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In this work, the potential of automated microscale process sequences for the evaluation of recombinant biocatalysts and the use of microwell data to inform larger scale operations is examined. As a model bioconversion, the cyclohexanone monooxygenase (CHMO) Baeyer-Villiger oxidation of a range of cyclic ketone substrates was examined cyclopentanone, cyclohexanone and bicyclo 3.2.0 hept-2-en-6-one. Three whole cell biocatalysts were evaluated which included two strains of E. coll. TOP10 pQR239 and JM107 and A. calcoaceticusNCIMB 9871. Initial studies to establish the microwell process used the well-characterised E. coli TOP 10 pQR239 biocatalyst that was previously prepared in our laboratory. It was shown that quantitative and reproducible data could be obtained for fermentation, enzyme induction and bioconversion operations carried out in 96 Deep Square Well (96 DSW) plate formats. It was possible to produce up to 5.8 gocw-l" of the E. coli biocatalyst and support growth rates up to 0.55 h". The biocatalyst had a specific CHMO activity of 11 U.gocw"1 and could catalyse the conversion of bicyclo 3.2.0 hept-2-en-6-one at an initial rate of product formation of 62 pmoLl min*1 with high yields of up to 92%. The microscale process sequence was then operated in an automated fashion using a Multiprobe II EX liquid handling robot (Packard Instrument Company, Meriden, Connecticut, US). The process performance of the three biocatalysts was then examined following a combinatorial approach in which biocatalyst was produced at initial glycerol concentrations of 10 and 20 in fermentation and, following induction (when required), bioconversion rates were measured for each of the three ketone substrates at initial concentrations of 0.5 g.l"1 and 1.0 g.l"1. The E. coli TOP10 pQR239 biocatalyst showed the best overall performance and the results obtained were comparable to those already reported in the literature. Finally, the ability of microwell results to be predictive of larger scale operations was examined. Given the demand for molecular oxygen at both, the fermentation and bioconversion stages, the oxygen mass transfer coefficient, kLa, was considered as a basis for scale-up. ki,a values in both shaken microwell plates and a 2 1 stirred bioreactor were determined over a range of operating conditions. Values in the range 15 - 188 h"1 could be obtained at both scales. Processes carried out at the two scales at matched a values showed excellent agreement both in terms of the quantitative values of the results obtained and the way in which the process performance varied with kia. The results obtained in this thesis further show the potential of automated microwell experimentation to support rapid and more cost effective bioprocess development.

In order to realise the potential of embryonic stem (ES) cells as a regenerative medicine, it is crucial that economical, robust and scalable bioprocesses be established. Because bioprocesses irrevocably define the safety and efficacy of any biologically derived product, an understanding of the the impact of the engineering environment on ES cells is sought. This thesis uses murine ES cells as a mimic for ES cell types that will be used in cell based regenerative medicine applications to examine the bioprocessing impact of centrifugal recovery cells. A micro scale-down approach was used to examine the effects of centrifugal force, centrifugation time and process temperature on both the yield and biological characteristics of cells subjected to batch centrifugation. When subjected to centrifugation, mES cell loss and cell damage does not appear to occur during the settling or cell pelleting. In general, 5-25% of cells are lost during pellet resuspension to recover the centrifuge cells. The level of cell loss is determined by a combination of centrifugal force, centrifugation time and process temperature. The extent of damage of the remaining cells (i.e. cells not lost during resuspension) is minimised at lower processing temperatures. It is hypothesised that at low processing temperatures, cell loss is minimised due to weak cell-to-cell contact and are thus less susceptible to damage caused by the shear environment generated to disperse the collected cell pellet. The concept of Windows of operations was also applied to evaluate an optimal set of centrifuge operating conditions that results in minimal cell loss and cell damage. The process visualisation tool indicates that operating the centrifuge at 5-9 mins x 300-500 g will result in maximum cell recovery at 4, 21 and 37oC process temperatures. The influence of centrifugation on the biological characteristics of mES cells revealed changes in proliferative capacity, pluripotency and differentiation status when exposed to varying levels of centrifugal force. mES cells exposed to increasing levels of centrifugal force up to 2,000 g progressively lost pluripotency. The pluripotency potential of cells exposed to 3,000 g of centrifugal force was not significantly different from un-centrifuged mES cells. Differentiating mES cells exposed to increasing levels of centrifugal force exhibited increased cell proliferation and a possibility of early induction of endoderm and mesoderm differentiation. Although limited in some areas, the results strongly suggest that restricting exposure to no more than low levels of centrifugal force is necessary to safeguard the stability of the desired mES cell characteristics. Overall, the insight gained from the work accomplished serves to create and establish an awareness of the challenges faced within the arena of whole cell bioprocessing for regenerative medicines.

