Oligosaccharide Characterisation

The post-translational modification of proteins plays a key role in development of therapeutically useful biomolecules. Moreover, as a number of biopharmaceutical products come to the end of their patent life there is opportunity to develop replacement / substitute products. These products, often termed ‘biosimilars' or ‘biobetters', frequently rely on post-translational modifications such as protein N-Linked glycosylation to achieve better performance or a point of difference.

Oligosaccharide Characterisation

N-linked Glycosylation

N-linked glycosylation describes attaching a complex sugar (oligosaccharide) via an amide bond to an asparagine (Asn) residue of a defined tripeptide sequence (Asn-X-Ser/Thr). X can be any amino acid except proline.

Key then to the development and production of any therapeutic protein is full knowledge of exactly where on the protein the glycosylation has taken place. After all, any given protein may contain single or multiple occurrences of the Asn-X-Ser/Thr sequence, and glycosylation may occur at none, some or all of these sites.

The drug discovery/submittal process requires a great deal of information to be submitted about the nature and positions of the oligosaccharides attached to proteins. It is worth noting at this point that the oligosaccharide structures are not single moieties. They may differ in sialic acid and galactose content, the numbers of antennae or branches, the degree and type of fucosylation, and whether the oligosaccharide is high-mannose, hybrid or complex-. All of these factors conspire to add to the complexity of information that must be obtained, and the skill required of the analyst.

Analysis of N-linked glycosylation by chromatographic methods

Oligosaccharide analysis may involve several varied and complex methods such as high performance liquid chromatography, mass spectrometry, lectin-based analysis, enzyme digestions, chemical methods etc.

In most cases, it is first necessary to release the oligosaccharides from the protein. Enzymatic release is the preferred method, using peptide N: glycosidase F (PNGase F) which removes all N-linked oligosaccharides from mammalian proteins. Most biopharmaceuticals are currently produced using mammalian cell lines, meaning PNGase F should be appropriate in all cases. However, should there be a core-fucose residue attached via an α1-3 linkage, as found in plant and some insect oligosaccharides, PNGase F will not work. In this case, peptide: N-Glycosidase A (PNGase A) must be used. The oligosaccharides are further purified, prior to subsequent analysis, to remove any salt, detergent and protein contamination.

HPAEC, HILIC, HIAX Chromatography

HPAEC

High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) is a popular method for the analysis of oligosaccharides. It allows for easy separation of neutral from charged, sialic acid-containing oligosaccharides. Moreover, the charged oligosaccharides are separated on the basis of the number of charged residues per oligosaccharide and the underlying branched structure. However, it is difficult to interface the HPAEC-separated oligosaccharides to a mass spectrometer to obtain both mass and sequence information for oligosaccharide characterisation. This is due to the HPAEC eluent containing high pH and non-volatile salts.

Increasing sensitivity

By linking fluorescent molecules to the core-GlcNAc residue (formerly attached to the protein backbone Asn residue of the oligosaccharide) it is possible to increase the sensitivity of the technique. A number of different fluorescent reporter groups are reported in the literature and these influence sensitivity, choice of column, efficiency of separation and mass spectrometric analysis. By use of volatile HPLC solvents and the methods of separation outlined below, it is possible to interface the HPLC systems to mass spectrometers to increase the amount of information obtained. This information includes the mass of the individual oligosaccharides and the potential structural sequence of the oligosaccharides.

HILIC

The most popular technique used to analyse fluorescently derivatised oligosaccharides is hydrophilic interaction liquid chromatography (HILIC). This is a form of HPLC routinely performed using an amine- or amide-derivatised column matrices on HPLC systems with fluorescence detection. The retention times of the oligosaccharides are usually compared to that of an external standard, and expressed in terms of glucose unit (GU) values. Recently, it has become possible to perform the same analysis using an internal standard method that provides more accurate GU values. The GU values obtained with different column matrices may be used to generate oligosaccharide retention chromatography databases. With the recent advent of ultra-performance liquid chromatography (UPLC), or ultra-high-pressure liquid chromatography (UHPLC), the separation time has been decreased and peak resolution has dramatically increased when using HILIC-based separations.

Analysis using HILIC may also be performed in combination with weak anion-exchange (WAX), strong anion-exchange (SAX), porous graphitized carbon (PGC) or reverse-phase (RP) separations. This combination of techniques greatly increases the ease of analysis and the number of different structures that can be observed.

Mixed-mode

It is possible to combine different chromatographies, hydrophilic interaction and anion-exchange, using a single column or a tandem column arrangement (so-called mixed-mode chromatography). It is also now possible to obtain GU values following hydrophilic interaction anion-exchange chromatography (HIAX Chromatography) using a Dionex AS11 column. This offers the benefit of HPAEC-like separation (neutral from charged, sialic acid-containing oligosaccharides) but using volatile HPLC solvents. Additionally, HIAX may be coupled to mass spectrometry to obtain oligosaccharide mass and sequence information.

Enzyme digests

HPLC coupled to mass spectrometry permits the analysis and assignment of many oligosaccharide structures. However, confirmation of sequence information must be obtained in tandem with specific enzyme digests. The enzymes, or glycosidases, remove specific monosaccharides, e.g. galactose, mannose, fucose etc., which are attached via specific linkages to the parent oligosaccharide. The enzymes may be used singly or in combinations. The products of the enzyme digests are subjected to further HPLC analysis. Where monosaccharide(s) have been removed, a shift in retention time/GU should be observed. Combining the results of multiple enzyme digests/HPLC analysis permits assignment/confirmation of oligosaccharide structure.

Conclusion

The analysis of N-linked oligosaccharides is not easy but a number of different technologies can be employed to obtain the information required for submissions and regulatory approval. The most sensitive methods involve derivatising the free oligosaccharides with a fluorescent reporter molecule, which then influence exactly how best to proceed with the analysis. However, there is no single technique that provides all the required information and a combination of HPLC, mass spectrometry and enzyme digests will be needed.