High throughput DNA single cell analysis of CHO-K1 cell surface glycosylation using lectin probes.
Ferreira, Flavio
(2020)
High throughput DNA single cell analysis of CHO-K1 cell surface glycosylation using lectin probes.
PhD thesis, Dublin City University.
Biological glycosylation is the process which adds specific sugars to other sugars, proteins and lipids. Protein glycosylation is one of the most important post-translational modifications, which occurs in more than half of all proteins present in the human body. Abnormal glycosylation has been demonstrated to be linked to many different diseases due to alterations associated with protein folding and biological function. Therefore, glycosylation is absolutely essential for the correct structure, function and stability of important proteins.
Surface glycosylation patterns play a key role in the modulation of the immune responses which are mediated by carbohydrate-binding proteins called Lectins. Such biomolecules are typically highly selective for specific glycan structures, making them extremely useful for glycan variation investigation.
A rapid and accurate bioanalytical method to detect early unhealthy cell signs during a bioprocess is a current issue facing the industry. It is widely known that as cells become stressed or diseased the earliest changes that occur are in cell surface glycosylation.
CHO cells are the host cell of choice of the rapidly emerging biopharmaceutical industry for the production of glycoprotein therapeutics. Hence, this research work investigated the interaction between lectin probes with the membrane glycoconjugates of CHO cells subjected to different levels of spent medium, temperature and CO2.
High throughput DNA single cell analysis using flow cytometry allowed the determination of cell surface glycosylation variation in response to the stressors. Cells subjected to different levels of spent medium had their cell surface glycosylation profile most affected in relation to cells subjected to temperature and CO2 alteration. Fucose and N-Acetylglucosamine were identified as key glycans changing on the cell surface.