Carbohydrate Analysis

N-linked Oligosaccharide Profiling

Oligosaccharide profiling methods are the most common method for lot-to-lot comparisons of glycosylation. They are also the most common carbohydrate analysis for release testing. N-linked oligosaccharides are released from the protein enzymatically and a profile (sometimes also referred to as a fingerprint or map) is generated using one of the following methods for separation.

Fluorescent
In this method, the released oligosaccharides are labeled at their reducing end (the end that was attached to the protein) with the fluorophore 2-aminobenzoic acid (AA). The labeled oligosaccharides are then separated by HPLC. We have chromatographic separations that have been optimized for neutral oligosaccharides or pools of oligosaccharides with different levels of sialylation. This method is a very sensitive method for picking up lot-to-lot differences in glycosylation and works well as a release test.

MALDI-TOF MS
In this method, the released oligosaccharides are permethylated and then the pool is analyzed by mass spectrometry. The masses of the oligosaccharides are then used to determine the oligosaccharide structures present in the pool.

HPAEC-PAD
This is a traditional method of oligosaccharide profiling. In this method, the released oligosaccharides are separated by HPLC and detected by electrochemical detection. Electrochemical detection (pulsed amperometric detection or PAD) is a very sensitive method of detecting underivized oligosaccharides. We have chromatographic separations that have been optimized for neutral oligosaccharides or pools of oligosaccharides with different levels of sialylation. Although this method is a very sensitive method for picking up lot-to-lot differences in glycosylation the fluorescent method is more reproducible and robust.

O-linked Oligosaccharide Profiling

O-linked oligosaccharides must be released from the glycoprotein chemically since there is no enzyme available that releases all the different possible O-linked oligosaccharides from a glycoprotein. Unfortunately, the chemical release methods that only release O-linked oligosaccharides destroy the reducing end of the oligosaccharide so it is not possible to label with a fluorophore.

O-linked oligosaccharides are released from the protein chemically and a profile (sometimes also referred to as a fingerprint or map) is generated using one of the following methods for separation.

HPAEC-PAD
In this method, the released oligosaccharides are separated by HPLC and detected by electrochemical detection. Electrochemical detection (pulsed amperometric detection or PAD) is a very sensitive method of detecting underivized oligosaccharides.\

MALDI-TOF Mass Spectrometry
In this method, the released oligosaccharides are permethylated and then the pool is analyzed by mass spectrometry. The masses of the oligosaccharides are then used to determine the oligosaccharide structures present in the pool.

N and O-linked Oligosaccharide Profiling

By using a chemical method that releases both N and O-linked oligosaccharides we are able to generate a profile that includes both forms of oligosaccharides. The released oligosaccharides are labeled at their reducing end (the end that was attached to the protein) with the fluorophore 2-aminobenzoic acid (AA). The labeled oligosaccharides are then separated by HPLC or analyzed by MALDI-TOF Mass Spectrometry.

Sialic Acid Analysis

This assay quantifies the two most common sialic acids: N-acetylneuraminic acid (NeuAc/NANA) and N‑glycolylneuraminic acid (NeuGc/NGNA) on glycoproteins. Sialic acids need to be analyzed separately from neutral monosaccharides because they are more acid labile than neutral monosaccharides and are destroyed by the acid hydrolysis conditions required to release neutral monosaccharides from the glycoprotein.

Samples are hydrolyzed in mild acid and then analyzed by HPAEC (high pH anion-exchange chromatography) using electrochemical detection. The sialic acids are quantified relative to standard curves of each sialic acid. The data are usually expressed as moles sialic acid/mole protein.

Neutral Monosaccharide Composition

This analysis measures the most common neutral monosaccharides (fucose, N-acetylglucosamine, N-acetylgalactosamine, mannose and galactose) in samples although we are also able to quantify additional monosaccharides such as glucose and xylose. We have separate SOPs that have been optimized for MAbs or general glycoproteins.

Samples are hydrolyzed in acid and then analyzed by HPAEC (high pH anion-exchange chromatography) using electrochemical detection. The monosaccharides are quantified relative to standard curves generated for each monosaccharide. The data are usually expressed as moles monosaccharide/mole protein.

Mannose 6-Phosphate Analysis
This assay measures mannose 6-phosphate (Man 6-P/M6P) on recombinant glycoproteins. Mannose 6-phosphate is released from glycoproteins using acid hydrolysis. Man 6-P is then identified by HPLC (high performance liquid chromatography) and measured by comparing the response in the sample to a standard curve of Man 6-P.

Gal Alpha Gal Analysis
This assay measures galactose linked α1-3 to another galactose molecule on recombinant glycoproteins and other glycans. Galactose is released, enzymatically using an enzyme specific for galactose in α1-3 linkage and then free galactose is identified by HPLC (high performance liquid chromatography) and measured by comparing the response in the sample to a standard curve of galactose.

Mannose 6-Phosphate Analysis

This assay measures mannose 6-phosphate (Man 6-P/M6P) on recombinant glycoproteins.
Mannose 6-phosphate is released from glycoproteins using acid hydrolysis. Man 6-P is then
identified by HPLC (high performance liquid chromatography) and measured by comparing the
response in the sample to a standard curve of Man 6-P

Gal Alpha Gal Analysis

This assay measures galactose linked α1-3 to another galactose molecule on recombinant glyco-
proteins and other glycans. Galactose is released, enzymatically using an enzyme specific for
galactose in α1-3 linkage and then free galactose is identified by HPLC (high performance liquid
chromatography) and measured by comparing the response in the sample to a standard curve of
galactose.

Identification of Oligosaccharide Structures

It is sometimes necessary to identify the oligosaccharide structures on your glycoprotein. This can be accomplished by isolating the different oligosaccharides (often by collecting peaks from an oligosaccharide profile) and characterizing them using one/more of the following methods:

MALDI-TOF MS. To determine the mass of the oligosaccharide.

Glycosidase sequencing. To selectively release monosaccharides from the oligosaccharide. This can also be used to determine their linkages.

Neutral monosaccharide and/or sialic acid analysis. To determine the monosaccharides present in the oligosaccharide and/or their ratios.