Development of a pilot-scale bacterial fermentation for plasmid-based biopharmaceutical production using a stoichiometric medium

The recognition of the potential efficacy of plasmid DNA (pDNA) molecules as vectors in the treatment and prevention of emerging diseases has birthed the confidence to combat global pandemics. This is due to the close-to-zero safety concern associated with pDNA vectors compared to viral vectors in cell transfection and targeting. Considerable attention has been paid to the potential of pDNA vectors but comparatively less thought has been given to the practical challenges in producing large quantities to meet current rising demands. A pilot-scale fermentation scheme was developed by employing a stoichiometrically-designed growth medium whose exceptional plasmid yield performance was attested in a shake flask environment for pUC19 and pEGFP-N1 transformed into E. coliDH5α and E. coliJM109, respectively. Batch fermentation of E. coliDH5α-pUC19 employing the stoichiometric medium displayed a maximum plasmid volumetric and specific yield of 62.6 mg/L and 17.1 mg/g (mg plasmid/g dry cell weight), respectively. Fed-batch fermentation of E. coliDH5α-pUC19 on a glycerol substrate demonstrated one of the highest ever reported pilot-scale plasmid specific yield of 48.98 mg/g and a volumetric yield of 0.53 g/L. The attainment of high plasmid specific yields constitutes a decrease in plasmid manufacturing cost and enhances the effectiveness of downstream processes by reducing the proportion of intracellular impurities. The effect of step-rise temperature induction was also considered to maximize ColE1-origin plasmid replication.

Enhancing methacrylate-monolith-based downstream processes to champion plasmid DNA production

Increasing numbers of preclinical and clinical studies are utilizing pDNA (plasmid DNA) as the vector. In addition, there has been a growing trend towards larger and larger doses of pDNA utilized in human trials. The growing demand on pDNA manufacture leads to pressure to make more in less time. A key intervention has been the use of monoliths as stationary phases in liquid chromatography. Monolithic stationary phases offer fast separation to pDNA owing to their large pore size, making pDNA in the size range from 100 nm to over 300 nm easily accessible. However, the convective transport mechanism of monoliths does not guarantee plasmid purity. The recovery of pure pDNA hinges on a proper balance in the properties of the adsorbent phase, the mobile phase and the feedstock. The effects of pH and ionic strength of binding buffer, temperature of feedstock, active group density and the pore size of the stationary phase were considered as avenues to improve the recovery and purity of pDNA using a methacrylate-based monolithic adsorbent and Escherichia coli DH5α-pUC19 clarified lysate as feedstock. pDNA recovery was found to be critically dependent on the pH and ionic strength of the mobile phase. Up to a maximum of approx. 92% recovery was obtained under optimum conditions of pH and ionic strength. Increasing the feedstock temperature to 80°C increased the purity of pDNA owing to the extra thermal stability associated with pDNA over contaminants such as proteins. Results from toxicological studies of the plasmid samples using endotoxin standard (E. coli 0.55:B5 lipopolysaccharide) show that endotoxin level decreases with increasing salt concentration. It was obvious that large quantities of pure pDNA can be obtained with minimal extra effort simply by optimizing process parameters and conditions for pDNA purification.

Process engineering aspects of plasmid-based biopharmaceuticals production : tackling the threatening vaccine shortages to prevent global pandemics

Infectious diseases such as SARS, influenza and bird flu have the potential to cause global pandemics; a key intervention will be vaccination. Hence, it is imperative to have in place the capacity to create vaccines against new diseases in the shortest time possible. In 2004, The Institute of Medicine asserted that the world is tottering on the verge of a colossal influenza outbreak. The institute stated that, inadequate production system for influenza vaccines is a major obstruction in the preparation towards influenza outbreaks. Because of production issues, the vaccine industry is facing financial and technological bottlenecks: In October 2004, the FDA was caught off guard by the shortage of flu vaccine, caused by a contamination in a US-based plant (Chiron Corporation), one of the only two suppliers of US flu vaccine. Due to difficulties in production and long processing times, the bulk of the world's vaccine production comes from very small number of companies compared to the number of companies producing drugs. Conventional vaccines are made of attenuated or modified forms of viruses. Relatively high and continuous doses are administered when a non-viable vaccine is used and the overall protective immunity obtained is ephemeral. The safety concerns of viral vaccines have propelled interest in creating a viable replacement that would be more effective and safer to use.

