The worldwide increase in antibiotic resistant bacteria has been identified as a major concern to the continued provision of healthcare. The lack of new antibiotic discoveries and the diminishing number effective against multi-drug resistant bacteria has increased efforts to identify new natural antimicrobials and to use synthetic biology to engineer artificial ones. Lantibiotics, a class of ribosomally synthesized antimicrobial peptides, with a broad spectrum of activity, have been identified as potential candidates to combat such bacteria. However no clinical lantibiotic products currently exist, owing to low product titres and lack of a suitable manufacturing process. Initial studies on the lantibiotic gallidermin have managed to increase product titre by modifying the producer strain, Stapylococcus gallinarum Tϋ3928, to synthesize the biologically inactive precursor, pregallidermin. This helps overcome product auto-toxicity and may be a generic route to enhancing the production of novel engineered lantibiotics. This thesis investigates the scalability of a pregallidermin production process to determine whether it could serve as a production platform for novel lantibiotics. Initially, fed-batch cultures were performed in 7.5 L stirred tank bioreactors to characterise the parameters necessary for achieving high cell density. These identified aeration, agitation and nutrient feed rates as important parameters to optimal product formation. The fed-batch culture was repeated at pilot scale (30 L) under the same process conditions, yielding 0.7 g.L-1 of pregallidermin. A scalable downstream process was also developed and evaluated. The overall process flowsheet comprised of pregallidermin capture on the hydrophobic resin Amberlite XAD-7, followed by cation exchange chromatography to purify the pregallidermin. This was then trypsinised to release the required gallidermin. The final purity of gallidermin was 70 % (w/w) with an overall process yield of 13.5 %. Using this downstream process sequence 1.9 g (0.09 g.L-1) of pregallidermin was isolated from the 30 L fermentation. Confirmation of the identity of the purified pregallidermin and gallidermin was obtained by HPLC and mass spectrometry. Microscale methods were developed to optimise the downstream process and determine product physico-chemical properties with a view to informing process design. These determined that higher purity gallidermin recovery was possible with the optimisation of the XAD-7 adsorption/desorption. Optimal conditions for pregallidermin adsorption, resin wash and pregallidermin elution from resin were determined using the microscale methods. A redesign of the downstream process based on optimised pregallidermin capture increased overall recovery of mature gallidermin 3-fold to 0.3 g.L-1. In summary, this work has demonstrated a novel process for lantibiotic production and purification at pilot scale. The process is sufficiently generic that it could serve as a manufacturing platform for production of next generation engineered lantibiotics. It has also demonstrated the utility of microscale methods in optimising the production process.

A specific challenge to the translation of cell therapies and stem-cell derived products is the ability to develop and manufacture such products in a cost-effective, scalable and robust manner. To this end, this thesis investigates the creation and application of a set of computational tools designed to aid bioprocess design decisions for cell therapy and stem-cell derived research products. The decision-support tools comprise advanced bioprocess economics models with databases tailored to cellular products. These are linked to Monte Carlo simulation for uncertainty analysis and techniques to identify optimal bioprocess designs that include brute-force search algorithms, an evolutionary algorithm, and multi-attribute decision making analysis. A trio of industrially-relevant case studies is presented within this thesis, along with an additional study included in the appendices of this work, in order to demonstrate the applicability of the decisional tools to bioprocess design for different cell therapies (allogeneic, human embryonic stem cell-derived retinal pigment epithelial (RPE) cells for macular degeneration, allogeneic CAR-T cells for oncology) and induced pluripotent stem cells (iPSCs) for drug discovery applications. Questions tackled included manual versus automated production, costeffective inflection points of planar vs microcarrier-based bioprocess strategies, and the identification optimal process technologies for an allogeneic CAR-T cell therapy based on both qualitative and quantitative attributes. The analyses highlighted key bioprocess economic drivers and process bottlenecks. Furthermore, the Monte Carlo simulation technique was used in order to capture the effects of the inherent uncertainty associated with cell therapy bioprocessing on manufacturing costs and process throughputs. Future process improvements required to create financially feasible bioprocesses were also identified. This thesis presents the application of a series of decisional tools to bioprocess design problems and demonstrates how they can facilitate informed decisions regarding cost-effective process design in the cell therapy sector.

