Berry, Peter (2023) An investigation into approaches and pathways relevant to cellular flocculation in CHO cells. PhD thesis, Dublin City University.
Abstract
Over the last two decades, viable cell concentrations in industrial processes have reached multiples of 107 cells/mL at harvest. Given that many production facilities were built to handle viable cell concentrations in the order of 106 cells/mL, cell concentrations of this magnitude have the potential to cause problems during primary recovery activities as existing processes/equipment may not be able to efficiently remove cells from these high cell concentration processes. Flocculation, the ability of cells to clump together is one solution to this issue. The objective of this thesis was to engineer a flocculating CHO cell line. Three distinct approaches are described. The first approach was the cloning, in CHO cells of four eukaryotic proteins known to promote flocculation. The second approach used inorganic cations as flocculating agents and the third approach utilised proteomics to investigate cellular adhesion in CHO cells. The nature of the proteins investigated in this thesis lead to challenges in achieving ectopic expression in CHO cells. While the addition of FeCl3, MnCl2 and CaCl2 did result in improved rates of sedimentation of CHO cells compared to untreated controls without any negative impact on a human antibodies structural integrity, the antibody yield at 62% for MnCl2,72% FeCl3 and 76% for CaCl2 was not commercially viable. A differential expression proteomic approach identified a cohort of proteins involved in the catabolism of amino acids, the catabolism of fatty acids, cholesterol metabolism, protein processing in the endoplasmic reticulum and glycolysis/gluconeogenesis which were significantly enriched following the adaptation from attached to suspension growth. Additionally, the thesis proposes a potential mechanism which CHO cells developed to combat increased cellular stress during this adaptation process. This thesis is an describes the first steps towards engineering a flocculating CHO cell line suitable for use with current commercial processes.
Metadata
Item Type: | Thesis (PhD) |
---|---|
Date of Award: | March 2023 |
Refereed: | No |
Supervisor(s): | Barron, Niall, Racher, Andrew and O'Gorman, Donal |
Subjects: | Biological Sciences > Biochemistry Biological Sciences > Biology Biological Sciences > Biotechnology Biological Sciences > Molecular biology |
DCU Faculties and Centres: | DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology Research Initiatives and Centres > National Institute for Cellular Biotechnology (NICB) |
Funders: | Lonza |
ID Code: | 28007 |
Deposited On: | 03 Apr 2023 09:03 by Donal O'gorman . Last Modified 03 Apr 2023 09:03 |
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