For many years, molecularly imprinted polymers (MIPs) have been described as "plastic antibodies", yet even some modern examples cannot approach the true binding affinity and specificity of monoclonal antibodies. This project sought to include within the imprinted site a short peptide sequence isolated from a phage display library with high affinity and specificity. A system is hypothesised in which synergism between the robust nature of the polymer and the binding affinity and specificity of the peptide may be exploited. Peptide phage display is a technique that can rapidly enrich binding peptides from a combinatorial library of over 109 unique moieties. Initial studies attempted to isolate peptides with high affinity and specificity to propranolol from this library. However, when several methodologies failed to demonstrate any binding effect, a peptide was selected from the literature that had been found to bind the fluorophore Texas Red. The peptide was immobilised to a Merrifield Resin support, its binding properties thoroughly assessed, and a polymerisation protocol was developed using living radical polymerisation. Preliminary studies suggested that when peptide-functionalised resin was washed in ethanol, no binding to Texas Red was evident, whereas once a protective polymer shell was formed, the peptide retained a binding conformation and affinity for Texas Red was slightly increased. This was, however, at the expense of binding capacity, which fell dramatically. Whilst the evidence presented here is by no means complete, it provides proof-of-principle for a functional peptide- molecularly-imprinted polymer. Further work in this area may lead to the development of a truly biomimetic artificial antibody: the plastibody.

The aim of this study is to understand what type of surface texture and chemistry after laser processing will have an effect on the biocompatibility of Ti-6AI-4 V. A number of laser techniques have been investigated to modify the surface properties including texturing, coating and heat treating. The biocompatibility of the samples was investigated by observing the apatite growth and monitoring osteoblasts (bone cell) attachment. Coating a 5 μm layer of Ca2Si04 by using a combined high power diode laser/sol-gel method on Ti-6Al-4V substrate resulted in increased cell attachment by 58% compared to the uncoated substrate. Laser oxide growth and micro-crack formation with minimized surface structuring was applied by Nd:YAG laser which resulted in increasing the formation of apatite (improved biocompatibility). Surface micro structuring was performed by using Nd:YAG, Excimer and Nd:YV04 lasers. The effect of structures with different sizes including micro-holes (larger than bone cell size), micro-grooves and micro-cones (in the range of bone cell size) on bone cell attachment is investigated. The effect of chemical modifications with and withouth structuring is also studied. Results indicated that laser processing improved the biocompatibility in all cases. Nd:YAG micro-texturing to produce micro-size holes (diameter-127 μm, centre to centre spacing-465 μm) with reduced chemical modifications, increased the osteoblast attachment on the substrate by 64% compared to the untreated surface. Producing micro-grooves and submicron ripples on Ti-6AI- 4V substrates by using a KrF excimer laser resulted in 20% increase in osteoblast attachment (50 μm groove width). Micro-cones and chemical modifications generated by Nd:YV04 laser resulted in the highest cell attachment (213%). This was repeated a number of times to provide statistical confidence of the results. The best results, for the first time, showed higher cell attachment than an untextured tissue culture plastic substrate. Immunoflorence microscopy indicated that osteoblasts created larger focal contacts on structures which showed highest cell attachment. This work was carried out in collaboration between the Laser Processing Research Centre (LPRC) of the Faculty of Engineering and Physical Sciences and the Faculty of Life Sciences, The University of Manchester.

No description available.