The mechanisms by which mesenchymal cells can repair the infarcted myocardium are still unclear and are made more challenging by the fact that hMSCs engraft in the myocardium for only a short amount of time. This thesis first examined the ability of commercial bone marrow (BM) MSCs expanded in low serum (< 5% FBS medium), umbilical cord (UC) MSCs and individual donor BM-MSCs to perform biologically relevant functions. The characterization studies showed that the differently-sourced hMSCs successfully underwent tri-lineage differentiation and displayed similar expression levels of positive MSC markers. Additionally to this, there was a tendency for cells at increased in vitro age to display reduced expression of CD105. Biologic priming of cells using sJag1 typically enhanced MSC attachment to fibronectin, although to varying degrees in the different MSC types. In addition, vascular support assays revealed that MSCs displayed pericyte-like behaviour lining the outside of the vessels and bridging in between endothelial cells during network formation. Assessment of how bioprocess parameters affect vascular tubule formation revealed that economic benefits can be derived by using lower volumes of alternative matrix substrates. In addition, automated counting tools achieved an unbiased measurement compared to manual counting processes. Finally, from a perspective of the vascular endothelial cells used in the assays, it was possible to extend their use an additional 50%, from passage 10 to passage 15 before losing functional capacity. With further work, these assays could be optimized for high-throughput screening and be used in industry as surrogate tests for quality control (QC) hence enabling the advancement of well-characterized cell therapy products.

The aim of this project is to establish Roseobacter marine bacteria and Trypanosoma brucei (T. brucei) protozoa as synthetic biology chassis. This work addresses the gap within synthetic biology resulting from the limited choice of host cells available for use in practice. This was done by developing synthetic bacterial and trypanosomal genetic regulatory networks (GRNs) which function as an oscillator as well as by developing the necessary protocols and set-ups to allow for the analysis of GRN dynamics within the host. Roseobacter clade bacteria are naturally found in diverse oceanic habitats and have an important ecological role in balancing global carbon levels. This makes Roseobacter an ideal chassis for future efforts to apply synthetic biology to bioremediation and geo-engineering challenges. The aim of this investigation was to establish straight-forward molecular biology procedures in Roseobacter bacteria followed by characterisation and modelling of an E. coli oscillator in Roseobacter. Results showed that Roseobacter synthetic biology is non-trivial. Protozoa are exploited as host cells for industrial production of biotherapeutics due to fast doubling times and host proteins' mammalian-like post-translational glycosylation. As an established model organism for studying protozoa, T. brucei provided a test case for establishing synthetic biology in this phylum for the first time. T. brucei is highly divergent from eukaryotes commonly used in synthetic biology and possesses a sophisticated genomic machinery to evade host immune systems. The establishment of standard synthetic biology approaches in mathematical modelling and gene network design in T. brucei will underpin application of synthetic biology to enhance the industrial capability of the protozoa as a chassis and to probe its pathobiology. This investigation involved design and assembly of a Goodwin oscillator, followed by characterisation and modelling of the network and the development of a novel experimental set-up for live-cell imaging of single motile trypanosomes. Results showed that T. brucei is a promising novel synthetic biology chassis.

