Glycan Analysis Information

Glycan and Glycoprotein analysis

Glycans serve a variety of structural and functional roles in membrane and secreted proteins, with the majority of proteins undergoing some degree of glycosylation during their synthesis. Regulatory agencies worldwide, including the FDA and EMA, are increasing the demands placed upon manufacturers to comprehensively analyze the glycosylation of their therapeutics, and also to demonstrate how process can affect glycan composition.

Changes in the glycosylation pattern of protein biotherapeutics have been shown to impact their half-life, stability, safety and efficacy. In general, glycans are divided into two main groups; O-linked and N-linked glycans. O-linked glycosylation involves the attachment of oligosaccharides to serine or threonine amino acid residues through an oxygen atom, and N-linked glycosylation involves the attachment of oligosaccharides to asparagine amino acid residues through a nitrogen atom.

More than 60% of therapeutic proteins are post translationally modified following biosynthesis by the addition of N- or O-linked glycans. Glycosylation of biotherapeutics can be influenced by a multitude of process related factors, such as pH, carbon source, dissolved oxygen, temperature during manufacture, and by the choice of expression system.

Whilst glycosylation is the most common post-translational modification of proteins, it is also the most demanding from an analytical point of view. In order to maintain consistent biotherapeutic glycosylation patterns, efficient manufacturing processes and effective glycan characterization are required. The complete analysis of a glycoprotein provides information on the primary structure of the oligosaccharides, as well as their variation at individual glycosylation sites being achieved through a number of different analytical approaches.

To view the products and workflows related to glycan analysis please visit our Glycan Analysis product page.

What is my glycosylation pattern? Intact glycoprotein analysis


Intact glycoprotein profiling is used to ascertain the pattern and degree of glycosylation. Due to the heterogeneity of the attached glycan moieties, intact glycoprotein profiling is best performed using high resolution accurate mass (HRAM) mass spectrometry (MS), together with chromatographic separation to gain full insight into the various glycoforms present on a protein or biotherapeutic.

Where do my glycans reside? Glycopeptide profiling


Glycan analysis can also be performed at the peptide level, with the goal of obtaining both glycan composition and peptide sequence at the site of glycosylation.

Which glycans are in my drug and what is their concentration? Glycan structure determination


Monitoring of specific glycan species or determination of relative quantities of a particular set of glycans provides important information for the development of biotherapeutics. Due to the complexity of glycan structures, quantitation and identification is performed upon release of glycans from the protein. N-linked glycans are released by enzymatic treatment, whereas O-linked glycans need to be released by chemical methods, as no enzyme exists for this purpose. Glycans have no chromophore and therefore have a poor response with conventional LC-UV detection; as such, they are commonly labeled with fluorescent tags prior to high sensitivity analysis by LC-fluorescence detection, the most common label being 2-aminobenzamide (2-AB). MS has emerged as one of the most powerful tools for glycan structure elucidation. However, as most glycans do not ionize efficiently 2-AB labeling can also be performed to improve sensitivity.

Labeled glycan protocols in AppsLab Library of Methods

Get instant access to over a dozen IC and UHPLC methods for 2-AA, 2-AB labeled N-glycans from therapeutic proteins. Or access our latest featured application from NIBRT Ireland on fast profiling of the N-glycan population in biotherapeutic antibodies by UHPLC-FLD with MS confirmation.

 Labeled N-glycan AppsLab methods
 Featured NIBRT application

What is the composition of sugars in my drug entity? Monosaccharide and sialic acid analysis


Monosaccharide composition, namely fucose, galactosamine, glucosamine, galactose, glucose and mannose, is routinely determined as the number and composition of sugar units bound to the protein and can impact the efficacy of biotherapeutics. Monosaccharides are weak acids and can be separated using anion-exchange chromatography under basic conditions. Samples are acid hydrolyzed to release monosaccharides and analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) after chromatographic separation.

Featured glycan analysis information learning content

White paper

Carbohydrate analysis, also known as glycosylation analysis, glycan analysis, or sometimes simply as sugar analysis, is of growing importance to sciences as diverse as pharmaceutical drug development, cancer research, stem cell research and biofuels development.

