The production of therapeutic proteins with complex post-translational modifications is typically performed with suspension adapted mammalian cells, in stirred tank bioreactors (STRs). In this environment cells are exposed to hydrodynamic forces derived from both agitation and aeration. An improved understanding of these forces and the resulting cellular response is therefore critical. This thesis has characterised the hydrodynamic conditions within a STR using computational fluid dynamics and investigated the lethal and sub-lethal effects of this environment on CHO cells during the production of a monoclonal antibody. The hydrodynamic forces varied within the vessel, with the maximum forces being present in the impeller region. Energy of dissipation rates obtained from single and multiphase models were found to be higher than those previously reported. Having gained understanding of the hydrodynamic forces within the bioreactor, the environment was changed in a systematic manner. To create a bubble and bubble free environment within the STR direct gas sparged and silicone membrane aerated systems (SMAS) were used. When cells were subjected to the stress of gas-liquid interfaces they entered apoptosis at an earlier stage of cell culture, had decreased F-actin intensity and a modified cell morphology. This had implications on cell strength and impurity release during primary recovery. The application of fed-batch mode and mild hypothermic conditions were shown to be beneficial; delaying the onset of lethal and sub-lethal effects, enabling higher cell densities to be obtained, prolonging the culture duration and increasing product titre. The vital role of shear protectant agents at gas-liquid interfaces was highlighted; in absence of Pluronic F-68 cell death occurred within 24 hours of inoculation, but growth proceeded when using the SMAS. The glycosylation profile was not significantly affected by the STR environment, with harvest point being shown to have a greater impact on the relative abundance of different glycoforms.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626396 |
| Date | January 2013 |
| Creators | Velez Suberbie, L. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1410199/ |
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