How to characterize antibody glycosylation by HILIC-MS

Mar 4. 2019


How do I characterize the N-glycosylation of my antibody?

What is N-glycosylation and how do I characterize the N-glycosylation of my antibody? Is it possible to compare batch-to-batch variations of antibody glycosylation?


Glycosylation is a post-translation modification (PTM). It occurs when a carbohydrate moiety (glycan) is added to the protein. Since there are different types of glycosylations, you distinguish them by the glycan linkage site, how the glycan is branched, the glycan length and the form of glycan linked to the protein [1, 2].

N-linked glycosylation occurs when glycans binds to asparagine’s amino group. This type of glycosylation is crucial for protein folding, stability and solubility. It also helps with cell-cell signaling and much more [2, 3].

Many antibodies are N-linked glycoproteins. Thus a comprehensive analysis of your antibody’s structure should include a detailed look at the N-glycan structure [3, 4, 5].

The HILIC-MS based approach – robust and thorough antibody glycosylation analysis

For a comprehensive characterization of N-glycosylation of a monoclonal antibody (mAb) you must conduct analysis at different levels [2, 3].

The typical four complementary HILIC MS-based analysis techniques, which we suggest, include:

  • Top-down analysis. Based on accurate mass measurement of the intact mAb.
  • Middle-up approach. Based on accurate mass measurement of the mAb subunits (LC, Fd´, and scFc).
  • Bottom-up characterization of the N-glycopeptides.
  • Free N-glycan analysis.

A combination of these analyses provides a full picture of your antibody’s N-glycan structure.

NB: For a detailed analysis of glycans you must produce a pure antibody sample in at least 500 µg quantities.

Glycosylation analysis

Benefits of HILIC-MS analysis for antibody glycosylation assessment

The workflow provides a reproducible, accurate, sensitive and fast glycan assessment. Therefore, it is very useful for routinely monitoring of critical glycoforms. This also includes those of low abundance at intact protein, subunits, peptides, and glycan levels.

The HILIC solution for antibody glycosylation analysis is particularly beneficial to the biopharmaceutical industry. They thus use it for control of glycoprotein product quality and consistency. The method also makes it easy to compare batch-to-batch and process modifications. You can even use the analysis to compare different batches on a quantitative level [2].

Need help with your antibody characterization?

Visit the Alphalyse website for more information on glycosylation analysis and antibody glycosylation.

We can also help with your antibody analysis, e.g determine the molecular weight, sequence, precise amount, drug-to-antibody ratio and impurities (host cell proteins) in your antibody sample.


[1]         Varki et al: “Essentials of Glycobiologi, 2nd Edition“, Cold Spring Harbor Laboratory Press, 2009

[2]         Dalziel et al: “Emerging principles for the therapeutic exploitation of glycosylation.“, Science2014

[2]         Imperiali et al: “Effect of N-linked glycosylation on glycopeptide and glycoprotein structure.“, Current Opinion in Chemical Biologi1999

[4]         Crispin et al: Targeting host-derived glycans on enveloped viruses for antibody-based vaccine design.“, Current Opinion in Virology2015

[5]         Razaghi et al: “Improved therapeutic efficacy of mammalian expressed-recombinant interferon gamma against ovarian cancer cells.“, Experimental Cell Research2017

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