Glycosylation analysis

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

Technical details, sample info, experts etc.

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 service:

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.

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Protein characterization for optimization of manufacturing processes.

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de-novo sequencing application note

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).

Separation of mAb subunits

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.

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
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:

Li Peng Lundgren

PhD in Protein Chemistry

Fen Yang Reske-Nielsen

PhD in Protein Chemistry

Stine Thyssen

PhD in Health Science

Glycosylation analysis

Determine the glycan structure and heterogeneity

Determine the glycan structure and heterogeneity

Select from the following service:

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

The process:

Learn more:

Glycan analysis by HILIC-MS

Start the collaboration

Ask a question or get a quote

Set up a phone meeting

Testimonials

"We are very pleased Alphalyses high-quality tests. We can ship them hundreds of samples at once and always receive the analytic results shortly after."

"Not only do we gain access to their hands, but we also get to pick their brains for mass spectrometry knowledge."

Knowledge center

More information

Learn more in these blog posts and application note:

Introduction to glycoylation analysis

Intact protein analysis

Glycopeptide analysis

Sample requirements for glycosylation analysis

Purify the protein/peptide

Put the protein sample into microcentrifuge tube

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

Meet the experts