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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