Glycan biomarker discovery: Method development and application to inflammatory bowel disease

The overarching goal of this study was to develop and implement methodologies for the comprehensive analysis of glycoprotein glycosylation. This research is aimed at supporting the biopharmaceutical industry and advancing the discovery of glycan biomarkers for human diseases, with a specific focus on inflammatory bowel disease (IBD). The project encompasses six chapters: Introduction (Chapter 1): This chapter provides an overview of protein glycosylation and emphasizes its significance in both health and disease. Analytical Technologies Review (Chapter 2): This section reviews the latest advancements in analytical technologies used for high-throughput glycan analysis, particularly those involving automation. Automated Permethylation Workflow (Chapter 3): Chapter 3 is dedicated to the development, optimisation, and validation of an automated and high-throughput permethylation derivatisation workflow. This workflow is designed to analyse glycosylated biopharmaceuticals, ensuring the stability of sialic acids and enabling the detection of neutral and acidic glycans using MALDI-TOF-MS analysis. The chapter also presents quantification data for permethylated IgG4 monoclonal antibodies and highly sialylated recombinant human erythropoietin samples, comparing them with traditional 2-AB labelled UHPLC profiles. Application to Complex Tissue Glycans (Chapter 4): Chapter 4 demonstrates the practical application of the automated permethylation technique to complex and heterogeneous mouse tissue glycans. It includes a comparison with the gold standard manual permethylation technique, highlighting the speed, reliability, and robustness of the newly developed automated approach for analysing complex biological materials. Serum N-Glycan Signatures in IBD (Chapter 5): This chapter focuses on the use of serum N-glycan signatures to predict treatment escalation in IBD. It involves the characterisation of serum samples from a large cohort of individuals and highlights the prediction accuracy of medical treatment escalation based on procainamide-labelled released glycans using UHPLC. The findings are validated in an independent replication cohort, underscoring their significance in IBD management. General Discussion and Future Perspectives (Chapter 6): The final chapter provides a comprehensive discussion that ties together the results presented in the thesis. It also addresses current technological challenges and outlines future perspectives for clinical and biopharmaceutical glycomics. Overall, this thesis contributes to the advancement of analytical techniques for glycoprotein glycosylation analysis, with practical applications in biopharmaceutical development and disease biomarker discovery, particularly in the context of IBD.