This thesis describes three applications of the theory of continuous autonomous dynamical systems. The focus of the thesis is on qualitative, as opposed to numerical, analysis. The applications examined are biological and chemical, and as such there are signicant uncertainties in any mathematical representation of them. While the qualitative relationships that dene a biological or chemical system may be well understood, it is often dicult to obtain accurate measurements of the parameters that govern each interaction, due to inherent variability and/or experimental constraints. For this reason, a model that avoids dependence on numerical values while still accurately reecting the qualitative structure of the system it represents is potentially of use in gaining a greater understanding of how the system can behave. Conversely, if a purely qualitative model allows certain behaviour that is never experimentally observed, this may highlight the importance of certain parameter values for the system's real world behaviour. The rst application presented is a model of electron transport in mitochondria, the second is a model of an inter-cellular gap junction, and the third represents a set of reactions occurring in a continuous ow stirred tank reactor. For each application, a reasonable set of qualitative assumptions is found under which there is a unique steady state to which all initial conditions converge, regardless of precise numerical values. Uniqueness of steady states is proved using results on the injectivity of functions, and degree theory. The convergence criteria are constructed using two dierent areas of dynamical systems theory. The rst of these is the theory of monotone ows, while the second is a group of results known as autonomous convergence theorems. The theory of monotone ows is fairly well known, and relies on nding conditions under which trajectories of a dynamical system preserve a partial ordering, thereby limiting the possibly asymptotic behaviour of the system. The autonomous convergence theorems appear much less well known; they work by nding a norm under which trajectories approach each other, either in phase space or in a related exterior algebra space. Both theories are discussed in detail, along with some extensions.

The enzyme transketolase (TK; E.C. 2.2.1.1) from Escherichia coli occupies a pivotal place in metabolic regulation. TK catalyses the interconversion of sugars by transferring a two-carbon ketol unit from a ketose donor substrate to an aldolase acceptor substrate. It is also an important biocatalyst in stereo-specific carbon-carbon bond synthesis with potential industrial application for the synthesis of pharmaceuticals, agrochemicals and fine chemicals. Although many useful reactions have been reported for TK, many of the substrates and products are unstable or insoluble at the pH or temperature for which the enzyme has optimum activity. Understanding the structural stability of transketolase under bioprocess conditions will improve our capacity to comprehend and ultimately to engineer it to make it work in a broader range of pH or temperature to potentially help in the reduction of process time and to increase the quality and solubility of products. In this research I characterised the early events on the urea denaturation pathway of E. coli transketolase, providing new insights into the mechanisms of enzyme deactivation that occur under biocatalytic conditions. Equilibrium denaturation measurements by fluorescence intensity and circular dichroism (CD), combined with size-exclusion and dynamic light scattering studies, have revealed three transitions in the denaturation pathway for holo-TK. The first step, at low urea concentration corresponds to the local restructuring of the thiamine diphosphate (TPP) binding-sites. Next, the dissociation of the TPP cofactors and partial loss of secondary structure produces a form, which is most consistent with a partially denatured dimeric enzyme. While the enzyme is deactivated initially by changes in structure associated with the cofactors, this event does not release the cofactor from the enzyme, consistent with the intermediate formed during the reconstitution of holo-TK from apo-TK. Improvement of biocatalytic processes using TK over prolonged reaction times would, therefore, need to address the formation of this cofactor-associated intermediate state. Equivalent results were also observed with a high throughput microplate-based fluorescence method that uses less enzyme and time. The equilibrium denaturation of holo and apo-TK at different temperatures and pH was also investigated for further insights into the enzyme stability and to provide a benchmark for assessing any future enzyme variants with altered pH or temperature optima. In an effort to enhance the stability of the enzyme I subsequently used bioinformatical, statistical and multivariate analyses of protein sequences and associated properties to determine the most likely residues to affect temperature and pH optima in biocatalysis. The outcome of this first parametric statistical analysis (Pearson’s r test) rendered 20 different points. Promising mutation points were selected based on the correlation coefficient (r) results taken into account a level of significance α = 0.05. TK E. coli selected points were then mutated and screened by site-saturated mutagenesis (SSM) and automated techniques respectively. Finally alternative statistical correlation methods were examined, including a non-parametric statistical analysis (Kendall’s τ or tau test), principal component analysis (PCA) and partial least square (PLS), for their potential to generate TK mutant variants with either enhanced pH or temperature stability.