Biorefineries allow for the sustainable production of higher value products from biomass. In addition to bioethanol, they can produce added value chemicals and pharmaceutical intermediates from isolated component compounds such as sugars. Sugar beet pulp (SBP) is a high volume, low value by-product from sugar beet processing with a low lignin and a high carbohydrate content, making it an attractive biomass feedstock for biorefinery processing. The pectin fraction of SBP can be isolated via steam explosion, which, after complete acid hydrolysis, gives a hydrolysate rich in monosaccharides: primarily L-arabinose (Ara) and D-galacturonic acid (GA), with some D-galactose (Gal) and L-rhamnose (Rha). Isolation of these sugars is therefore a critical step in realising an integrated, whole crop biorefinery. Currently, little work has been reported on the separation and utilisation of SBP hydrolysates. The aim of this thesis is to establish novel, scalable separation processes for the isolation of the component monosaccharides from crude hydrolysed sugar beet pulp pectin. Centrifugal partition chromatography (CPC) is a liquid-liquid separation technique with no solid stationary phase and offers an alternative to traditional resin-based chromatographic techniques. As such it can more easily cope with crude feedstreams such as hydrolysates. Hydrophilic ethanol : ammonium sulphate two-phase systems were examined based on monosaccharide partition coefficients and phase settling times. An ethanol : aqueous ammonium sulphate (300 g L-1 ) (0.8:1.8 v:v) system was chosen for CPC separations of the crude SBP hydrolysate and was shown to be capable of removing the coloured contaminants and isolating three sugar fractions in a single step: Rha, Ara and Gal, and GA. The separation was optimised and the throughput was increased by maximising the sample loading. Operation in an elution-extrusion mode allowed for reproducible separations in 100 min without additional column regeneration. The process was scaled up from a 250 to a 950 mL column providing a final throughput of 1.9 gmonosaccharides L -1 column h -1 using the crude SBP. The following purities and recoveries of the three main fractions were achieved: Rha at 92% purity and 93% recovery; Ara at 84% purity and 97% recovery; and GA at 96% purity and 95% recovery. Simulated moving bed (SMB) allows for continuous chromatographic separations using multiple columns, improving separation performance and throughputs. Isolation of Ara from the neutral sugars Gal and Rha was performed with resins and conditions screened on single columns leading to the selection of a Dowex 50W X8 resin in the Ca2+ form. SMB separation using 8 columns was performed in the 4-zone and 3-zone setups and achieved 94% purity with 99% recovery at a throughput of 4.6 gmonosaccharides L -1 column h -1 with a synthetic mixture of the neutral sugars (Ara, Gal and Rha). However, equivalent separations could not be achieved using the crude SBP hydrolysate which needed pretreatment before SMB. Decolourisation with activated carbon was able to remove 97% of the coloured contaminants with sugar losses of 15% (w/w) in a batch process demonstrated to 50 mL scale. Anion exchange chromatography using a Dowex 1x8 resin was then found to be capable of isolating GA from a synthetic crude mixture of GA and neutral sugars with a dynamic binding capacity of 1.31 mmol mL-1 resin. However, further work is needed to enable this anion exchange step to achieve satisfactory separations with the decolourised crude hydrolysate. The isolated neutral sugars, after GA removal, can be processed on the SMB with comparable separation performance and throughput to a mixture of neutral sugars prepared without GA. In summary, this thesis presents two possible process paths each with their own benefits and drawbacks. CPC is capable of processing the crude SBP hydrolysate directly, isolating the sugars and removing the coloured contaminants in a single step. However, Ara co-elutes with Gal providing a stream that is only 84% pure. In SMB, the potential throughputs and separation performance are higher, however, this could only be experimentally demonstrated with synthetic crude mixtures of sugars and not with the crude SBP hydrolysate. Further pretreatment or SMB method development would be required in order to process the crude hydrolysate, and the resulting multistep processes may reduce the overall viability. Overall this thesis demonstrates two feasible approaches to the preparative scale separation of SBP pectin hydrolysates and supports development of an integrated SBP biorefinery.

No description available.

