Wu, AE-T;
(2007)
Examination of transient transfection as a potential means of recombinant protein production.
Doctoral thesis , UCL (University College London).
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Abstract
The application of transient transfection to the production of recombinant proteins in mammalian cell culture is examined in this thesis. Transient transfection is able to rapidly produce significant amounts of recombinant protein in a matter of weeks. This allows the early product development, such as preclinical testing and characterization, to be performed independently of the lengthy cloning process that can take up to 6 months. Selection of cell line (CHO), culture conditions (serum-free) and transfection agent (polyethylenimine) were chosen with a focus on industrial relevance. Physicochemical characterisation of PEI-DNA complexes formed in physiological buffer (150 mM NaCl) and culture media (DMEM and CHO-S-SFM II) showed that positive zeta potential during formation lead to higher transfection efficiency. Particle studies showed that initial aggregation during formation was reversed when complexes were diluted in media during transfection. DNA uptake is influenced by culture media in which the transfection takes place. Complexes formed in 150 mM NaCl had the highest transfection efficiency. Examination of process variables showed that complexes prepared in 150 mM NaCl were less sensitive to variations in formation incubation time than complexes prepared in culture media. The presence of conditioned media was determined to be detrimental to transfection efficiency and introduced more variability. Studies carried out into the effect of the volume fraction of PEI-DNA complex solution showed that 10% v/v gave better transfection efficiency. Additions to agitated microwells showed a transition in mixing time and mixing regime between shaking speeds of 200-250 rpm, mixing times were observed to decrease by two orders of magnitude. This was further showed by observations from still images of high-speed camera footage of increased deformation in the liquid surface between these shaking speeds. Scale-up of transient transfection for recombinant protein production across three geometries (24-well plates, shake flasks and bioreactor) gave comparable SEAP expression levels of 2.4- 2.8 mg/l, thus showing that transient transfection is a scalable process Microwells are, hence, a suitable platform for process development, and productivity is translatable to larger scales. Overall this work has determined that with adequate characterisation of process parameters, transient transfection is a rapid tool for the production of recombinant proteins.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | Examination of transient transfection as a potential means of recombinant protein production |
| Identifier: | PQ ETD:593331 |
| 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/1446005 |
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