Glycan Standards

Ludger has a range of system suitability standards, process controls and reference standards for different applications including the analysis of sialic acids, monosaccharides, glycan profiling and characterisation. We provide a variety of unlabelled N-glycans (N), O-glycans (O), oligosaccharides, glycoproteins / glycopeptides and glycan library standards. Furthermore, our catalogue of standards also includes a large range of N- and O- glycan standards fluorescently labelled with 2-aminobenzamide (2-AB), 2-AA (2-aminobenzoic acid), procainamide, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) and permethylated glycan standards. We also provide quantitative glycan and glycopeptide standards under the brand name Ludger BioQuant.

A key component in a well-designed analytical strategy is the inclusion of standards.

Standards: i) help with characterisation, ii) allow quantitation, and iii) provide vital information on the performance of an analytical process.
Example: in a QbD framework, glycan standards that match the GCQAs which have been identified for optimised drug function can be used to ensure that the analytical methods chosen for characterisation provide the necessary data required to monitor the drug realisation process

The data acquired when using standards allow the analyst or reviewer to judge whether a specific stage in the process or the sample itself is in or out of specification.
Glycan standards are used for different applications during glycan analysis and can be broken down into four broad categories: system suitability standards, process standards, reference standards, and quantitative standards.

Workflow:

Ludger Glycan Standards Workflow

The Standards Table below shows which standards can be used for best practice during the analysis of sialic acids, monosaccharides, N-glycans and/or O-glycans.
These fall into the following categories;

  1. System suitability standards enable an analyst to test the holistic functionality of an analytical system (e.g. chromatographic, mass spectroscopic and/or CE) and evaluate whether it is adequate for its intended use.

  2. Process standards or process controls are used to verify that part of or an entire process has worked correctly. There are four main categories for processes standards in glycoanalysis: release, labelling, release followed by labelling and exoglycosidase sequencing

  3. Reference standards allow for characterisation by comparison. This can be accomplished by the direct comparison of the chromatographic or electrophoretic retention time of an unknown to that of a standard whose structure have been fully characterised. Additionally, primary assignment of unknown structures can be accomplished by comparison of their GU (Glucose unit) values (obtained using glucose homopolymer (GHP) standard) with glycans whose GU values are in databases or in the literature.

  4. Quantitative glycan standards are used to determine the absolute amount of an analyte in a sample. They can also be used to quantify the efficiency of a process.
Ludger Glycan Standards and Controls Table

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Ludger Standards Reference Table


Ludger Glycan Standards and Controls Table

Click to view our

Glycan Standards and Controls Table


List of Standards:

(click on each to expand)

N-glycans

The N-glycans that are available are found in most biological and biopharmaceutical glycoproteins and can be grouped into the following categories: oligomannoses, and complex glycans that are fucosylated, sialylated, and di-,tri-and tetra-antennary. The glycans are available as molecules with a free reducing terminus (aldol), or chemically derivatized. The common chemical derivatives of N-glycans are fluorescently labelled glycosides (2-AA, 2-AB, APTS, procainamide, etc.), or permethylated glycans.

Native glycans are generally suitable process controls for labelling methods using reductive amination with fluorescent tags or when performing derivatizations like permethylation or ethyl esterification.



O-glycans

The O-linked glycans are linear or biantennary and contain an initial GalNAc residue. O-Glycans are grouped into eight core structures, however, the most common O-glycans generally belong to core 1 and 2. These glycans are provided with a free reducing terminus (aldol), or are derivatized as a fluorescently labelled glycoside. The fluorescent labels that are commonly used are 2-AA and 2-AB.



N-Glycan Libraries

Glycan libraries are made up of a mixture of species from a specific type of glycoprotein or as panels with common characteristics. The following is an example of glycan libraries produced from a specific glycoprotein: N-glycans released from human IgG antibody glycoprotein. This library contains fucosylated and bi-antennary glycans with variable sialylation.



O-Glycan Libraries

Glycan libraries are made up of a mixture of species from a specific type of glycoprotein or as panels with common characteristics. The following is an example of glycan libraries produced from a specific glycoprotein: O-Glycans released from fetuin glycoprotein (purified from fetal calf serum). This library contains mainly core 1 and core 2 O-glycans with variable sialylation.



LC Calibration Standard

A Glucose Homopolymer (GHP) ladder can be used as an external calibration standard during the analysis of glycans by (U)HPLC. This standard gives a characteristic ladder profile from monomeric glucose to approximately 20-mers of glucose oligosaccharides. The elution position of each peak in this ladder is expressed as a glucose unit (GU) and is used to assign GU values to peaks in the released glycan pool by comparison. These GU values are reproducible and predictive as each monosaccharide in a glycan contributes a set increment to the GU value. This allows for primary assignment of structure by comparison of GU values for unknown glycans with glycan standards whose GU values are in databases or reported in the literature.



Glycoproteins

Glycoproteins are used as process controls for glycan release, and consecutive release followed by labelling. Glycoprotein standards that are widely available are IgG, mAbs and fetuin. These are useful during endoglycosidase release (N-glycans), hydrazinolysis, Orelea or beta elimination (generally O-glycans) and acid release (during monosaccharide/sialic acid analysis). Fetuin, IgG and mAbs can also be used as process controls for proteolytic digestion during glycopeptide mapping.



Glycopeptides

Glycopeptides are used as process controls for glycan release, and consecutive release followed by labelling. They are useful during both endoglycosidase release (N-glycans) and acid release (during monosaccharide/sialic acid analysis). They can also be used as purified standards during glycopeptide mapping.



Monosaccharide and Sialic Acid Standards

Monosaccharides, such as hexoses, hexosamines and sialic acids, are suitable fluorescent labelling controls for their respective analyses.



Quantitative Glycan and Glycopeptide Standards

Ludger’s range of quantitative standards are called BioQuant standards. BioQuant standards are designed to fit simply into your analysis workflow and you treat them as you would any other sample.

Ludger has integrated BQ quantitative standards into many of our analytical processes for glycan analysis and has set acceptance criteria. This allows the analyst to judge the results and know if they are truly in or out-of-specification.



Exoglycosidase Testing Standards

Native or fluorescently labelled glycans are common process controls when using exoglycosidase sequencing for structure elucidation. Your choice of standard will depend on the analytical detection method and which exoglycosidases are being used. These process controls allow you to assess if the exoglycosidase is cleaving the right monosaccharide with a specific glycosidic linkage.