Currently the purification of monoclonal antibodies for therapeutic purposes is reliant on protein A affinity chromatography. The rapid growth of this class of therapeutic and their high value makes the understanding of protein A chromatography an important target. There is a range of commercially available protein A chromatography media. The main differences between these media are the support matrix type, the pore size, the particle size, the amount of ligand attached to the matrix and the kind of protein A modification. The differences in these factors give rise to differences in compressibility, chemical and physical robustness, diffusion resistance and binding capacity of the adsorbents. The ideal media would have high specificity, high mass transfer and binding capacity, low non-specific adsorption and ligand leakage, incompressibility, resistance to alkaline condition for sanitization, chemical stability and cost effectiveness. Current resins offer a compromise, which balances what is achievable in respect of these features giving rise to an array of different solutions. Measurement of these parameters is often complex and agreed standards have yet to be determined. The objective of this study is to further develop understanding of these measurements for the assessment of the matrix performance. This thesis employs a suite of techniques to characterise commercial and prototype adsorbents. The adsorbents that will be looked at are MabSelect (GE Healthcare), MabSelect Xtra (GE Healthcare), Prosep Ultra (Millipore), Protein A immobilised on 4CL Sepharose (GE Heatlhcare) in house and a prototype adsorbent with a Protein A mimic ligand (Millipore). Both down-scaled techniques of fixed bed chromatography, together with supporting analysis of equilibrium and dynamic behaviours are used. The latter will adopt standard and novel ‘wet chemistry’ approaches together with the increasingly adopted techniques of laser scanning confocal microscopy. Experiments are carried out using hIgG to study the static capacity, adsorption equilibrium and dynamic capacity of adsorbents. Other techniques will be used to study the kinetic uptake and desorption rates of adsorbents in different conditions. A novel approach using confocal microscopy is used to further understand the adsorption behaviour of individual beads of different sizes. The main results that were drawn from these techniques are that MabSelect Xtra had the highest static capacity of 61.8mg/ml. It also showed the highest dynamic capacity at 2 mins, 4 mins and 8 mins residence time (0.66cm Omnifit column, bed height 6cm) when compared to other adsorbents. This is mainly due to the more porous nature of the MabSelect Xtra beads, which increased the surface area available for Protein A ligand immobilisation. From the adsorption equilibrium data the Kd values ranged from 181nM to 36nM. Such low values are expected by affinity adsorbents such as these. The uptake rate curves were similar for all the adsorbents. Hence the difference in particle size, pore size, the type of ligand or the material of the adsorbent itself did not have an effect on the uptake rate when carried out in a batch mode. A similar behaviour was shown for the desorption curves. The confocal analysis using a flow cell showed that all the adsorbents showed a shrinking core effect except for the prototype where the hIgG didn’t penetrate into the bead and was only attached to the surface of the bead. It was found that the adsorption rate to the centre of each bead was linear. The different particle sizes within any particular type of matrix and also across different matrix did not result in different diffusion rates. From the adsorption curves produced it was seen that smaller beads reached saturation much faster than larger beads at any given time. This technique can have great benefits in understanding how individual beads of different adsorbents behave in different circumstances.

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