Further development of discrete computational techniques for calculation of restricted diffusion propagators in porous media

Magnetic resonance is a well-established tool for structural characterisation of porous media. Features of pore-space morphology can be inferred from NMR diffusion-diffraction plots or the time-dependence of the apparent diffusion coefficient. Diffusion NMR signal attenuation can be computed from the restricted diffusion propagator, which describes the distribution of diffusing particles for a given starting position and diffusion time. We present two techniques for efficient evaluation of restricted diffusion propagators for use in NMR porous-media characterisation. The first is the Lattice Path Count (LPC). Its physical essence is that the restricted diffusion propagator connecting points A and B in time t is proportional to the number of distinct length-t paths from A to B. By using a discrete lattice, the number of such paths can be counted exactly. The second technique is the Markov transition matrix (MTM). The matrix represents the probabilities of jumps between every pair of lattice nodes within a single timestep. The propagator for an arbitrary diffusion time can be calculated as the appropriate matrix power. For periodic geometries, the transition matrix needs to be defined only for a single unit cell. This makes MTM ideally suited for periodic systems. Both LPC and MTM are closely related to existing computational techniques: LPC, to combinatorial techniques; and MTM, to the Fokker-Planck master equation. The relationship between LPC, MTM and other computational techniques is briefly discussed in the paper. Both LPC and MTM perform favourably compared to Monte Carlo sampling, yielding highly accurate and almost noiseless restricted diffusion propagators. Initial tests indicate that their computational performance is comparable to that of finite element methods. Both LPC and MTM can be applied to complicated pore-space geometries with no analytic solution. We discuss the new methods in the context of diffusion propagator calculation in porous materials and model biological tissues.

Endotoxin removal : a critical step in vaccine production

A major drawback to the immunological potency of conventional vaccines, resulting in reduced level of immune responses, tissue injury, shock and high cytotoxicity, thus making their applications contraindicated in immunodeficiency diseases, is the presence of high contaminant concentrations in vaccine titers. Vaccine contamination arises from the simultaneous occurrence of competitive pathways resulting in the formation of other bio-products during cellular metabolism aside the pathways necessary for the production of vaccine molecules. One of such vaccine contaminating molecules is endotoxins which are mainly lipopolysaccharides (LPS) complexes found in the membrane of bacterial cell wall. The structural dynamics of these molecules make their removal from vaccine titers problematic, thus making vaccine endotoxin removal a major research endeavour. This presentation will discuss a novel technique for reducing the endotoxin level of vaccines. The technique commences with the disentanglement of endotoxin-vaccine molecular bonding and then capturing the vaccine molecules on an affinity monolith to separate the vaccine molecules from the endotoxins.

Dancing as Knowledge Production/Dancer as Researcher in Practice-led Research

This chapter discusses research undertaken for a PhD in dance, highlighting the oscillating and ambivalent nature of practice-led research methods. Holding an unusual position within the field of practice-led research in dance, the researcher adopted the role of dancer within four creative processes led by four choreographers. Although instigating and leading this research, the author produced new knowledge of dancing practices whilst being directed creatively by the choreographers in the research environment. In line with many definitions of practice-led research, the methods used were emergent from the research arena and responsive to the requirements of the project. Deeply imbricated in the research environment, the researcher used her embodied self as a research tool, as the one who participated, discovered and recorded. This outsider/insider role of researcher/participant produced a series of meta-positions, leading to a mixed-modal outcome that encompassed live performance, a movement treatise and a written thesis.

Bio-hydrogen production from food waste through anaerobic fermentation

In order to protect our planet and ourselves from the adverse effects of excessive CO2 emissions and to prevent an imminent non-renewable fossil fuel shortage and energy crisis, there is a need to transform our current ‘fossil fuel dependent’ energy systems to new, clean, renewable energy sources. The world has recognized hydrogen as an energy carrier that complies with all the environmental quality and energy security, demands. This research aimed at producing hydrogen through anaerobic fermentation, using food waste as the substrate. Four food waste substrates were used: Rice, fish, vegetable and their mixture. Bio-hydrogen production was performed in lab scale reactors, using 250 mL serum bottles. The food waste was first mixed with the anaerobic sewage sludge and incubated at 37°C for 31 days (acclimatization). The anaerobic sewage sludge was then heat treated at 80°C for 15 min. The experiment was conducted at an initial pH of 5.5 and temperatures of 27, 35 and 55°C. The maximum cumulative hydrogen produced by rice, fish, vegetable and mixed food waste substrates were highest at 37°C (Rice =26.97±0.76 mL, fish = 89.70±1.25 mL, vegetable = 42.00±1.76 mL, mixed = 108.90±1.42 mL). A comparative study of acclimatized (the different food waste substrates were mixed with anaerobic sewage sludge and incubated at 37°C for 31days) and non-acclimatized food waste substrate (food waste that was not incubated with anaerobic sewage sludge) showed that acclimatized food waste substrate enhanced bio-hydrogen production by 90 - 100%.

