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."
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 can also be performed at the peptide level, with the goal of obtaining both glycan composition and peptide sequence at the site of glycosylation.
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.
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.
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.
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.
Glycosylation is a key intracellular process that involves interactions of various enzymes and substrates.
A fully integrated workflow for glycan profiling and structural characterization of fluorescently labeled N-glycans released is demonstrated in this application.
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.
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.
|Application Compendium||Monosaccharide and Sialic Acid Determinations in Biosimilars Using HPAE-PAD||Ion Chromatography||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 Note||Comparison of Therapeutic Antibody Originator and Biosimilar Glycosylation Using an Integrated Glycan Labeling Solution||HPLC / UHPLC||2018|
|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|
|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||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||Life Science Mass Spec||2015|
|Webinar||Challenges in Glycomics and Glycoproteomics||Life Science Mass Spec||2015|
|Webinar||Life Science Mass Spec||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|
|Application Note||Subunit Analysis Approach for the Determination of Fucosylation Levels in Monoclonal Antibodies||HPLC / UHPLC||2018|
|Application Note||A Quick and Accurate Comparison of Biosimilar and Originator Biotherapeutics||HPLC / UHPLC||2018|
|Application Note||HPAE-PAD method for determination of Hib capsular polysaccharide content||HPLC / UHPLC||2021|
|Application Note||An Improved Method for Galactosyl Oligosaccharide Characterization||HPLC / UHPLC||2022|