In 2015, an estimated 425 million people were chronically infected with Hepatitis B, Hepatitis C or Human Immunodeficiency Virus. Many of these infections are preventable but remain undiagnosed and entirely off the record. This is driving new technologies in the space of vaccines and diagnostics. A current bottleneck in making vaccines accessible is the ease and cost of manufacturing, which depends significantly on adequate process control technology. Major limitations of current diagnostic tools concern their speed, ease-of-use and cost. In short, there is a lack of biosensing tools which are fit for use in point-of-care settings as well as for quality control in viral vaccine manufacturing. The aim of this thesis is to explore the potential for micro- and nanotechnologies to fight infectious diseases, with a specific focus on new vaccines and mobile phone connected tests. The first chapter explores the potential of dielectrophoresis in microfluidic channels for rapid biophysical characterisation of whole virus in vaccine development. The first bioapplication of dielectrophoretic sensing based PAT and surface conduction of bovine Herpes virus is reported. The second chapter is concerned with innovative paper microfluidic diagnostics, which are inherently low cost and can be read out by a smart phone app. Capture ligands were sourced and characterised to detect acute Hepatitis B and C biomarkers as well as first key demonstrations of a new sensitivity enhancement strategy using dendrimers to detect down to low ng/ml level within 2 minutes. The third chapter evolved out of a collaboration with our industrial partner OJ-Bio and contains a proof of concept for detection of Hepatitis C biomarkers on a surface acoustic wave immunosensor. Each chapter highlights a landmark step in the development of different biosensor approaches, while also demonstrating what some of the real challenges are before they can be adopted into either clinical or industrial use. Our findings afford important insights into how these technologies can be further developed to become the next generation of biosensors, to help diagnose more people, monitor outbreaks of infectious diseases better and widen access to affordable vaccines.

In this thesis, effects of stable concentration of key growth factors on retinal differentiation of human induced pluripotent stem cells (hiPSCs) were investigated in a microfluidic culture device (MFCD). In vitro culture dishes such as flasks and well-plates lack microenvironmental controls required for maintaining stable culture conditions. Unlike cell culture dishes, microfluidic devices provide greater microenvironmental controls resulted from shorter characteristic length and use of laminar flow. It is hypothesised that using the MFCD, a flow rate can be found which keeps key growth factors in retinal differentiation culture in steady-state. Subsequently, steady-state concentration of growth factors would result in upregulation of retinal progenitor (Pax6, Lhx2, Six6 and VSX2/Chx10) and precursor markers (Crx and Nrl). Initially, degradation and consumption of growth factors (DKK-1, Noggin, IGF-1 and bFGF) used in retinal differentiation were studied to establish an order of importance. These findings along with dimensionless ratios, Péclet and Damköhler numbers, were utilised to establish a perfusion rate of 130 μL/h. At this flow rate growth factors DKK1 and Noggin were delivered to the cells in steady-state conditions. A second perfusion culture with the same media exchange rate as the static culture at flow rate of 5.2 μL/h was added to act as a second control. Perfusion cultures were performed for 5, 10, and 21 days (n=3). The MFCD with higher flow rate showed significantly higher expression of markers Crx (p < 0.05) and Rhodopsin (compared to lower MFCD, p < 0.05) on day 21. The MFCD with lower flow rate, showed significantly higher expression of Pax6 (p < 0.05), Vsx2/Chx10 (p < 0.01) and Crx (p < 0.05) on day 5, Nrl (p < 0.01) on day 10 and Six6 (p < 0.05) on day 21. This was the first continuously perfused long-term (21 days) retinal differentiation of hiPSCs in a microfluidic device, and results illustrated the importance of steady-state conditions in stem cell bioprocessing.