In the UK, public access to emerging biotechnologies is largely regulated by three government bodies that operate at arm's length from democratically elected representatives: the Human Fertilisation and Embryology Authority (HFEA), the National Institute for Health and Care Excellence (NICE), and the Medicines and Healthcare Products Regulatory Agency (MHRA). Purposefully situated beyond traditional lines of electoral accountability, how can these independent bodies (commonly referred to as 'quangos') conceptualize and demonstrate their accountability to the public in regulating technically complex and morally controversial technologies? After articulating this general theoretical tension between independent and accountable decision-making in the arm's length context, I illustrate how it generates distinct, practical challenges within the HFEA, NICE and the MHRA. To navigate this general tension and address its practical ramifications, I invoke political theories of public reason and deliberative democracy, as well as recent attempts to apply these theories to the governance of biotechnologies. Through a critical analysis of these accounts, I articulate a new framework for 'public accountability as reciprocity' in the arm's length context. Its four, process-oriented conditions, of 1) reciprocal reasoning, 2) acceptance of deliberative disagreement, 3) voting, and 4) fostering the means of deliberative resistance, affirm the goal of identifying mutually acceptable reasons for collective decisions, but take seriously the fact that characteristics of emerging biotechnologies will often confound such attempts. Taken together, these conditions provide a more theoretically robust and normatively legitimate approach than existing accounts, notably 'accountability for reasonableness.' Next, I translate the theoretical claims of this framework into practice, illustrating how its conditions may be fruitfully applied towards the resolution of the practical challenges identified within the HFEA, NICE, and MHRA. In the HFEA, public accountability as reciprocity provides a robust approach to moral reasoning in the body's deliberations and resituates the role of public consultation. In NICE, it suggests a justifiable and properly tailored role for patient involvement in technology appraisals. Finally, in the MHRA, it explains why a growing number of devices defy the institution's traditional assessments of safety and effectiveness and offers reforms to its regulatory boundaries. I conclude with a set of policy recommendations for creating and operating arm's length bodies, as well as an account of broader contributions to the literature on public reasoning, the concept of 'ethics expertise,' and the practice of patient and public involvement.

The Electromagnetic Acoustic (EMA) technique is a novel multi-modal technique for medical imaging. It is sensitive, in principle, to contrast in mechanical properties and electrical properties and has potential in a number of applications such as breast tumour detection where there will be contrast between diseased and healthy tissue and high intensity focused ultrasound monitoring, where there will be contrast when tissue is ablated. A complete computational model for the EMA imaging is developed. The model considers the linear or nonlinear propagation of ultrasound in soft tissue, the dynamic response of the viscoelastic soft tissue to acoustic radiation force (ARF) stimulation and scattering of electromagnetic waves with and without the Doppler effect. The suitability of the EMA imaging for breast tumour detection is evaluated, modelling a tumour as a spherical inclusion in an infinite homogeneous background tissue with clinically relevant material properties. The results show that variations of the mechanical properties of underlying healthy breast tissue and tumour tissue in clinically feasible range should result in a change in the amplitude of the first Doppler component (FDC) of up to 50%, and varying the electrical contrast leads to a change in the ratio of the FDC and unshifted component (UC) of less than 1 dB. The relative difference between the first Doppler component and the unshifted component is greater than 68 dB and therefore the frequency demodulation may pose a significant challenge if EMA imaging is used for breast tumour detection. The feasibility of using the EMA imaging for real-time monitoring of High Intensity Focused Ultrasound (HIFU) therapy is also investigated. Simulations conducted with realistic liver tissue properties show that the induced Doppler effect in the scattered EM wave is not well correlated with the growth of thermal lesion, therefore it is unlikely to be a good indicator of the lesion size. EMA imaging may not be appropriate for monitoring HIFU therapy.

Microbial fuel cells (MFCs) have been used in a variety of applications to date, however the power output of an MFC is its biggest limitation. The power output of an MFC can be improved by improving the electron transfer from the bacteria to the electrode. This project was interested in improving the electron transfer using gold nanoparticles. Gold nanoparticles were incorporated in MAgarose which showed good conductive properties and when used in a bio-electrochemical cell with Shewanella oneidensis MR-1 as the bacteria, a current enhancement of ~ 29 times was recorded when compared to plain veil. Gold macrostructures were also electrodeposited in thiolated agarose gels and used as electrodes in the bio-electrochemical cell, but the current enhancement was not very significant compared to the control and reached a saturation point over time. Thiol group functionalised PAMAM dendrimer protected gold nanoparticles, were attached to So surface and used as the biocatalysts in a bio-electrochemical cell. The current recorded was lower than the control. This was attributed to a weak biofilm formation on the electrode because of gold nanoparticle attachment. The data obtained in this thesis suggests GelAuNP to be the most promising way forward of achieving a high power output from an MFC.

