Prediction of transgenic tobacco plant processing properties
by ultra scale down and physical property measurement for
monoclonal antibody production.
Doctoral thesis, UCL (University College London).
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 IgG1 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 (mIgG). 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 -20oC 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 dryfreeze- 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 intracellularly 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. Moreover, despite the fact that the default pathway of IgG is secretion and thus, can be secreted into the medium within a hydroponic system for example, the secretion rate is slow and there is likely to be a large medium requirement. A substitute for this is mechanical root breakage in order to generate higher antibody yields. A novel approach is described here with the capability of determining the force magnitude for breaking single plant roots. Roots were taken from transgenic tobacco plants, expressing a secreted monoclonal antibody. They were divided into four key developmental stages. A novel micromanipulation technique was used to pull to breakage, single tobacco roots in buffer in order to determine their breaking force. A characteristic uniform step-wise increase in the force up to a peak force for breakage was observed. The mean breaking force and mean work done were 101mN and 97μJ per root respectively. However, there was a significant increase in breaking force from the youngest white roots to the oldest, dark red-brown roots. We speculate that this was due to increasing lignin deposition with root stage of development (shown by phloroglucinol staining). No significant differences between fresh root mass, original root length, or mean root diameter for any of the root categories were found, displaying their uniformity, which would be beneficial for bioprocessing. In addition, no significant difference in antibody yield from the different root categories was found. These data demonstrated that it is possible to characterise the force requirements for root breakage and should assist in the optimisation of recombinant protein extraction from these roots. Following on from this, the determination of the minimum energy required for monoclonal antibody (MAb) extraction from transgenic tobacco roots is desirable, in order to optimise product yield. Mechanical breakage of transgenic tobacco roots (expressing a MAb) in a scalable laboratory scale mechanically stirred shear device was assessed. The resulting experimental data together with a mathematical model was used to estimate root tensile strength and this was compared with the mean tensile strength of such roots as previously determined by a micromanipulation technique where the scale corresponds to a single 1cm root section. Breakage of multiple roots (~100) in a small-scale mechanically stirred scalable shear device was investigated. Size distributions of the roots when passed through the breakage device at a rotational speed of 75s-1 were obtained as a function of time. It is probable that root fragmentation in such a mechanically stirred shear device is due to root-impeller collisions. Assuming this mode of breakage, a relationship between the equilibrium mean length of the root debris and the critical physical parameters affecting it was established using a theory of failure based on a maximum strain energy criterion. Data on the stable size of the root debris agreed well with the model based on the maximum energy criterion. The data further suggest that root breakage within this device was approximately a first-order process. The model suggested that knowledge of root tensile strength was important since it directly determined the minimum energy required to break the transgenic roots. A macroscopic estimation of the relative root tensile strength by using experimental data within the suggested model gave an estimated tensile strength of 2200 ± 600 kPa (average ± SEM) which agreed well with the tensile strength measured by micromanipulation 2400 ± 200 kPa. Thus, measurement of root tensile strength by micromanipulation appeared to be a valid means of finding the minimum energy required for modelling breakage in a macroscopic mechanical shear device.
|Title:||Prediction of transgenic tobacco plant processing properties by ultra scale down and physical property measurement for monoclonal antibody production|
|Additional information:||Authorisation for digitisation not received|
|UCL classification:||UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering|
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