Tait, AS;
(2005)
PEI-mediated transient gene expression in cell culture for the rapid production of therapeutic proteins.
Doctoral thesis , UCL (University College London).
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Abstract
The aim of this study was to investigate the feasibility of using transient gene expression in mammalian cell culture for the production of recombinant therapeutic proteins. This would allow rapid production of candidate molecules for pre-clinical trial testing and characterisation. For unsuccessful candidates, this would remove the time consuming and costly requirement of producing a stable cell line. For effective candidates, the removal of the immediate requirement of producing a stable cell line could reduce development times by up to 6 months. This thesis describes the development of a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells (CHO-S) for the large-scale production of recombinant therapeutic proteins. Optimisation was done in a serum free environment, to make the process industrially applicable. However, removal of serum was found to dramatically affect the cell specific growth rate and transient transfection expression rate. This problem was negated by the addition of bovine serum albumin, and a concentration of 6 mg L"1 was used in subsequent optimisations. The PEI mediated transfection process was optimised with respect to PEI nitrogen to DNA phosphate ratio and the plasmid DNA mass to cell ratio, using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI nitrogen to DNA phosphate molar ratio of 10:1 and 5 jug DNA per 106 cells. It was found that PEI is cytotoxic to CHO-S cells, and the final concentration of PEI in the growth medium appears to be the critical factor. At high PEI concentrations, cell growth is reduced, which is coupled with a reduction in the transgene expression. Therefore at ratios greater than 10:1, or DNA concentrations higher than 5 ng DNA per 106 cells, cell growth rate, and therefore transgene expression was lower. This suggested that transient gene expression is linked to cell growth, and therefore cell division. Cell cycle blocking experiments demonstrated that transition into mitosis is a pre-requisite for efficient transient gene expression.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | PEI-mediated transient gene expression in cell culture for the rapid production of therapeutic proteins |
| Identifier: | PQ ETD:593189 |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. Third party copyright material has been removed from the ethesis. Images identifying individuals have been redacted or partially redacted to protect their identity. |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1445865 |
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