Endoglycosidases are enzymes that cleave intact glycans from glycoproteins. These enzymes are highly stable in clean preparations with no glycerol, NaCl, or other additives such as EDTA. All enzymes are tested for the absence of proteolytic or unexpected glycosidic activity.

Endoglycosidases are some of the most widely used enzymes for the analysis of glycoproteins. These enzymes release intact glycans from glycoproteins, while exoglycosidases release individual monosaccharides (i.e. sialic acid, galactose, β-N-acetylgucosamine, mannose, etc.).

PNGase F is commonly included in the group of enzymes due to its similar activity of cleaving intact N-linked glycans, though it is actually an amidase. It cleaves between the asparagine amino acid and the first β-N-acetylglucoseamine sugar (GlcNAc) of the chitobiose core of N-linked glycans. The Endo F enzymes and Endo H cleave N-linked glycans between the first and second GlcNAc, leaving a charged residue which aids in increasing solubility of the deglycosylated protein, reducing the potential for precipitation of the protein aggregates. PNGase F cleaves all N-linked glycans, while Endo H and the Endo F enzymes remove specific types of N-linked glycans.

Also included in this classification of enzymes is O-Glycosidase, which removes the Core 1 O-Linked glycan (Gal-β(1-3)GalNAc-α) from serine or threonine amino acids. Often times O-linked glycans contain additional monosaccharides linked to the galactose in both branched and linear linkages, requiring the use of exoglycosidases such as sialidase and galactosidase to remove the additional sugars.

PNGase F (Peptide N Glycosidase F) and recombinant PNGase F

PNGase F is suitable for release of all types (high-mannose, hybrid and complex) N-linked glycans from glycoproteins and glycopeptides. PNGase F will not remove oligosaccharides containing α(1-3) linked core fucose commonly found on plant glycoproteins.

Table below lists and compares the PNGase F enzyme kits offered at Ludger.

Cleave specific terminal-monosacharides from glycans
Allows detailed structural characterisation of glycans (monosaccharide type, linkage and sequence)
All enzymes are tested for the absence of proteolytic or unexpected glycosidic activity

Example Enzyme Sequencing:

Workflow for exoglycosidase sequencing of glycans based on HPLC/LC-MS detection:

For purification of glycans after chemical and enzymatic release.

For purification of glycans after labelling.

Ludger Velocity Vacuum Manifold System

Ludger Velocity Vacuum Manifold System – Use of a vacuum manifold for sample clean up speeds up processing times. Ludger’s Velocity vacuum manifold system is compatible with cartridges or plates, and is a valuable tool for your lab.

The system can be used with individual clean up cartridges (e.g. LC-T1-A6 or LC-A-24) or 96 well plate clean up devices (e.g. LC-EC50-96, LC-EXO-96, LC-PBM-96, LC-PROC-96 or LC-PERMET-96).

Use of a vacuum manifold for sample clean up speeds up processing times. Ludger’s Velocity vacuum manifold system is compatible with cartridges or plates, and is a valuable tool for your lab.

The options available are summarised in the Table below:

LudgerTag Glycan Labeling Kit reagents are purified to analytical grade and are dispensed and sealed under clean, inert atmospheres. The ampoules are pre-cleaned by pyrolysis at 500°C then opened just before dispensing and sealing under oxygen-free dry nitrogen. These controls ensure that your analyses work properly each and every time.

2-AB and 2-AA are used in HILIC-(U)HPLC, WAX-HPLC, and MALDI-MS assays. They are the most referenced glycan label for biopharmaceutical analysis. 2-AA is also used for monosaccharide analysis.

Fluorescent labelling methods for glycoprofiling of therapeutic glycoproteins are well established and aid subsequent separation and quantitation using a range of techniques. These include high performance liquid chromatography (HPLC and UHPLC), capillary electrophoresis (CE), and mass spectrometry. Of the fluorescent labels available, 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA) are most widely used for oligosaccharide profiling. Existing 2-AA and 2-AB labelling kits use sodium cyanoborohydride as a reducing agent during glycan labelling.

LudgerTag™ 2AB and 2AA Glycan Labelling Kits with Safer Reductant Fluorescent

Fluorescent labelling methods for glycoprofiling of therapeutic glycoproteins are well established and aid subsequent separation and quantitation using a range of techniques. These include high performance liquid chromatography (HPLC and UHPLC), capillary electrophoresis (CE), and mass spectrometry.

Of the fluorescent labels available, 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA) are most widely used for oligosaccharide profiling. Existing 2-AA and 2-AB labelling kits use sodium cyanoborohydride as a reducing agent during glycan labelling. This reagent is toxic so a fume cupboard should be used during handling. To conform with emerging Health and Safety regulations we are now replacing these with our new VP glycan kits that use picoline borane which is a significantly safer reductant. This technology, developed in collaboration with Leiden University Medical Centre, The Netherlands, has now been patented

The VP kits have the same glycan labelling performance as the traditional kits but are safer, more convenient (fewer vials to mix) and the reagents are more stable.