The need to speed up bioprocessing and to enhance process understanding of the heat extraction process, for the recovery of periplasmic Fab’ from E.coli cells, has led to the development of two workable scale-down models that are capable of predicting the performance of the lab scale and pilot scale process. In this work, a design of experiment (DoE) study was initially conducted at the 2L scale to identify the effect of key parameters such as heating duration (6-14 hours), heating temperature (55-65°C) and specific power input (0.05-0.41W L-1) on the heat extraction process. The results showed that extraction temperature and duration had the most impact on the process whereas specific power input had no significant impact in the range studied. Fluid dynamic studies were conducted on the 2L vessel, and on a scale-down 20mL vessel and shaken 24-well deep square-well (DSW) plate, in order to assess mixing performance under various operating conditions. Mixing time studies were performed on all three models using the dual indicator system for mixing time (DISMT). The mixing time curves between the 2L and 20mL vessel were well matched over the same range of specific power input values and the results showed that mixing time stayed relatively constant for both scales above a specific power input value of 0.05W L-1. Particle image velocimetry (PIV) experiments were also conducted on the 20mL vessel in order to visualise flow patterns and analyse fluid velocity. The data indicated that flow patterns were fully formed at 300rpm (9 x 10-4W L-1) and that velocity stabilised after 0.05W L-1. Mixing time studies in the DSW plate showed that turbulent mixing was achieved above 500rpm. Process verification studies were performed between the two scale-down models and the 2L vessel, and the performance was compared using Fab’ titre, total protein concentration, dsDNA concentration, HCP profiles and Fab’ profiles. The results demonstrated that both models were capable of correctly mimicking the performance in the 2L vessel for a variety of different experimental conditions, using feed material from different batches and using different E.coli strains and Fab’ types, provided that the heating profiles were matched and there was sufficient turbulent mixing at all scales. The results also agreed with data from the DoE and fluid dynamic studies, thus establishing the 20mL vessel and 24-well DSW plate as two feasible scale-down models for the periplasmic heat extraction process.

Currently, the production of mAbs in mammalian cells plays a significant part in the biopharmaceutical industry. Consequently, the increased market has led to a need for increased production volumes and a drive to improve process efficiency. One of the key parameters that has a significant impact on the cell growth and metabolism, mAb productivity and quality is the pH. The understanding of the response of mammalian cells to pH perturbations is critical. Therefore, a scale-down model of the pH effects on an industrial GSCHO cell line (CHO-CY01) growth and mAb (IgG4) production was developed. A scale translation criteria of matching mixing times (< 5 ) and energy dissipation rates (10 −3 ) was found to be more reliable to set the microwell plates (24-SRW MTPs) and shake flasks as a scale-down models of a 5L-STR bioreactor CHO-CY01 cell line bath and fed-batch cultures. A high-throughput online multi-parametric analysis method of mammalian cell cellular activity at 24-SRW MTP was developed, where the Presens 24-SRW MTPs were exclusively used for the online monitoring of the process parameter. Therefore, it was possible to evaluate the effects of different 2 and pH profiles created with 2/pH shifts on the CHO-CY01 cell line kinetics. A culture pH higher than 7.50 caused an excessive lactate production. While, culture pHs between pH 6.70 - 6.80 inhibited the cell growth and lactate production, pH 6.60 – 6.70 caused an increase in ammonium production and pH < 6.60 triggered cell apoptosis. Therefore, it became important to identify the lactate and ammonium inhibition kinetics. It was found that an initial lactate concentration of 4.08 −1 and an initial ammonium concentration of 18.1 −1 reduced to half the specific growth rate. Ultimately, the optimal pH for IgG4 production was at pH 6.85 ± 0.02, where the maximum specific antibody production rate (10.50 10−9 −1 −1 ) with the minimal lactate production was reached.

Four million surgeries involving bone grafting or bone substitutes for the treatment of bone defects are performed yearly worldwide. However, limited donor tissue availability, pain and the risk of infection and immune rejection, have led to the development of alternative strategies for bone repair. Tissue engineering represents an alternative to current treatments as it consists of using a biomaterial scaffold alone or in combination with proteins, genes or cells, as a bioactive implant to stimulate bone repair. Microspherical scaffolds have been proposed as a potential modular unit for bone tissue engineering applications as their shape could facilitate filling of irregular shaped defects. Furthermore, microspheres could be used as a support for ex vivo expansion of adherent cells as well as a carrier to directly deliver cells to the defect site. In this study, the use of phosphate glass microcarriers for bone tissue engineering applications was investigated. As this material is completely soluble and non-toxic, it can be implanted in the patient together with cells. Furthermore, the tuneable glass composition can be easily engineered to induce specific structural and biological properties. Here, the effect of culturing MG-63 and hBM-MSCs on titanium-doped phosphate glass microspheres containing increasing concentration of cobalt (0, 2 and 5%) was investigated, as these ions have been shown to induce osteogenesis and angiogenesis, respectively. Furthermore, as part of this study a novel perfusion microfluidic bioreactor was fabricated to culture cells on microspheres under perfusion and to enable parallel screening of multiple culture variables. Cells proliferation on the microspheres as well as secretion of ECM proteins in response to the substrate was observed over time, thus confirming the biocompatibility of all compositions tested. Upregulation of osteogenic markers by MSCs also occurred in response to the microspheres in the absence of exogenous supplements. However, this effect was suppressed when cobalt was added to the glass composition. On the other hand, while cobalt doping was found to induce key angiogenic responses (i.e. VEGF secretion), this did not translate into improved functional vascularization in comparison to the cobalt-free microspheres. Successful MSCs culture on the microspheres within the microfluidic reactor was achieved and it was possible to efficiently quantify functional outputs, such as the expression of ECM proteins as a function of microspheres substrates and nutrient feeds under perfusion. In conclusion, titanium-doped phosphate glass microspheres were identified as a potential substrate for bone tissue engineering applications in terms of MSCs expansion and differentiation, as well as to support endothelial cells migration towards the scaffold and vessel formation, while additional doping with cobalt was not found to improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled to identify optimal parameters for perfused cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing.

