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.

ICH (Q6B) recommends 6 test approaches for characterization and confirmation of biological products: "For glycoproteins, the carbohydrate content and structure (neutral sugars, amino sugars, and sialic acids) is determined."

 Explore the workflows


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.

Glycan analysis poses major challenges for the biopharma industry. How can new technology lighten the load?

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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.

Glycosylation plays an important part in protein structure and function. Factors such as cell age, culture conditions, and purification affect the nature of protein glycosylation.

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Featured glycan analysis information learning content

Applications compendium

HPAE-PAD is a well-established method for carbohydrate analysis, separating carbohydrates through specific interactions between the hydroxyl and carboxyl groups of carbohydrates based on charge, size, composition, isomerism, and linkage.

Application update

Glycosylation is a key intracellular process that involves interactions of various enzymes and substrates.

Application note

A fully integrated workflow for glycan profiling and structural characterization of fluorescently labeled N-glycans released is demonstrated in this application.

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

iBook

Learn about our five simple glyco-analysis workflows in this interactive iBook for iPad; filled with product information, videos, editorials and webinars.

 Download the iBook

Poster note

This ASMS poster demonstrates that HPAE-PAD/MS is an excellent tool to profile and annotate complex mixture of native glycans released from different glycoproteins.

 Download the poster note


Featured videos


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Type Title Product focus Year
Application Compendium Monosaccharide and Sialic Acid Determinations in Biosimilars Using HPAE-PAD Ion Chromatography 2015
Application Note Ligand Binding Mass Spectrometric Immunoassay (LB-MSIA™) Workflow with Deglycosylation for Therapeutic Antibodies MSIA 2015
Application Note HILIC – an alternative separation technique for glycopeptides HPLC / UHPLC 2017
Application Note Automated Glycan Structural Isomer Differentiation Using Bioinformatics Tool Life Science Mass Spec 2011
Application Note Comprehensive protein glycosylation comparison of an innovator monoclonal antibody to a candidate biosimilar by HILIC UHPLC analysis HPLC / UHPLC 2017
Application Note Evaluating Protein Glycosylation in Limited-Quantity Samples by HPAE-PAD Ion Chromatography 2015
Application Note Fast profiling of the N-glycan population in biotherapeutic antibodies by UHPLC-FLD with MS confirmation HPLC / UHPLC 2017
Application Note High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) Analysis of Mannose-6-Phosphate Ion Chromatography 2010
Application Note Integrated 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 Spectrometer Workflow 2014
Application Note Label-Free Analysis by UHPLC with Charged Aerosol Detection of Glycans Separated by Charge, Size, and Isomeric Structure HPLC / UHPLC 2015
Application Note Separation of 2AA-Labeled N-Linked Glycans from Glycoproteins on a High Resolution Mixed-Mode Column Chemistries and Consumables 2013
Application Note Separation of 2AA-Labeled N-Linked Glycans from Human IgG on a High Resolution Mixed-Mode Column Chemistries and Consumables 2013
Application Note Separation of 2AB-Labeled N-Linked Glycans from Bovine Fetuin on a Novel Ultra High Resolution Mixed-Mode Column Chemistries and Consumables 2013
Application Note Analysis of Gentamicin Using a pH Stable Specialty Column for Aminoglycoside Antibiotics Separation Chemistries and Consumables 2015
Application Note Analysis of Spectinomycin Using a pH Stable Specialty Column for Aminoglycoside Antibiotics Separation Chemistries and Consumables 2015
Application Note The Determination of Carbohydrates, Alcohols, and Glycols in Fermentation Broths Ion Chromatography 2013
Application Note An ultrafast, batch-to-batch comparison of monoclonal antibody glycosylation HPLC / UHPLC 2017
Application Notebook High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) - Carbohydrates Analysis Application Notebook Ion Chromatography 2014