 Download the white paper

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TypeTitleProduct focusYear
Application CompendiumMonosaccharide and Sialic Acid Determinations in Biosimilars Using HPAE-PADIon Chromatography2015
Application NoteHILIC – an alternative separation technique for glycopeptidesHPLC / UHPLC2017
Application NoteAutomated Glycan Structural Isomer Differentiation Using Bioinformatics ToolLife Science Mass Spec2011
Application NoteComprehensive protein glycosylation comparison of an innovator monoclonal antibody to a candidate biosimilar by HILIC UHPLC analysisHPLC / UHPLC2017
Application NoteEvaluating Protein Glycosylation in Limited-Quantity Samples by HPAE-PADIon Chromatography2015
Application NoteFast profiling of the N-glycan population in biotherapeutic antibodies by UHPLC-FLD with MS confirmationHPLC / UHPLC2017
Application NoteHigh Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) Analysis of Mannose-6-PhosphateIon Chromatography2010
Application NoteIntegrated LC/MS Workflow for the Analysis of Labeled and Native N-Glycans from Proteins Using a Novel Mixed-Mode Column and a Q Exactive Mass SpectrometerWorkflow2014
Application NoteLabel-Free Analysis by UHPLC with Charged Aerosol Detection of Glycans Separated by Charge, Size, and Isomeric StructureHPLC / UHPLC2015
Application NoteSeparation of 2AA-Labeled N-Linked Glycans from Glycoproteins on a High Resolution Mixed-Mode ColumnChemistries and Consumables2013
Application NoteSeparation of 2AA-Labeled N-Linked Glycans from Human IgG on a High Resolution Mixed-Mode ColumnChemistries and Consumables2013
Application NoteSeparation of 2AB-Labeled N-Linked Glycans from Bovine Fetuin on a Novel Ultra High Resolution Mixed-Mode ColumnChemistries and Consumables2013
Application NoteAnalysis of Gentamicin Using a pH Stable Specialty Column for Aminoglycoside Antibiotics SeparationChemistries and Consumables2015
Application NoteAnalysis of Spectinomycin Using a pH Stable Specialty Column for Aminoglycoside Antibiotics SeparationChemistries and Consumables2015
Application NoteThe Determination of Carbohydrates, Alcohols, and Glycols in Fermentation BrothsIon Chromatography2013
Application NoteAn ultrafast, batch-to-batch comparison of monoclonal antibody glycosylationHPLC / UHPLC2017
Application NoteComparison of Therapeutic Antibody Originator and Biosimilar Glycosylation Using an Integrated Glycan Labeling SolutionHPLC / UHPLC2018
Application NotebookHigh-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) - Carbohydrates Analysis Application NotebookIon Chromatography2014
Application UpdateImproved Separation of Aminobenzamide (2-AB)-Labeled N-glycans from Human α1 Acid-Glycoprotein for Analysis by HPAE-FLDIon Chromatography2016
Application UpdateRapid Screening of Sialic Acids in Glycoproteins by HPAE-PADIon Chromatography2011
AppsLabIn-depth protein glycosylation comparison of a biosimilar candidate by hydrophilic interaction UHPLCChemistries and Consumables2016
AppsLabA fast separation of 2-AB labelled N-Glycans from polyclonal human serum IgGChemistries and Consumables2016
AppsLabA high resolution separation of 2-AA derivatized N-Glycans from a commercial chimeric IgG1 monoclonal antibody (Infliximab)Chemistries and Consumables2016
AppsLabUltrafast hydrophilic interaction UHPLC analysis of 2-AA labelled N-glycans from monoclonal antibodies (mAbs)Chemistries and Consumables2016
ArticleGlycobiology ToolsLife Science Mass Spec2016
BrochureCarbohydrate analysis with HPAE-PADIon Chromatography2013
BrochureThermo Scientific Glycan Analysis for Biotherapeutics: Join the Sweet Revolution in BiopharmaceuticalsWorkflow2015
eBookGlycan Analysis Workflows for BiotherapeuticsWorkflow2015
InfographicTop Four Things to Know about Glycan Analysis by Mass SpectrometryLife Science Mass Spec2013
PosterMiddle-down Approach for Monitoring Monoclonal Antibody Variants and DeglycosylationChemistries and Consumables2016
Poster NoteOrbitrap Based Mass Spectrometric Characterization of Antibody Drug Conjugates Engineered through Antibody GlycansLife Science Mass Spec2015
Poster Note2-Amino Benzamide Labeling of Oligosaccharides: How Much Sialic Acid Is Lost?Ion Chromatography2014
Poster NoteAn Ultra High Resolution Column and Mass Spectrometer for isomeric Separation and the Structural Identification of Labeled N-linked GlycansChemistries and Consumables2014
Poster NoteDirect Measurement of Sialic Acids Released From Glycoproteins, by High Performance Liquid Chromatography and Charged Aerosol DetectionHPLC / UHPLC2016
Poster NoteHPAE-FLD Method for Separation of Human α1 Acid-Glycoprotein and Bovine Fetuin 2-Aminobenzamide (2AB)-Labeled OligosaccharidesIon Chromatography2016
Poster NoteLabel-Free Analysis by UHPLC with Charged Aerosol Detection of Glycans Separated by Charge, Size, and Isomeric StructureHPLC / UHPLC2015
Poster NoteLabel-Free Profiling of O-linked Glycans by UHPLC with Charged Aerosol DetectionHPLC / UHPLC2016
PresentationAnalytical Strategies for Studying Glycosylation of BiopharmaceuticalsWorkflow2016
PresentationSix analytical strategies for studying glycosylation of biopharmaceuticalsWorkflow2016
Technical NoteGlycoprotein Monosaccharide Analysis Using HPAE-PAD with Eluent GenerationIon Chromatography2016
Technical NoteGlycoprotein monosaccharide analysis using HPAE-PAD with manually prepared eluentIon Chromatography2017
Technical NoteHPAE-PAD N-linked oligosaccharide profiling of IgGIon Chromatography2017
Technical NoteReview of peer-reviewed HPAE-PAD glycoprotein oligosaccharide analysis scientific literature evaluating electrochemical response.Ion Chromatography2013
Technical NoteUHPLC Analysis of 2-AB-labeled Dextran Ladder and Assignment of Glucose Units to Unknown GlycansChromatography Data Software2014
Technical NoteUHPLC Analysis of 2-Aminobenzamide-Labeled Glycans with the Vanquish Flex SystemHPLC / UHPLC2015
WebinarIon Chromatography2013
WebinarLife Science Mass Spec2015
WebinarChallenges in Glycomics and GlycoproteomicsLife Science Mass Spec2015
WebinarLife Science Mass Spec2015
White PaperHPAE-PAD for the Analysis of CarbohydratesIon Chromatography2013
White PaperOrbitrap Fusion MS for Glycan and Glycopeptide AnalysisLife Science Mass Spec2015
Application NoteSubunit Analysis Approach for the Determination of Fucosylation Levels in Monoclonal AntibodiesHPLC / UHPLC2018
Application NoteA Quick and Accurate Comparison of Biosimilar and Originator BiotherapeuticsHPLC / UHPLC2018
Application NoteHPAE-PAD method for determination of Hib capsular polysaccharide contentHPLC / UHPLC2021
Application NoteAn Improved Method for Galactosyl Oligosaccharide CharacterizationHPLC / UHPLC2022

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