Over the past decade the bioprocessing industry has embraced and led the creation of a number of initiatives to drive process development and optimisation. There has consequently been an increased demand for tools capable of meeting the requirements of these initiatives. Spectroscopic monitoring systems such as Near infrared (NIR), Mid infrared (MIR) and Raman can provide a number of substantial advantages over more conventional off-line monitoring methods, normally applied to bioprocessing. With these techniques the bioprocessing industry has means of meeting these demanding challenges. The aim of study was to determine the feasibility of using NIR, MIR and Raman spectroscopy as a combined ‘toolkit’ to correlate changes in metabolic profile to spectral changes, within low passage Chinese Hamster Ovary cell cultures. The target metabolites focused upon in the spectroscopic analysis for this study were glucose and lactate. Conventional off-line techniques were used to investigate any correlation between spectral changes and those in the reference data. A design of experiment (DoE) approach was used to identify the optimum preprocessing techniques of the data and to build more accurate process trajectory models. These models were subjected to both an internal and external validation to guarantee the reliability of the results. Feasibility of data fusion, to create a single ‘fused’ dataset from the three spectroscopic techniques was also assessed, to produce a model with lower errors of prediction that the individual parts. The other area investigated was the characterisation of low passage number cultures in various media and process formats, which was of interest to the industrial collaborators. To test this three media; CD CHO, CD OptiCHO and Dynamis were provided by Thermo Fisher Scientific. Currently there is no system which utilises this toolkit with a combined DoE and data fusion strategy, within the bioprocessing industry. This research demonstrates a gap in the industry and a novel approach as to how tackle process monitoring. The results in this project demonstrate the varying degrees of success the spectroscopic techniques across all process formats and media. The NIR proved to be the most successful at modelling the target metabolites, with MIR being unsuccessful and Raman only being able to detect but not model the metabolites. This research provides an indicator of media suitability in low passage number cell cultures, by comparing the batch culture processes. The CD CHO media proved to be the best of the three tested media, based upon the cell density, viability and mAb titre produced. Overall this study represents a valuable stage in the progression towards real time monitoring of biomanufacturing processes and development of tailored low passage number cell culture media. However there are areas of this study where further investigation could be improved.

Aromatic compounds are among the most persistent and prevalent toxic pollutants in the environment. Biotransformation and bioremediation processes can convert these compounds into non-toxic compounds and valuable products. The purple phototrophic bacterium Rhodopseudomonas palustris (R. palustris) has the ability to degrade a wide range of aromatic compounds and the identification of several nitroreductases, nitrilases and a nitrile hydratase (NHase) in the genome sequence indicates an ability to degrade nitroaromatic and nitrile compounds respectively. The candidate nitroreductases RPA1711 and RPA4285 were recombinantly produced in E.coli and the purified proteins were assayed for their ability to reduce the nitro-groups of ligands to amino groups by UV-Spectrophotometry. Michaelis-Menten constants (Km) for RPA1711 of 0.21 mM and 0.13 mM were obtained for 2,4 Dinitrotoluene (2,4-DNT) and 2,6-Dinitrotoluene (2,6 DNT) respectively, while RPA4285 had higher Km values with 2,4-DNT of 0.76 mM and with 2,6-DNT of 0.41 mM. On the other hand, nitriles were utilised by nitrilases and a nitrile hydratase and both aromatic and aliphatic nitriles were degraded. The RPA0599, RPA1563 and RPA4166 nitrilases were purified and their activities were determined by the release of ammonia using the continuous coupled assay, phenol-hypochlorite, ophthalaldehyde (OPA) methods. 4-cyanopyridine was a preferred substrate. Furthermore, a NHase of two subunits RPA2805-2806 and NHase with activator (P14K) RPA2805-RPA2806-RPA2807 was also purified and characterised. Benzonitrile was a preferred substrate for the NHase. Exploring the ability of R. palustris to degrade nitroaromatic and nitrile pollutants might help improve bioremediation biotechnology.