Chiral amino alcohols are industrially valuable intermediates used in the synthesis of many pharmaceuticals and fine chemicals. Transamination (TAm) is not only one of the most interesting industrial enzymatic reactions on its own nowadays but also the second enzymatic step of the de novo pathway studied by our research group (Bioconversion-Chemistry and Engineering Interface Programme-BiCE) for the synthesis of chiral amino alcohols. In this thesis, firstly a study on the kinetic modelling of TAm reactions was presented. Kinetic behaviour of two ω-TAm reactions employing different amino acceptors but the same amino donor was investigated. Using a Genetic Algorithm, kinetic models could be determined with only 10-12 progress curves for both reaction schemes without having any experimental data for the reverse reaction. Also, a sensitivity analysis on the kinetic parameters of both reactions was carried out. Having gained enough insight into the kinetic modelling of TAm reactions, two different sequential enzyme reactions leading to the synthesis of 2-amino-1,3,4- butanetriol (ABT) were studied. The first one involved three different enzymes, namely TAm (pQR977), Transketolase (TK) and TAm (pQR801). 100% conversion was proven possible with both experimental and simulation results in a 28 hour-long fed-batch reaction. Although, the concentrations tested in the experiments were low, this three-step sequential enzyme reaction showed great promise for further being optimized for industrial applications. The second one was a recycling sequential enzyme reaction involving three different enzymatic reactions taking place simultaneously. Two of the reactions were catalyzed by TAm (pQR1021) and one of them was catalyzed by TK. With the help of second TAm reaction not only desired product was synthesized but also one of the intermediary precursors was recycled within the system. The long term goal of this project was the integration of de novo pathways leading to the synthesis of chiral amino alcohols into host cell metabolism. Thus, the recycling sequential enzyme reaction employing only two different enzymes and two different substrates was found promising for achieving this goal. With both experimental and simulation results the favorability of this reaction was demonstrated and low amounts of ABT (~6 mM) was synthesized in a 8-hour long reaction using purified enzymes. One of the precursors in both sequential enzyme reactions was serine. In the final results chapter, a metabolic modelling methodology was used for identifying optimal metabolic engineering strategies, namely enzyme knock-outs and enzyme expression level modulations, for the overproduction of serine by employing a partial mechanistic model of central carbon metabolism of Escherichia coli. Simulation results showed 89% increase in serine synthesis in the case of doubling the expression level of SerSynth, a hypothetical enzyme for the synthesis of serine from 3-phosphoglycerate.

Over the last few decades, developing reconstructed dermis with the potential of replacing the injured dermis is highly explored. Even though a range of dermal scaffolds are available commercially (Integra® and Matriderm®), none have shown the potential to regenerate injured skin similar to normal skin. Commercially available dermal scaffolds have a range of limitations such as wound contraction, scar formation and poor integration with the host tissue. Designing three dimensional scaffolds using tissue engineering approaches involves optimising the architectural design of the scaffold to promote cell adhesion, proliferation and differentiation to repair the injured tissue. The aim of this thesis was to design a foam-based dermal replacement fibrin-alginate scaffold using surfactants as a tool to introduce gradient porosity within the scaffold (pore range 20 - 270 μm). Initially, a range of non-ionic sugar surfactants were tested on the basis of foaming capacity, stability and their effect on the structure of the fibrin-alginate scaffold. Fibrinalginate scaffolds manufactured using a combination of three sugar based surfactants were highly porous with gradient pore structure, which was confirmed using microscopic techniques. The suitability of this scaffold for the repair of full thickness skin wounds was evaluated using microscopic characterization, viscoelastic measurements, in vitro biocompatibility using human dermal fibroblasts and in vivo testing using a porcine model. Further, this thesis has sought to examine and discuss the challenges faced during the translation phase of the fibrin-alginate technology from the R&D laboratory to a GMP facility for commercial purposes. Moreover, this study also investigated combining the fibrin-alginate technology with synthetic polymers like polydimethylsiloxane and poly(ε-caprolactone) for developing composites with applications for chronic wounds and non-union fractures respectively. A novel immunoassay was designed to comprehend the interfacial binding between the protein and the synthetic polymeric counterpart. This study has increased the current understanding of the use of surfactants on the structural behaviour of foam based protein scaffolds.

