title: Development of Tools for Glycan Analysis and Quantitative Sialic Acid Glycomics
Creators: name: Allen, Matthew
Publisher: name: The Open University
Year: 2022

description: Glycans are molecules consisting of glycosidically linked monosaccharides, ubiquitous in biological systems forming the glyco portion of many glycoconjugates including glycoproteins and glycolipids. The variety in the glycan structure dictate the wide variety of roles these glycans play. These include protective, stabilising and organisation functions. In addition glycans present on biotherapeutics affect the stability and efficacy of the drugs due to the sialylation of the glycan and therefore biotherapeutic glycans must be characterised as their presence is regulated by drug agencies. The inherent structures of glycans hinders their detection by traditional methods and therefore a derivative of the glycan are often analysed to overcome the poor detection. One of these derivatization methods is where a chemical label is added to the reducing end of the glycans by reductive amination to facilitate detection, however, these modifications prevents further uses of glycans which due to the difficulty of glycan synthesis is a drawback of these workflows. Glycan analysis is commonly performed in hydrophilic interaction mode HPLC, however this method of HPLC suffers from many limitations due to the mechanism of retention relying on an immobilised layer of water. Hydrophilic interaction HPLC suffers from poor peak shape, difficult method development and long equilibration times. The analysis of glycans is also complicated by the class of monosaccharide, sialic acid which is common on the non-reducing end of the glycans. Sialic acid play many roles in biotherapeutic clearance. For these reasons sialic acid analysis is also performed as the derivative due to the inherent negative charge of sialic acid. Derivatisation is performed with 1,2-diamino-4,5-methylenedioxybenzene (DMB), however this label is associated with poor stability and samples require analysing as soon as possible after labelling. This thesis presents potential solution to these observations. The synthesis of two multifunctional glycan labels containing additional groups which show similar HILIC mode HPLC analysis to currently used labels for the analysis of glycans meaning these labels can be employed in glycan analysis workflows to provide further uses. The further uses investigated in this research examined the ability for the carbohydrates labelled with the alkyne multifunctional labels to react with an azide conjugation partners to give a more sensitive molecule to improve detection and characterisation. This research also investigated the ability for the analysis of glycans via reverse phase HPLC methods to overcome the common limitations of hydrophilic interaction mode HPLC. The reverse phase HPLC of labelled carbohydrates resulted in the production and optimisation of eight reverse phase HPLC methods capable of analysing derivatised glycans from a variety of sources which may be used as alternatives to hydrophilic interaction HPLC for the analysis of glycans. Finally, this thesis trialled ortho-phenylenediamine derivatives in the quinoxaline derivatisation of sialic acids in an effort to find a possible alternative to DMB in the analysis of sialic acid. These labels were employed in the analysis of Neu5Ac and were observed to provide sufficient detection and resolution for the labelled sialic acid in reverse phase HPLC. These alternatives however were found to be inferior to DMB which displayed similar stability and superior detection sensitivity.