The LudgerTag DMB Sialic Acid Labeling Kit has been developed for the qualitative analysis of sialic acids.

The kit contains reagents for the release of sialic acids from glycoproteins. Released sialic acids are conjugated with DMB dye by an amination-cyclisation reaction. The kit contains reagents and materials for up to 22 samples including the sialic acid reference panel, and the N-acetyl neuraminic acid and N-glycolyl neuraminic acid standards.

Workflow:

DMB Sialic Acid Labeling Kit are designed to satisfy the following regulatory requirements:

• Overall degree of drug sialylation – absolute quantitation of sialic acid residues per molecule (nmol/mg protein).
• Relative quantities of Neu5Ac:Neu5Gc.
• Identification and relative percentage of O-acetylated sialic acids.

The LudgerTag V-tag labeling kit is designed for the fluorophore labeling of enzymatically digested peptides and glycopeptides followed by enrichment of the labeled glycopeptides. The V-tag labeled glycopeptides are analyzed using MALDI-MS and HPLC/UHPLC

Ludger V-Tag glycoprofiling technology is a cost-effective, GMP-validated, and rapid tool for mAb developers and biosimilar manufacturers. Used for mAb quality control and biosimilarity validation throughout the drug lifecycle.  Easy to integrate into your peptide mapping. it provides glycan identity, quantitation, attachment site and site occupancy.

Monosaccharide analysis is a regulatory requirement laid out in the ICH Q6B guidelines for characterisation of biopharmaceuticals. This information can be used at all stages of drug development as a method of determining the type of glycosylation (N-linked and/or O-linked) and the extent to which glycosylation has occurred. It can also be used to demonstrate consistency between batches for QC lot release during the manufacturing process.

Ludger’s monosaccharide analysis kit is easy to use, reliable and can be used for quantitative routine monosaccharide analysis. (Kit sufficient for 96 analyses).

• Release neutral and amino monosaccharides from your glycoprotein samples by mild acid hydrolysis
• Fluorescent labelling of released monosaccharides with 2-aminobenzoic acid (2AA)
• Relative quantitative analysis of 2AA-labelled monosaccharides compared to standards (GlcN, GalN, Gal, Man, Glc, Fuc, Xyl) by (U)HPLC analysis

2-AA labeled glycans (2-aminobenzoic acid) are considered to be a superior standard for glycan analysis due to the higher sensitivity of detection as compared to 2-AB labeled glycans. Labeled glycans may be analyzed by high-sensitivity fluorescence detection or monitoring of UV-absorbance during various chromatographic and structure sequence analyses, as well as mass spectrometry. UHPLC, HPLC, ESI-MS, MALDI-MS and CE methods have been developed and are commonly used in the analysis of therapeutic glycoproteins such as EPO and antibody-based bio-therapeutics. Methods include ion exchange, normal phase, reverse phase, and size exclusion chromatography.

The 2-AA labeled glycans are supplied as 100 pmols dried form from an aqueous solution. They are stable at least 5 years as supplied and after reconstitution. Typically the 2-AA labeled glycans are reconstitute with 100 µls of the buffer solution being used, yielding a concentration of 1 pmol/µl. It is recommended to dissolve the dried glycans in the aqueous solution first (ammonium formate). 35ul + 65ul ACN will make 100 pmol in 100ul concentration.

2-aminobenzamide, 2-AB labeled glycans are one of the most referenced glycan standards for biopharmaceutical analysis. Labeled glycans may be analyzed by high-sensitivity fluorescence detection or monitoring of UV-absorbance during various chromatographic and structure sequence analyses, as well as mass spectrometry. UHPLC, HPLC, ESI-MS, MALDI-MS and CE methods have been developed and are commonly used in the analysis of therapeutic glycoproteins such as EPO and antibody-based bio-therapeutics. Methods include ion exchange, normal phase, reverse phase, and size exclusion chromatography.

The 2-AB labeled glycans are supplied as 100 pmols dried form from an aqueous solution. Typically the 2-AB labeled glycans are reconstitute with 100 µls of the buffer solution being used, yielding a concentration of 1 pmol/µl. It is recommended to dissolve the dried glycans in the aqueous solution first (ammonium formate). 35ul + 65ul ACN will make 100 pmol in 100ul concentration.

They are stable at least 5 years as supplied and after reconstitution.

APTS labeled glycans (9-Aminopyrene-1,4,6-trisulfonic acid) are commonly used as standards for the analysis of glycoproteins by cappilary electrophoresis (CE). The fluorescent APTS molecule confers a strong negative charge to the APTS labeled glycans resulting in rapid separation via capillary electrophoresis.

