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.
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."
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.
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.
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.
Featured glycan analysis information learning content
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.