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Proteomic profiling of phosphoproteins and secreted proteins from mammalian cell lines in order to gain insights into factors affecting cellular growth and recombinant protein production

Power, Martin (2015) Proteomic profiling of phosphoproteins and secreted proteins from mammalian cell lines in order to gain insights into factors affecting cellular growth and recombinant protein production. PhD thesis, Dublin City University.

Abstract
Chinese hamster ovary (CHO) cells are one of the most commonly used cell lines in the production of biopharmaceuticals. The reduction of culture temperature during the exponential phase of a culture is a strategy that is commonly employed in the bioprocessing industry to increase process yield. Lower culture temperature results in a marked reduction in cell growth, increased specific productivity and prolonged cell viability. In order to understand the mechanisms involved at the post-transcriptional level in the cellular response to temperature-shift a phosphoproteomic analysis of SEAP secreting CHO cells was conducted. Using a liquid-chromatography mass spectrometry-based label-free approach in conjunction with Gallium, Iron and Titanium phosphopeptide enrichment strategies 1,307 unique phosphopeptides (1,480 phosphosites) were identified. Gene Ontology analysis revealed enrichment of pathways involved in protein synthesis and cell cycle progression. 92 phosphopeptides were determined as being significantly differentially-expressed 36hrs post temperature-shift, including translation initiation factors EIF5B, EIF4G3, Transcription activator BRG1 and the tumour suppressor protein, Protein NDRG1. Such proteins are potentially involved in controlling growth and recombinant protein production in CHO cells. While the effects of temperature-shift can result in an increase in overall product yield, it can also have implications for the extracellular-milieu from which the product must be purified. The second aspect of this thesis identifies changes in the host cell protein (HCP) profile of an IgG-producing and non-producing cell line over time in culture under both temperature-shifted and non-temperature-shifted conditions. This includes the identification of proteases (Cathepsin B, Cathepsin D, MMP9) and glycosidases (beta-galactosidase and α-N-acetylgalactosaminidase), that could negatively impact product quality. In addition, proteins that could enhance cell growth such as vascular endothelial growth factor isoforms A and C and Hepatomaderived growth factor were also identified. It was also found that the cell line and culture conditions used both impacted on the HCP profile generated.
Metadata
Item Type:Thesis (PhD)
Date of Award:November 2015
Refereed:No
Supervisor(s):Meleady, Paula and Clynes, Martin
Uncontrolled Keywords:Chinese hamster ovary cells; CHO cells; Process yield
Subjects:Biological Sciences > Biotechnology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:20570
Deposited On:24 Nov 2015 14:53 by Paula Meleady . Last Modified 03 Aug 2021 15:12
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