Development and characterisation of recognition molecules for the specific identification of non-human glycans

Glycosylation is an enzymatic process through which glycans attach to proteins, lipids and other organic molecules with huge structural diversity. This is a common post-translational modification found on proteins and cell surfaces and has significant effects on cellular functions, such as, cell adhesion, migration, differentiation, inflammation, immunity, structural folding and bimolecular interactions. Glycans of non-human origin, that are not naturally synthesised or expressed in humans but are introduced into the body through dietary intake or through biological therapeutic drugs that have been derived in non-human production systems have the potential to elicit an immune response. These responses can vary from minor inflammation to anaphylaxis. There is a constant need for the identification and characterisation of specific recognition molecules that can be incorporated into cost effective and high-throughput methods to specifically detect and quantify non-human glycans. These assays may have utility in development and quality control of recombinant biopharmaceuticals that express these molecules. The development and characterisation of recognition molecules to two important non-human glycans: Galactosyl-α-(1, 3)-Galactose (Gal-α-(1, 3)-Gal) and N-glycolylneuraminic acid (Neu5Gc) are described here. Gal-α-(1, 3)-Gal is immunogenic in humans and is directly involved in hyper-acute rejection during xenotransplantation. Recombinant therapeutic molecules generated in non-human cell culture systems such as CHO, NS0 and SP2/0 cell lines can potentially contain Gal-α-(1, 3)-Gal. The structural basis of the interaction of the anti-Gal-α-(1, 3)-Gal epitope with a previously developed scFv recombinant antibody fragment was analysed and key binding regions and properties among the amino acid residues critical in the recognition and binding of scFvs' against the target carbohydrate epitopes were determined. Like Gal-α-(1, 3)-Gal, Neu5Gc can also be added to recombinant biopharmaceutical molecules when using CHO, NS0 and SP2/0 cell lines. There is also evidence that Neu5Gc-containing glycoconjugates occur frequently in cancer patients, and that tumour associated Neu5Gc facilitate tumour progression and inflammation. There are very few recognition molecules produced with high specificity for Neu5Gc that can be used to assay for this molecule. Aptamers are DNA or RNA oligonucleotides which have the capacity to bind to a variety of molecules with high affinity and specificity. Here I describe the development of DNA aptamers that show high affinity and specificity allowing for discrimination between the non-human Neu5Gc from the human Neu5Ac form. These molecules will have great application in biopharmaceutical production as part of a quality control workflow in allowing for the identification of potentially harmful molecules.

Funder

Science Foundation Ireland (SFI)

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Attribution-NonCommercial-NoDerivs 3.0 Ireland

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University of Galway Theses (PhD Theses)