Improvements in fermentation processes, coupled with the rising demand for biologies, are requiring that new ways in which therapeutics are recovered and purified be identified. Column-based approaches are constrained by the need for pre-treatment which leads to decreases in process yield and throughput. By contrast batch adsorption has the potential to handle high solids concentrations and can eliminate the need for pre-treatment. It may offer a viable, robust and simple means to improved process yields and throughput and this forms the central theme to this work. The thesis presents a scale-down evaluation of the use of both column and batch approaches based upon the adsorption of an antibody fragment (Fab') using ion exchange media. The particular Fab' used throughout this study was found to bind to cell debris and therefore removal of cell debris had a significant impact on the total process yield. The recovery of the Fab' from unclarified feedstocks, through the use of packed bed columns and stirred tank batch adsorption, was investigated, as was the impact of feedstock ionic strength and solids concentration. The use of homogenisation and periplasmic heat lysis as distinct product release routes were also investigated and compared via a series of Windows of Operation. Isotherm data was used to develop an empirical model capable of predicting process performance. From this the effect of cell disruption and feedstock properties, as well as other factors such as the length of the lysis procedure and the number of adsorption cycles, were modelled and the data used to generate Windows of Operation, identifying feasible operating conditions. The limitations of using packed bed columns for the recovery of Fab' from an unclarified E. coli feedstock are discussed. Batch adsorption was found to offer a viable alternative to packed bed chromatography. An analysis of the commercial and regulatory aspects of the use of batch adsorption as an alternative to conventional packed bed adsorption concludes the thesis.

This thesis illustrates the need for biologies manufacturing processes to be considered as a whole, as the operation of the upstream process can greatly alter the performance of downstream steps. It develops methods to allow this to be done at minimal cost and with the minimum requirement for process material. The industrial bioprocess for antibody fragment (Fab9) production in E. coli is used as the case study. Two variations of the antibody production process were examined. In the first lactose was used both to induce Fab' production and to provide the post-induction energy source. Cell growth was controlled by phosphate limitation. The second process used isopropyl p-D-l-thiogalactopyranoside (IPTG) for Fab' induction, with a glycerol feed providing energy post-induction. Both variations produced high cell density broths (ca. 20% solids w/v), and industrially relevant quantities of Fab' (ca. 1 g/L). However, the IPTG-induced fermentation was chosen for detailed study as it was the most controllable and reproducible of the two. The fermentation was investigated together with the initial unit operations of broth centrifugation, heat/chemical Fab9 extraction, and spheroplast centrifugation. Methods were developed to allow all aspects of the two centrifugation steps in the initial process to be mimicked at small-scale. Loss of cell integrity upon entry and exit to the industrial centrifuge was mimicked through the use of a rotating disc and capillary jet device respectively. Clarification was mimicked through the adaptation of an existing method to high cell density feeds ( > 10% w/v) by the addition of a dilution step and a mathematical correction based on the Richardson-Zaki equation. Dewatering of the solids cake was predicted to within 8% at small-scale using a novel method that maintains industrial spin speed, spin time, and height of the sediment cake at millilitre-scale. All small-scale methods developed were verified at pilot-scale. The heat/chemical Fab' extraction step was mimicked in microfuge tubes. Outputs, in terms of % total protein and Fab' release, were shown to be within 6% of pilot- scale results in all cases tested. Any differences between pilot- and small-scale performances were shown not to be statistically significant. Product quality, as assessed by SDS-PAGE, was shown to be very similar to pilot-scale. As the periplasmic extraction was identified as the step most detrimental to overall yield of the initial DSP the microfuge method was used in a 2-factor statistical design of experiments study to analyse the robustness of the extraction procedure. The process was then considered as a whole. Post-induction glycerol feed rate was varied and the impacts upon subsequent processing analysed using both the methods developed and pilot-scale data. This demonstrated the usefulness and accuracy of the small-scale methods developed, and also showed that optimizing the feed rate for maximum Fab' production during fermentation did not result in optimum Fab9 production after initial DSP. Instead a higher feed rate should be used to produce cells with weaker periplasmic membranes to facilitate more effective Fab9 release upon heat/chemical extraction. The thesis concludes with a discussion of the business relevance of the small-scale methods developed. Their general applicability for candidate screening, process development and process understanding is suggested, and the regulatory issues relevant to their use are explored.