The majority of products made by microalgae and requiring extraction before use are restricted, commercially, by the high cost of the harvesting methods employed. Although considerable progress has been made in biofuel development there are still relatively few studies on the initial downstream processing stages. Ultra-scale down (USD) approaches have previously been established to study the impact of the engineering environment on biopharmaceuticals; they are valuable because they enable study of a wide range of operating parameters using minimal quantities of material and resources. The aim of this project is therefore to establish a USD platform for the rapid evaluation of pre-treatment and recovery operations for microalgae downstream processes. The first objective was to explore flocculation as a pre-treatment step. Flocculation is a difficult process to operate reproducibly, hence standardization of flocculation conditions becomes vital in order to characterize and quantify process performance. A series of scale-down flocculation reactors were designed, characterised and scaled-up using Chitosan to flocculate heterotrophically grown Chlorella sorokiniana. These enabled flocs with defined particle size distributions to be consistently and reproducibly produced. An optimal Chitosan concentration of 9.9 ± 0.4 mg.g-1 of algal dry cell weight was determined. Scale-up of the flocculation process from the scale-down reactor (120 mL) to a 7.5L STR stirred tank reactor was achieved at a fixed impeller tip speed during flocculant addition and ageing (0.29 and 0.07 ms-1 respectively). Due to the complex nature of unit operations, it is generally difficult to obtain data at laboratory scale that closely reflects the performance of operations at pilot scale or beyond. For the second objective of this work, a USD model of a cross-flow filtration process for microalgal biomass recovery was established. This could accurately reproduce the flux-TMP (transmembrane pressure) profiles of lab-scale hollow fibre cartridges when operated at a defined shear rate. The benefits of flocculation on filtration performance include a reduction in membrane cleaning cycles and a 20% reduction in filtration time. Filtration results were also in good agreement between the two scales for both unflocculated and flocculated feeds. The USD method enabled a 14-fold decrease in the volume of material required. It also demonstrated the benefits of flocculation. And lastly, USD was achieved using a 14.5–fold reduction in membrane area at matched volume: surface area ratio. The third objective was to establish a USD centrifugation method using a rotating disk shear device to expose particles to hydrodynamic shear before centrifugal separation. Evaluation of the influence of flocculation on centrifugation efficiency showed the benefits of increased particle size on clarification. Clarification efficiencies exceeding 99% was obtained even at low centrifugal forces using an optimal Chitosan dosage. The USD findings were validated at pilot scale using a CARR PowerfugeTM centrifuge. Similar clarification performance was predicted using 2000-fold less broth volume than was required for the pilot scale study. Sonication and homogenization as small scale cell disruption options for lipid release of heterotrophically grown C.sorokiniana were explored. Comparison of the optimal conditions of the two methods showed cell disruption and lipid release were similar in both cases on a g.g-1 basis. Finally comparison of the transesterified material produced using either USD microfiltration or USD centrifugation steps for harvesting showed major differences in terms of yield of fatty acid methyl esters (FAME). USD methods for evaluation of primary recovery operations and their interactions appear particularly useful in microalgae bioprocess synthesis. This work is the first to evaluate the use of USD technologies with microalgal cells. It illustrates the power of small-scale mimics to enable rapid selection and optimisation of different process options and thereby rational selection of the overall bioprocess sequence.

Separation based on aqueous two-phase extraction (ATPE) is a promising downstream separation technology for the production of biological products. The advantages of using aqueous two-phase systems include but are not limited to easy scalability, ease of continuous operation and a favourable environment for biological compounds. One of the main challenges associated with aqueous two-phase systems is process development. This is in part due to the many factors which influence the separation of biological materials in such systems such as polymer and salt type, pH and charge. The large number of factors to consider makes the development of aqueous two-phase systems challenging due to the need to find a robust and efficient separation in a large experimental space. This work addresses this issue by considering the use of dynamic process models and high-throughput experimentation for the development of aqueous two-phase extraction processes for biological products. The use of a dynamic equilibrium stage process model to simulate aqueous two-phase extraction is considered in Chapter 3. The process model is capable of simulating various modes of operation; and both multi-cycle batch and continuous counter-current modes of operation are considered. The capabilities of the model are demonstrated using a case study separation of enzyme α-amylase from impurities in a PEG 4,000-phosphate aqueous two-phase system containing NaCl. The dynamic model allowed investigation into the impact of upstream process variability on a continuous counter-current extraction process. The development of aqueous two-phase systems requires detailed knowledge of the phase diagram. In Chapter 4, PEG 4,000-citrate aqueous two-phase system phase diagrams are determined using a combination of high-throughput screening and lab scale experiments. This involved the development of a systematic two-stage screening approach to determine the binodial curve location to a high accuracy using ~50% of the experimental resources that a single high-resolution screen would use. In addition, a novel method was developed to quantify uncertainty in the phase diagram due to the binodial curve location and tie-line fitting. The characterised phase diagrams were then used to estimate thermodynamic interaction parameters which are used in process models to describe phase equilibria. In Chapter 5, the simulation and high-throughput screening methods of Chapter 3 and 4 are combined to develop an aqueous two-phase extraction separation process. The approach is demonstrated by separating enzyme α-amylase from myoglobin in a PEG 2,000-phosphate aqueous two-phase system containing 6wt% NaCl. High-throughput experimentation is used to determine partitioning behaviour of α-amylase and myoglobin at different tie-line lengths and phase ratios. The experimental partitioning and phase diagram data was then used to simulate a counter-current extraction process. The insights gained using the process model allowed for better decisions to be made regarding selection, control and operation of aqueous two-phase separation equipment. Therefore, the combined approach of using process modelling and high-throughput experimentation allowed for greater amounts of process understanding to be gained for aqueous two-phase systems using limited resources where there is a large experimental space to be navigated.

