Mammalian cell technology, whilst a mature platform for monoclonal antibody production, remains poorly understood from a metabolic perspective. The metabolic activity of two cell lines through three scales of a commercial scale-up train, each a 10-fold increase on the previous volume. In large scale mammalian cell culture the key toxic by-products assessed and monitored are lactate and ammonia. Often no distinction between the two isoforms of lactate is made. Profiles of both D- and L- lactate at multiple scales and under a range of controlled physiochemical conditions are presented. D-lactate unlike L-lactate is not commonly re-utilised, and although during normal culture time frames it represents one tenth of total lactate, during L-lactate consumption it represents nearly 35 %. The behaviour of D-lactate is different from that of L-lactate with the level of one isoform changing whilst the concentration of the other remains constant. The main route for the creation of D-lactate in culture is the methylglyoxal pathway. Methylglyoxal (MG) requires a lengthy purification process for analysis; however the close association between D-lactate and methylglyoxal concentrations alleviates the requirement for direct measurement of extracellular MG concentration. High concentrations of free MG observed in the medium (up to 63 μM), the levels of MG observed are higher than those reported to have a toxic effect (Roy 2005). The physiochemical impact on culture performance is investigated, with cultures conducted under varying environmental conditions. The impact of dissolved oxygen tension and temperature on metabolism is investigated, with the result that changing DOT has a quantifiable impact on culture metabolism. The approaches developed were undertaken so with the understanding of trends within industrial practice towards low volume, high throughput culture systems. The assay techniques utilised are capable of being applied to an automated micro-scale analysis method in order to maintain a high level of data density.

Enterokinase (EK) is a serine protease used to cleave therapeutic recombinant proteins during downstream processing. It has been selected for the activation and cleavage of a range of proprietary fusion proteins developed by Syntaxin Ltd. Whilst EK is well suited to this role in regards to substrate specificity, it has drawbacks, especially when it comes to expression in bacterial cells. Expression of EK in bacterial cells is the preferred expression method for process optimisation but is problematic due to its preference for inclusion body formation. This project describes efforts for improving the solubility of EK in E. coli using different constructs and mutagenesis. A total of four constructs were tested with two found to be soluble and one partially soluble. Two of the constructs (D4K-EK & pelB-EK) were found to readily form inclusion bodies (IB). Refolding of these constructs was undertaken and optimised using DoE. Only the refolded pelB EK showed significant activity, but refolded activity was found to vary greatly based on IB quality. The partially soluble pelB-EK construct exports to the periplasm for activation and soluble expression and was chosen for mutagenesis studies to improve soluble expression. A rational design approach using a range of sequence and structural bioinformatics methods including the consensus sequence, Hotpatch and statistical coupling analysis were utilised to identify fifteen stabilising mutants and seven mutants designed to increase surface charge. Of these potential mutants, ten (five stabilising, five surface charge) were created and analysed for activity, soluble yield and changes to secondary structure. Seven of the ten mutants showed measurable activity. Of interest were the surface charge mutants, which helped improve the purified yield by up to 2.5 fold. Also of note was consensus mutant V30Q which helped improve activity of periplasmic EK by 4.3 fold, whilst A32S and A44G visibly improved the thermo-tolerance of EK.

This thesis presents a study of the interaction of medical gases nitric oxide, carbon monoxide and hydrogen sulfide with metal-organic framework materials. Most analysis is performed via single-crystal X-ray diffraction and Rietveld and pair distribution function analysis of powder X-ray diffraction data. A background to the field and the experimental methods used are described in Chapters 1 and 3. The use of a specially designed static environmental gas cell to assess the role of coordinatively unsaturated metal sites in nitric oxide storage in Co-CPO-27 via in situ single-crystal structure determination is described in Chapter 4. Nitric oxide was shown to bind to the Co-centre of the material in a bent geometry in an approximately 1:1 Co:NO ratio. A multi-technique study was conducted on the framework Cu-SIP-3 in Chapters 5 and 6, utilising both single-crystal X-ray diffraction and pair distribution function analysis to obtain complementary information about atomic movements during a thermally active single-crystal to single-crystal transition. These techniques were further applied during in situ gas-loading experiments on the same framework. Application of the pair distribution function technique to metal-organic frameworks is described in Chapter 7, where refinements of both known and unknown metal-organic framework structures are presented. Partial PDFs are used to determine the secondary building block of a new metal-organic framework and verify the structural solution determined from powder X-ray diffraction data. Chapter 8 presents the study of the M-CPO-27 isostructural series for the adsorption and release of hydrogen sulfide and carbon monoxide. Gas adsorption isotherms and release measurements are correlated with the structure of the Ni-CPO-27 hydrogen sulfide-adduct determined by both powder X-ray diffraction and differential pair distribution function methods which reveal the open-metal site as the primary adsorption interaction in the material. The hydrogen sulfide released from Zn-CPO-27 is determined to be biologically active through vasodilatation experiments.

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Turning cells into useful devices to perform unnatural functions creates the potential to permit the interface between biological organisms and electronics. In this thesis cell-based devices were designed and constructed to respond to either light or a specific chemical stimulus. Design constraints were defined by the eventual application of the device, a biohybrid robot. The enzyme endothelial nitric oxide synthase (eNOS) was chosen as a target for genetic engineering. Prior to constructing the device a suitable host for the engineered construct was selected. CHO-K1 cells were transfected with nitric oxide synthase and expression levels were characterized via flow cytometry and inhibitor studies. A novel method for the effective delivery of inhibitors was developed and applied to demonstrate that transfected eNOS was sufficiently expressed to produce a measurable output. In addition, a balance between the native nitric oxide production machinery of the cells and the transfected endothelial nitric oxide synthase was observed. Two systems were designed and constructed for stimuli responsive nitric oxide production. The first system was designed to produce nitric oxide in response to the presence of the antibiotic rapamycin. Chemical induced dimerization would bring the two separated domains of endothelial nitric oxide synthase into close enough proximity to re-establish protein function. The separate oxygenase and reductase domains were successfully amplified and subsequently fused with components of the chemically induced dimerization system. The second system involved fusing a domain from the plant gene Nhp1 (Light Oxygen Voltage domain - LOV) capable of harvesting a photon, with mouse endothelial nitric oxide synthase. This strategy aimed to hijack the wild type protein’s native electron transfer pathway. Manipulation was carried out in bacteria with subsequent transfection into CHO-K1 cells. Subsequent testing of nitric oxide production the mutant cells confirmed the optical sensitivity of the mutant eNOS. Moreover both LOV mutant cell lines were capable of fast response times and switching behaviour. The findings of this thesis demonstrate that genetic engineering of endothelial nitric oxide synthase is a suitable strategy for the controlled release of nitric oxide upon optical stimulation. Moreover the potential of an engineered cell to respond quickly to stimuli has been realized, comparing favourably to genetically engineered systems that rely on gene expression to elicit an output.

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