There are numerous potential advantages of producing significant quantities of a monoclonal antibody (MAb) via transgenic tobacco plants over other heterologous production systems, thus paving the way for new prophylactic and therapeutic applications within global human and animal health. However, current information on the key processing factors for large scale production of antibodies from transgenic plants is limited. This thesis presents the issues involved in the production of monoclonal antibodies in transgenic tobacco plants with a specific focus on initial extraction and aids the design and characterisation of an optimal small-scale extraction process using ultrascale down and micromanipulation techniques based on large-scale principals, in addition to offering different harvesting and extraction strategies dependent upon the specific target subcellular or tissue compartment. One of the preliminary objectives of this project was to examine methods for the initial extraction of recombinant IgGl antibodies from the leaf tissue of transgenic tobacco. Three different transgenic plant lines were investigated with the intention of establishing the parameters for optimal extraction of MAbs that reside in the apoplasm (IgG), endoplasmic reticulum (IgG-HDEL), or are bound to the plasma membrane (mlgG). For each transgenic line, seven techniques for physical extraction were evaluated. For IgG that is secreted and accumulated in the apoplasm, dry freeze-thaw (the freezing of leaf discs at -20 °C followed by room temperature thawing before buffer addition) was an appropriate technique for extraction of a high yield and a low release of native plant proteins from leaves in comparison to the other techniques investigated. In addition to lowering the downstream purification burden, the large-scale equipment involved in this step is likely to have a lower operating cost than a mechanical, energy-intensive grinding device. IgG-HDEL-expressing transgenic plants demonstrated an increase in IgG-HDEL yield with technique severity, demonstrating that harsher techniques such as dry-freeze-thaw followed by grinding were optimal. Conversely, the membrane-bound IgG required the leaf tissue to be ground in buffer that included a non-ionic detergent (Triton X-100), the optimal concentration of which was 0.1% (v/v). Grinding samples on ice or at room temperature was found to have no effect on IgG yield for all three MAbs. This indication of plant-derived IgG stability at room temperature is an obvious cost benefit at industrial scale. For all forms of the IgG, there was a wide variety of usable pHs (pH 5 to 7) with the exception of very low pHs (pH 3 and 4). Overall, an important finding of this study was that determining factors of optimal antibody extraction from plants had a direct influence on the initial choice of expression strategy, and thus it was essential to be addressed from the outset. In addition, a principally important consideration was the use of small scale techniques that were applicable to large scale purification. Another important factor of recombinant protein production in transgenic plants that is often overlooked is the initial bioprocesing step of harvesting. The major harvesting factors that need to be addressed are when to harvest, which part of the plant to harvest and how to harvest. Here some of these factors for the production of a secreted IgG and an intracellular retained form of this IgG in transgenic tobacco were addressed. Data analysis resulted in an interesting observation of plant wound response and its consequences for time-response IgG levels. The same monoclonal antibody (MAb), (Guy's 13 that acts against Streptococcus mutans, the main agent of tooth decay in the mouth) targeted to two different subcellular compartments, showed varying IgG response levels after wounding. In addition, there was a significantly different type of wound response and the subsequent IgG levels for young and old plants expressing the secreted form of IgG with a negative effect (IgG reduction) on young growing plants and a positive effect (IgG boost) in older plants. Additionally, for secreted IgG expressing plants, IgG strongly depended on plant age with the highest amount of IgG being found in young leaves of old plants and or young plants, but with a marked reduction in older tissue that was most likely due to senescence. In contrast, intracellularly retained IgG that accumulated in the endoplasmic reticulum was not significantly affected by mechanical wounding and its frequency or by overall senescence. In addition to transgenic tobacco leaves, tobacco roots were investigated as a potential source of the MAb. It was found that despite the focus of current related literature on recombinant protein recovery being from the leaves of whole transgenic tobacco plants, roots offer a promising alternative.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:625177 |
| Date | January 2008 |
| Creators | Hassan, S. Y. |
| 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/1444237/ |
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