Human induced pluripotent stem cells (hiPSC) have the potential to provide patient and disease specific cells for research and act as therapeutic agents in unlimited supply. To translate lab-scale research toward clinical applications we need to reduce variability from complex differentiation protocols which often include xenogeneic components. A move toward more defined culture systems will improve predictability and process control as well as reduce risks of exposure to animal pathogens. Cell therapy development, also requires flexibility in scalability as cell numbers for therapies vary greatly between disease indications. Improved control over the microenvironment at the lab scale would offer more defined parameters amenable to scale up with better flexibility, reduced waste and more precision. This thesis describes the use of forced aggregation to improve the initiation of differentiation combined with culture in pre-validated and scalable, 24 microwell plates on shaking platforms, for stem cell differentiation toward retinal lineages. Using an established hiPSC line (MSU001) we show using forced aggregation to form embryoid bodies (EBs) promotes efficient initiation of differentiation. We next determined 2 EB sizes (5K and 10K cells/EB) which improved initiation of retinal differentiation compared to scraped EBs as demonstrated by >3fold increase in expression of early eye field transcription factor Rax at day 3 of culture. The 5K and 10K EBs also facilitated the adaption of an adherent protocol for retinal differentiation to an orbital shaken suspension culture system. Size controlled EBs also enabled the selection of a permissive shaking speed (120rpm) suitable for orbital shaken culture for initiating retinal differentiation with 5K and 10K EBs. Furthermore orbital shaken culture enabled elimination of the undefined xenogeneic ingredients of Matrigel, from the culture system. This thesis demonstrates that combining size control for hiPSC derived EBs and orbital shaken culture is a feasible method for the initiation of retinal differentiation. By facilitating removal of xenogeneic materials from the culture system; the combination of orbital shaken culture with size controlled EBs may be of value to other complex stem cell differentiation systems to improve the initiation of differentiation whilst providing the potential for scalability.

This thesis aimed to design novel sensor proteins that can identify and measure various metal ions in vivo and in situ. Metal ions play key role in the metabolism of the cell, and monitoring of calcium has helped interrogate cellular processes such as fertilisation, contraction and apoptosis. Real-time monitoring of more divalent metal ions like zinc and copper is required to gain much needed insight into brain function and associated disorders, such as Alzheimer’s and Parkinson’s disease. Aequorin is a calcium-regulated photoprotein originally isolated from the jellyfish Aequorea victoria. Due to its high sensitivity to calcium and its non-invasive nature, aequorin has been used as a real-time indicator of calcium ions in biological systems for more than forty years. The protein complex consists of the polypeptide chain apoaequorin and a tightly bound chromophore (coelenterazine). Trace amounts of calcium ions trigger conformational changes in the protein, which in turn facilitate the intermolecular oxidation of coelenterazine and concomitant production of CO2 and a flash of blue light. Aequorin’s light emitting reaction can also be triggered by a range of other divalent and trivalent cations, leading however to significantly lower light yields. Based on aequorin’s promiscuity towards other ions, this project tested the hypothesis that aequorin’s preference for certain cations could be manipulated through mutations engineered in one or more of the three calcium-binding loops (EF-hands). In order to test the hypothesis, the following six stages were performed: cloning of the apoaequorin gene for expression in E. coli; development of a high-throughput assay for expression and measurement of bioluminescent activity in microwells; design of a library containing forty eight mutant variants of aequorin; screening of the library against seven metal ions; protein purification of wild-type aequorin and one selected mutant; analysis of activity and kinetics of purified wild type and one chosen mutant against all seven ions. This work produced mutants with shifted selectivity towards new metal ions at the cost of luminescence yield. The impact of mutations is analysed and it is suggested that one of the EF-hands (EF-I) is likely to serve as a gatekeeper to aequorin’s selectivity. It was also shown that at least one mutant utilised zinc ions (that wild type failed to utilise) to achieve low levels of bioluminescent activity.