In the biopharmaceutical industry, essentially, there are three components that play the main role in producing biopharmaceutical products, the host cell, the expression vector and the bioreactor and/or production environment. To produce the highly valued and desired products, the choice of a suitable host is one of the most important aspects. The host required is not only required to produce the desired product, but also needs to demonstrate robustness in a bioreactor system. Constantly facing challenges in a bioreactor, cells often undergo apoptosis, a well-known limiting factor in biopharmaceutical production, which ultimately leads to low yield of valuable protein(s). We have genetically engineered a CHO-K1 cell line to constitutively express human insulin-like growth factor-1 (IGF-1) and murine polyoma large T-antigen (PyLT-Ag) to generate Super-CHO and CHO-T respectively, two cell lines that can potentially serve different niches in the biopharmaceutical industry. In the first part of the project, we hypothesised that suspension-adapted Super-CHO and CHO-T cells are both resilient cell lines relative to the suspension-adapted CHO-K1 (designated as CHO-XL-99) when facing nutrient depletion, one of the most common problems in a bioreactor. Furthermore, in the second part of this project, the suspension-adapted CHO cell lines were also tested against a cytotoxic heavy metal, cadmium. Without the protection of the metal-resistance element, metallothionein, both Super-CHO and CHO-T cells were also challenged with cadmium to demonstrate their robustness over the parental cell line, CHO-XL-99. In the subsequent study, this project also focussed on the transfection efficiency of each parental and engineered CHO cell lines. Different strategies have been employed in the past in an attempt to improve productivity in the biopharmaceutical industry, from alterations in vector construction, improved culture condition, down to enhanced product recovery. However, the transfer and expression of the gene-of-interest (GOI) has still proven to be the limiting factor for achieving increased specific productivity. In an effort to improve transfection efficiency, strategies including cell cycle synchronisation and various transfection methods to deliver the GOI into the cells have been employed. Thus, the third part of this project has used synchronising agents in conjunction with commercially available lipid- and polymer-based reagents as delivery vehicle for the model protein, EGFP. The combination of cell synchronisation and transfection vehicle on transfection efficiency is studied here, in addition to their individual or collective effect on cell growth, apoptosis and viability. In summary, this project demonstrates the incidence of apoptosis in the cell culture induced by nutrient depletion and heavy metal, and that the use of transfection reagents solely, or in combination with synchronising agents also correlates with the increase of apoptotic indices in the cell culture. The use of the robust cell lines for transfection is an important aspect, and the balance between cell viability and the effort for augmenting transfection efficiency has to be met in order to achieve the maximum biopharmaceutical yields.
| Identifer | oai:union.ndltd.org:ADTP/215610 |
| Date | January 2007 |
| Creators | Wanandy, Nico Stanislaus, School of Biotechnology & Biomolecular Science, UNSW |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://unsworks.unsw.edu.au/copyright, http://unsworks.unsw.edu.au/copyright |
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