Record high titres of high value products have resulted from recent developments in cell culture production methods that are capable of producing high cell densities. These enhanced-titre, high cell density processes are thought to be a bottleneck for downstream processing due to the excessive solids content, product titre and impurity profile. This thesis explored these challenges and endeavours to find ways to overcome them. A mAb and an adenovirus have been used to represent two common biotech products, which represents the size range and complexity of biotech products used in these processes. High cell densities are challenging to clarify; however, this thesis shows that contrary to expectations from literature tangential flow microfiltration could be used to successfully clarify mAb harvests with cell densities up to 200 x 106 cell/ml, and could be successfully used for both mAbs harvest and more complex adenovirus harvests which contained lysed mammalian cells. In adenovirus harvests, high impurity concentrations are a particular challenge. This thesis shows that when dealing with high cell density harvests, using a DNA precipitation step with a variable detergent concentration linked to cell densities allows for predictable process performance. By being able to predict the range of the concentration of HC-DNA remaining after DNA precipitation, it is then also possible to design an AEX step which is capable of delivering robust performance. The economics of high-cell density processes was also investigated to better understand its impact on purification. High product concentrations present their own challenges, but increases in titre usually bring substantial reduction in production costs, sometimes is unexpected ways. Here we show for mAbs, increasing the titre also lead to increases in the dynamic binding capacity of both Protein A and cation exchange resins, which reduced the cost of goods.

This thesis details the development of a microbioreactor with application in synthetic biology. The aim was the development and engineering characterization of an instrumented microbioreactor system that could aid scientists in the phenotypic analysis of genetically engineered strains. An existing prototype of a microbioreactor was modified to improve mixing. The microbioreactor was then critically evaluated by: assessing two mixing methods, assessing the prototype against a second reactor design option, characterizing oxygen transfer and mixing, comparing performance with a commercially available minibioreactor, and successfully culturing the genetically modified, gram-positive bacterium, Staphylococcus carnosus using the chemostat mode of operation. Engineering characterization of the device was used to inform the selection of process conditions for chemostat studies. KLa values of ~ 113 h-1 and mixing times of ~ 1.2 s were achieved. Residence time distribution analysis demonstrated operation under nearly ideally mixed conditions with ~ 1% of stagnant volumes. Continuous fermentations using different dilution rates and glucose feed concentrations demonstrated the ability of the system to control the growth rate and create a controlled change in OD concentration, respectively. The oxygen uptake rate (OUR) was determined at two dilution rates, which are to the knowledge of the author, the first OUR values reported for Staphylococcus carnosus in a microchemostat. Overall, the results provided a more complete engineering understanding of the device, which will facilitate further improvement of the microbioreactor set up to create a high-throughput experimental platform capable of rapidly screening for growth conditions.

The past decade has seen an increasing focus on the issues surrounding climate change and this has triggered governments internationally to develop environmental legislation and policies for the energy-intensive industries (EIIs) that can help reduce their anthropogenic greenhouse gases (GHGs) emissions. The biopharmaceutical industry is a relatively new EII. As the industry matures, the level of environmental scrutiny is increasing. Therefore, there is a need for the development of a framework specific to this industry to help guide the selection of manufacturing and disposal routes that reflect the potential environmental impact. In this doctorate, a framework based on the life cycle assessment (LCA) tool was developed. The application of the framework for evaluating manufacturing and solid waste management alternatives is demonstrated via case studies that focus on production of therapeutic monoclonal antibodies using mammalian cell culture process at 200 L operational scale using either traditional or a hybrid based on a mix of traditional and disposable modes of production. The framework was employed to identify the process (whether traditional or hybrid) that contributes least to environmental impact, and also to identify the most suitable solid waste management method (landfill, incineration and pyrolysis). The life cycle inventory of the manufacturing processes, and the methodology used to obtain the inventory are presented. It is expected that this information will be beneficial for future studies in this area of research. The analysis also utilised sensitivity analysis studies to assess critically the uncertainties in the assumptions made in the case study. Finally, the application of the framework in evaluating the cumulative environmental impact, from manufacture in support of clinical stages up to production was assessed. Here, the focus was not only to evaluate the cumulative environmental impact, but also to explore the benefits of employing single-use technologies during clinical phase manufacture when developing a monoclonal antibody for therapeutic use. The work in this thesis highlights the benefits of adopting a consistent engineering framework to guide process and technology selections in the biopharmaceutical industry by improving the overall quality of decision-making. This in turn will help the industry to predict and to control their environmental performance.