Application Update Improved Separation of Aminobenzamide (2-AB)-Labeled N-glycans from Human α1 Acid-Glycoprotein for Analysis by HPAE-FLD Ion Chromatography 2016
Application Update Rapid Screening of Sialic Acids in Glycoproteins by HPAE-PAD Ion Chromatography 2011
AppsLab In-depth protein glycosylation comparison of a biosimilar candidate by hydrophilic interaction UHPLC Chemistries and Consumables 2016
AppsLab A fast separation of 2-AB labelled N-Glycans from polyclonal human serum IgG Chemistries and Consumables 2016
AppsLab A high resolution separation of 2-AA derivatized N-Glycans from a commercial chimeric IgG1 monoclonal antibody (Infliximab) Chemistries and Consumables 2016
AppsLab Ultrafast hydrophilic interaction UHPLC analysis of 2-AA labelled N-glycans from monoclonal antibodies (mAbs) Chemistries and Consumables 2016
Article Glycobiology Tools Life Science Mass Spec 2016
Brochure Carbohydrate analysis with HPAE-PAD Ion Chromatography 2013
Brochure Thermo Scientific Glycan Analysis for Biotherapeutics: Join the Sweet Revolution in Biopharmaceuticals Workflow 2015
eBook Glycan Analysis Workflows for Biotherapeutics Workflow 2015
Editorial A Sweet Revolution - Jonathan Bones, NIBRT Editorial Interview HPLC / UHPLC 2015
Editorial Glycopioneer Other 2015
Infographic Top Four Things to Know about Glycan Analysis by Mass Spectrometry Life Science Mass Spec 2013
Poster Middle-down Approach for Monitoring Monoclonal Antibody Variants and Deglycosylation Chemistries and Consumables 2016
Poster Note Orbitrap Based Mass Spectrometric Characterization of Antibody Drug Conjugates Engineered through Antibody Glycans Life Science Mass Spec 2015
Poster Note 2-Amino Benzamide Labeling of Oligosaccharides: How Much Sialic Acid Is Lost? Ion Chromatography 2014
Poster Note An Ultra High Resolution Column and Mass Spectrometer for isomeric Separation and the Structural Identification of Labeled N-linked Glycans Chemistries and Consumables 2014
Poster Note Development of a Rapid and Robust N-Glycan Profiling Method for Therapeutic Antibodies with UHPLC and CE Other 2016
Poster Note Direct Measurement of Sialic Acids Released From Glycoproteins, by High Performance Liquid Chromatography and Charged Aerosol Detection HPLC / UHPLC 2016
Poster Note HPAE-FLD Method for Separation of Human α1 Acid-Glycoprotein and Bovine Fetuin 2-Aminobenzamide (2AB)-Labeled Oligosaccharides Ion Chromatography 2016
Poster Note Label-Free Analysis by UHPLC with Charged Aerosol Detection of Glycans Separated by Charge, Size, and Isomeric Structure HPLC / UHPLC 2015
Poster Note Label-Free Profiling of O-linked Glycans by UHPLC with Charged Aerosol Detection HPLC / UHPLC 2016
Presentation Analytical Strategies for Studying Glycosylation of Biopharmaceuticals Workflow 2016
Presentation Six analytical strategies for studying glycosylation of biopharmaceuticals Workflow 2016
Technical Note Glycoprotein Monosaccharide Analysis Using HPAE-PAD with Eluent Generation Ion Chromatography 2016
Technical Note Glycoprotein monosaccharide analysis using HPAE-PAD with manually prepared eluent Ion Chromatography 2017
Technical Note HPAE-PAD N-linked oligosaccharide profiling of IgG Ion Chromatography 2017
Technical Note Review of peer-reviewed HPAE-PAD glycoprotein oligosaccharide analysis scientific literature evaluating electrochemical response. Ion Chromatography 2013
Technical Note UHPLC Analysis of 2-AB-labeled Dextran Ladder and Assignment of Glucose Units to Unknown Glycans Chromatography Data Software 2014
Technical Note UHPLC Analysis of 2-Aminobenzamide-Labeled Glycans with the Vanquish Flex System HPLC / UHPLC 2015
Webinar Analysis of N-Glycan Released from Biopharmaceutical Glycoproteins using HPAE-PAD Ion Chromatography 2013
Webinar Automating Mass Spectrometry Based Glycan Identification and Quantitation Using SimGlycan Software Life Science Mass Spec 2015
Webinar Challenges in Glycomics and Glycoproteomics Life Science Mass Spec 2015
Webinar Orbitrap Hits the Sweet Spot: Thermo Fisher Scientific Solutions For Glycan Analysis Life Science Mass Spec 2015
Webinar The Sweet Revolution in Glycan and Antibody Separations Workflow 2015
Webinar The Sweet Revolution in Mass Spectral Characterization of Glycosylated Antibodies Workflow 2015
White Paper HPAE-PAD for the Analysis of Carbohydrates Ion Chromatography 2013
White Paper Orbitrap Fusion MS for Glycan and Glycopeptide Analysis Life Science Mass Spec 2015

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