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Abstract

The structural elucidation of N-glycans represents an analytical challenge as N-glycans are diverse with more than 100,000 reported structures. N-Glycans are complex molecules, up to 5000 Da in size, and their functions are determined by their unique chemical structures. Effective and robust analytical methods are required to properly elucidate glycan structures, as even minute changes in branching or changing a single sugar moiety can have profound biological impacts. Rigorous analysis of the glycans on biopharmaceuticals is therefore essential to ensure therapeutic quality and safety. To aid research in this field the toolbox of
methods for analysis of N-glycans needs to be expanded. The glycobiology field has been severely hampered by a lack of accurate and solid analytical methods. I here demonstrate a new halogenated compound for labeling of N-glycans for analysis by Liquid chromatography-mass spectrometry (LC-MS). This method is 2-3 fold more sensitive than the previous state of the art using 2-Aminobenzamide (2-AB) or 2-Aminobenzoic acid (2-AA). This method is more sensitive and more precise, whether for monitoring glycosylation or for identifying unknown N-glycans. Glycoengineering using rational design of glycans creates immense possibilities. The possibility to make hormones, plasma proteins and antibodies with human glycosylation will make the next generation of biopharmaceutical drugs safer and more efficient. Here we demonstrate the entirely humanized N-glycans profile of two plasma proteins, which were made recombinantly in a Chinese hamster ovary (CHO) cell line. Both of these could potentially be used as a replacement in patients lacking a functional plasma protein. We furthermore demonstrate how glycans change by rational choice of cell line, how glycosylation changes over time in a fed-batch reactor and the combinatorial knock out of galactoses on N-glycans on proteins from a CHO cell. This has been possible because of the establishment of a stable and accurate analysis platform of N-glycans. The platform utilizes
high performance liquid chromatography to separate the N-glycans and their isomers with on-line mass spectrometry to verify the mass of the glycans to aid identification. The coming together of state of the art analytical methods with modern understanding and engineering possibilities in molecular biology will be of great importance in the years to come
Original languageEnglish
PublisherTechnical University of Denmark
Number of pages238
Publication statusPublished - 2019

Cite this

Hansen, A. H. (2019). N-Glycan analysis and engineering. Technical University of Denmark.