Assessment and application of clustering techniques to atmospheric particle number size distribution for the purpose of source apportionment

Long-term measurements of particle number size distribution (PNSD) produce a very large number of observations and their analysis requires an efficient approach in order to produce results in the least possible time and with maximum accuracy. Clustering techniques are a family of sophisticated methods which have been recently employed to analyse PNSD data, however, very little information is available comparing the performance of different clustering techniques on PNSD data. This study aims to apply several clustering techniques (i.e. K-means, PAM, CLARA and SOM) to PNSD data, in order to identify and apply the optimum technique to PNSD data measured at 25 sites across Brisbane, Australia. A new method, based on the Generalised Additive Model (GAM) with a basis of penalised B-splines, was proposed to parameterise the PNSD data and the temporal weight of each cluster was also estimated using the GAM. In addition, each cluster was associated with its possible source based on the results of this parameterisation, together with the characteristics of each cluster. The performances of four clustering techniques were compared using the Dunn index and Silhouette width validation values and the K-means technique was found to have the highest performance, with five clusters being the optimum. Therefore, five clusters were found within the data using the K-means technique. The diurnal occurrence of each cluster was used together with other air quality parameters, temporal trends and the physical properties of each cluster, in order to attribute each cluster to its source and origin. The five clusters were attributed to three major sources and origins, including regional background particles, photochemically induced nucleated particles and vehicle generated particles. Overall, clustering was found to be an effective technique for attributing each particle size spectra to its source and the GAM was suitable to parameterise the PNSD data. These two techniques can help researchers immensely in analysing PNSD data for characterisation and source apportionment purposes.

Investigation of fiducial marker and soft-tissue image guidance techniques in prostate radiation therapy

This thesis examines and compares imaging methods used during the radiotherapy treatment of prostate cancer. The studies found that radiation therapists were able to localise and target the prostate consistently with planar imaging techniques and that the use of small gold markers in the prostate reduced the variation in prostate localisation when using volumetric imaging. It was concluded that larger safety margins are required when using volumetric imaging without gold markers.

Multi-objective models and techniques for analysing the absolute capacity of railway networks

Railway capacity determination and expansion are very important topics. In prior research, the competition between different entities such as train services and train types, on different network corridors however have been ignored, poorly modelled, or else assumed to be static. In response, a comprehensive set of multi-objective models have been formulated in this article to perform a trade-off analysis. These models determine the total absolute capacity of railway networks as the most equitable solution according to a clearly defined set of competing objectives. The models also perform a sensitivity analysis of capacity with respect to those competing objectives. The models have been extensively tested on a case study and their significant worth is shown. The models were solved using a variety of techniques however an adaptive E constraint method was shown to be most superior. In order to identify only the best solution, a Simulated Annealing meta-heuristic was implemented and tested. However a linearization technique based upon separable programming was also developed and shown to be superior in terms of solution quality but far less in terms of computational time.

Optimising ketocarotenoid production in potato tubers : effect of genetic background, transgene combinations and environment

Astaxanthin is a high value carotenoid produced by some bacteria, a few green algae, several fungi but only a limited number of plants from the genus Adonis. Astaxanthin has been industrially exploited as a feed supplement in poultry farming and aquaculture. Consumption of ketocarotenoids, most notably astaxanthin, is also increasingly associated with a wide range of health benefits,as demonstrated in numerous clinical studies. Currently astaxanthin is produced commercially by chemical synthesis or from algal production systems. Several studies have used a metabolic engineering approach to produce astaxanthin in transgenic plants. Previous attempts to produce transgenic potato tubers biofortified with astaxanthin have met with limited success. In this study we have investigated approaches to optimising tuber astaxanthin content. It is demonstrated that the selection of appropriate parental genotype for transgenic approaches and stacking carotenoid biosynthetic pathway genes with the cauliflower Or gene result in enhanced astaxanthin content, to give six-fold higher tuber astaxanthin content than has been achieved previously. Additionally we demonstrate the effects of growth environment on tuber carotenoid content in both wild type and astaxanthin-producing transgenic lines and describe the associated transcriptome and metabolome restructuring.

Reducing nitrous oxide emissions while supporting subtropical cereal production in Oxisols

This is the first study to investigate alternative fertilisation strategies to increase cereal production while reducing greenhouse gas emissions from the most common soil type in subtropical regions. The results of this research will contribute to define future farming practices to achieve global food security and mitigate climate change. The study established that introducing legumes in cropping systems is the most agronomically viable and environmentally sustainable fertilisation strategy. Importantly, this strategy can be widely adopted in subtropical regions since it is economically accessible, requires little know-how transfer and technology investment, and can be profitable in both low- and high-input cropping systems.

Production of biodegradable nano and micro particles via ultrasonic atomization for biopharmaceutical delivery

Biopharmaceuticals have been shown to have low delivery and transformation efficiencies. To over come this, larger doses are administered in order to obtain the desired response which may lead to toxicity and drug resistance. This paper reports upon a continuous particle production method utilizing surface acoustic wave atomization to reliably produce micro and nanoparticles with physical characteristics to facilitate the cellular uptake of biopharmaceuticals. By producing particles of an optimal size for cellular uptake, the efficacy and specificity of drug loaded nanoparticles will be increased. Better delivery methods will result in dosage reduction (hence lower costs per dose), reduced toxicity, and reduced problems associated with multidrug resistance due to over dosing.