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Engineering of the Secretory Pathway of CHO Cells for Recombinant Protein Production: Manipulation of SNARE Proteins

Mozzanino, Théo (2018) Engineering of the Secretory Pathway of CHO Cells for Recombinant Protein Production: Manipulation of SNARE Proteins. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:73387)

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Abstract

The production of recombinant biotherapeutic proteins is usually achieved at an industrial scale using cultured cell systems with a number of host systems used including bacteria, yeast, insect cells and mammalian cells, depending on the needs of the protein to be produced. In the last 2-3 decades, the market for recombinant protein production has been dominated by monoclonal antibodies (mAbs), glycoproteins with complex post-translational modifications necessary for their therapeutic function. For the production of such molecules, mammalian cells and particularly Chinese hamster ovary (CHO) cells are often the host expression system of choice for their ability to correctly fold and assemble such complex molecules and to perform human-like glycosylation. The ability of CHO host cells to generate high levels (>5 g/L) of mAbs in particular has been enhanced and attained over the years via the implementation of different strategies including the design of media and feeding strategies, use of high throughput screening approaches to identify high producing cell lines, redesign of processes during bioprocessing, manipulation of genetic constructs to drive recombinant gene expression and protein engineering to improve CHO host cell productivity and product quality. Despite these approaches, there remains a desire to further improve the ability of CHO and other host cell systems to be improved to enhance their ability even further with regard to production and quality of biopharmaceuticals, particular more difficult to express molecules than 'standard' mAbs. One strategy that has been undertaken to enhance the cellular capacity for recombinant protein expression is cell line engineering, where a desired phenotype of the host is achieved by genetic manipulation. In this study, the secretory pathway of the CHO cell has been manipulated to investigate whether its engineering can improve the secretory capacity of two different CHO host cell lines under batch and fed-batch conditions. Specifically, members of the SNARE family, a family of proteins involved in the fusion machinery of vesicles which have been suggested to be a bottleneck in the secretory pathway, were ectopically over-expressed and the effect(s) of the overexpression on growth, culture viability and recombinant protein productivity of the CHO hosts determined. In a CHO-S cell line, overexpression of specific levels of syntaxin 17 (STX17) and SNAP29 fused to eGFP increased culture longevity and late culture viability, possibly through an impact on the autophagy pathway. Assessment of the impact of the ectopic expression of these SNAREs in CHO-S during transient expression of model proteins under batch culture conditions, IgG1 Adalimumab and the fusion protein Etanercept, showed an increase in titre up to 5-fold (STX17) and 2.4-fold (SNAP29) respectively. Specific levels of SNARE expression were required to observe a titre increase. Indeed, only cell lines overexpressing to a lower level of SNAP29 demonstrated an increased titre whereas a more linear effect was observed for cell lines expressing STX17, with higher STX17 expressing cell lines having a greater increase in titre than low STX17 overexpressers. Cells expressing STX18 also revealed an increase, up to 3-fold, in yield when producing Adalimumab, but not Etanercept, suggesting a molecule specific effect. Transferability of this approach to a second CHO host in the industrial environment was then investigated by engineering of an industrial host cell line,"Clone 27", a CHO-DG44 derived cell line. No increase in product titre was observed when the SNAREs were over-expressed in already high expressing recombinant mAb cell lines. The differences in the approaches and environment, as well as the intrinsic differences in the CHO host cell lines, might explain the divergence of observed effects between the cell lines. Nonetheless, a positive impact upon the overexpression of target SNAREs at specific levels was observed with regard to growth and productivity in CHO-S host cells suggesting SNARE manipulation was successful for the engineering of CHO-S cells. Moreover, the mechanisms by which the overexpressed SNAREs potentially elicit their changes in secretory phenotypes in the CHO-S host offers up new areas of interest for future cell line engineering strategies such as autophagy and mitophagy.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Smales, Christopher Mark
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 16 Apr 2019 12:27 UTC
Last Modified: 01 Jan 2020 00:00 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/73387 (The current URI for this page, for reference purposes)
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