphalyse provides a range of various analyses that can provide detailed information about protein and antibody glycosylations.

Intact protein analysis

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

HILIC glycosylation analysis

Separation of mAb subunits

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

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

Sample preparation

Sample requirements for protein glycosylation analysis

Protein analysis by mass spectrometry requires a pure protein in sufficient amounts to obtain good data. Samples must be 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 a 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 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?
Stine Thyssen

PhD in Health Science

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