The labeled glycans are supplied as 20 pmols dried form from an aqueous solution. They are stable at least 5 years as supplied and after reconstitution.

LudgerTag procainamide labeled glycans can be analyzed by UHPLC, HPLC, ESI-MS, and LC-ESI-MS methods. Because of its improved ionization efficiency compared to 2-AB labeling it can permit identification of minor glycans (<1% relative peak area) by ESI-MS.
Fluorescence response: Procainamide glycans show a much higher sensitivity as compared to 2-AB labeled glycans, providing peak heights three times higher than traditional 2-AB labeled glycans. The procainamide labeled sugars show comparable separation to HILIC-UHPLC assays with 2-AB.

Mass spectrometry response: Due to its high ionization efficiency, procainamide shows much higher sensitivities the seen with 2-AB. When analyzed using ESI-MS and ESI-MS/MS, procainamide produces twenty-two times higher peaks than 2-AB. This provides a great platform for the detection and identification on minor peaks by MS/MS analysis.

The labeled glycans are supplied as 20 pmols dried form from an aqueous solution. They are stable at least 5 years as supplied and after reconstitution.

Native glycans (N-Glycan Standards – unlabelled) are generally suitable process controls for labelling methods using reductive amination with fluorescent tags or when performing derivatizations like permethylation or ethyl esterification. We have a complete line of native glycan products for analytical markers including both N-linked and O-linked glycans.

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 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.

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.

O-glycans are present in biopharmaceuticals such as erythropoeitin (EPO), Etanercept (Enbrel) and human Factor VIII (FVIII).Core 1 and Core 2 O-glycans are also present in biopharmaceuticals such as erythropoeitin (EPO), Etanercept (Enbrel), humanFactor VIII(FVIII), Factor IX and insulin glargine.

The Ludger standards have a purity of >90% as assessed by HILIC-HPLC and can be used as internal references or system suitability standards for peak assignment during HPLC or UPLC analysis.

Glycan libraries are made up of a mixture of species from a specific type of glycoprotein or as panels with common characteristics. Our glycan libraries have been used in pharmaceutical and biotech companies around the world. They meet our rigorous standards of quality and contain no salts or other additives that may interfere with advanced analytical techniques such as mass spectrometry which require the highest levels of purity.

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-Glycans released from fetuin glycoprotein (purified from fetal calf serum). This library contains mainly core 1 and core 2 O-glycans with variable sialylation.

Part of Ludger’s range of System Suitability Standards and Controls, our BioQuant range has been designed for glycan quantitation.

Purity of BioQuant standards is determined as > 90% by UHPLC. Correct mass identity is assessed by MALDI mass spectrometry. The exact amount of material/concentration is determined by quantitative NMR (qNMR) and quantitative monosaccharide analysis (MA). Quantity values by qNMR and MA agree within 90-110%. MA is traceable to internationally accepted references from USP and dispensed using NIST traceable labware. qNMR is traceable to a NIST SRM traceable Certified Reference Material which has been analysed to the ISO 17025 standard.

A detailed Certificate of Analysis is given for each BioQuant standard which contains comprehensive documentation, lot-specific values, expiration date and storage information.

Sialic Acids (a family of derivatives of neuraminic acid) are usually found as terminal structures of both N-linked and O-linked glycans.

Being located at the terminal position of glycans, sialic acids are a likely point of contact for many glycoprotein interactions. They are also important for the stability and 3D conformation of glycoproteins and are involved in many biological interactions:

• Sialyation of IgG reduces ADCC and increases anti-inflammatory activity
• Sialic acids increase the serum half-life of glycoproteins by preventing uptake by the liver’s asialoglycoprotein receptor
• NGNA(Neu5Gc), a glycan not found in humans, can illicit an immune response and lead to increased neutralization of biopharmaceuticals.
• Cell line choice can greatly influence the type of neuraminic acids present on a biopharmaceutical, for example a large portion of the sialic acids on mouse IgG are often NGNA.

HPLC Calibration Standard
Glucose homopolymer ladder is derived from digested wheat starch used as a standard for sizing of glycans.

Glucose Homopolymer (GHP) – a System Suitability and a Reference Standard for Glycan Analysis using Liquid Chromatography

Standards containing the α-gal epitope are essential process controls when using an α-galactosidase. This gives confidence in the function of the enzyme and in the resulting characterization; being able to unequivocally and confidently identify an α-linked galactose from a β-linked galactose in a glycan structure has major implications for the safety of the glycoprotein therapeutic being studied.

For analysis of 2-AA & 2-AB labelled N- & O-glycans based on hydrophilicity which is mainly determined by size and in lesser degree arm specificity, and linkage.

For monosaccharide & sialic acid analysis based on hydrophobicity. LudgerSep R Buffer is formulated for monosaccharide analysis

For the charge profile analysis of 2-AA & 2-AB labelled glycans what is primary determined by the number of sialic acids per glycan