Characterisation of a harvesting step for monoclonal antibodies from mammalian cell culture

The focus of this research was defined by a poorly characterised filtration train employed to clarify culture broth containing monoclonal antibodies secreted by GS-NSO cells: the filtration train blinded unpredictably and the ability of the positively charged filters to adsorb DNA from process material was unknown. To direct the development of an assay to quantify the ability of depth filters to adsorb DNA, the molecular weight of DNA from a large-scale, fed-batch, mammalian cell culture vessel was evaluated as process material passed through the initial stages of the purification scheme. High molecular weight DNA was substantially cleared from the broth after passage through a disc stack centrifuge and the remaining low molecular weight DNA was largely unaffected by passage through a series of depth filters and a sterilising grade membrane. Removal of high molecular weight DNA was shown to be coupled with clarification of the process stream. The DNA from cell culture supernatant showed a pattern of internucleosomal cleavage of chromatin when fractionated by electrophoresis but the presence of both necrotic and apoptotic cells throughout the fermentation meant that the origin of the fragmented DNA could not be unequivocally determined. An intercalating fluorochrome, PicoGreen, was elected for development of a suitable DNA assay because of its ability to respond to low molecular weight DNA. It was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing pertinent monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 89.0 % of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples. Application of the fluorescence based assay resulted in characterisation of the physical parameters governing adsorption of DNA by various positively charged depth filters and membranes in test solutions and the DNA adsorption profile of the manufacturing scale filtration train. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. Production-scale centrifugation of harvest broth containing therapeutic protein resulted in the reduction of total DNA in the process stream from 79.8 μg m1-1 to 9.3 μg m1-1 whereas the concentration of DNA in the supernatant of pre-and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 μg m1-1 respectively. Hence the filtration train was shown to ineffective in DNA removal. Historically, blinding of the depth filters had been unpredictable with data such as numbers of viable cells, non-viable cells, product titre, or process shape (batch, fed-batch, or draw and fill) failing to inform on the durability of depth filters in the harvest step. To investigate this, key fouling contaminants were identified by challenging depth filters with the same mass of one of the following: viable healthy cells, cells that had died by the process of apoptosis, and cells that had died through the process of necrosis. The pressure increase across a Cuno Zeta Plus 10SP depth filter was 2.8 and 16.5 times more sensitive to debris from apoptotic and necrotic cells respectively, when compared to viable cells. The condition of DNA released into the culture broth was assessed. Necrotic cells released predominantly high molecular weight DNA in contrast to apoptotic cells which released chiefly low molecular weight DNA. The blinding of the filters was found to be largely unaffected by variations in the particle size distribution of material in, and viscosity of, solutions with which they were challenged. The exceptional response of the depth filters to necrotic cells may suggest the cause of previously noted unpredictable filter blinding whereby a number of necrotic cells have a more significant impact on the life of a depth filter than a similar number of viable or apoptotic cells. In a final set of experiments the pressure drop caused by non-viable necrotic culture broths which had been treated with DNase I or benzonase was found to be smaller when compared to untreated broths: the abilities of the enzyme treated cultures to foul the depth filter were reduced by 70.4% and 75.4% respectively indicating the importance of DNA in the blinding of the depth filter studied.

A greenhouse integrating desalination, water saving and rainwater harvesting for use in salt-affected inland regions

This study proposes a new type of greenhouse for water re-use and energy saving for agriculture in arid and semi-arid inland regions affected by groundwater salinity. It combines desalination using reverse osmosis (RO), re-use of saline concentrate rejected by RO for cooling, and rainwater harvesting. Experimental work was carried at GBPUAT, Pantnagar, India. Saline concentrate was fed to evaporative cooling pads of greenhouse and found to evaporate at similar rates as conventional freshwater. Two enhancements to the system are described: i) A jet pump, designed and tested to use pressurized reject stream to re-circulate cooling water and thus maintain uniform wetness in cooling pads, was found capable of multiplying flow of cooling water by a factor of 2.5 to 4 while lifting water to a head of 1.55 m; and ii) Use of solar power to drive ventilation fans of greenhouse, for which an electronic circuit has been produced that uses maximum power-point tracking to maximize energy efficiency. Re-use of RO rejected concentrate for cooling saves water (6 l d-1 m-2) of greenhouse floor area and the improved fan could reduce electricity consumption by a factor 8.

Solving Ratio-Dependent Predator-Prey System with Constant Effort Harvesting using Variational Iteration Method

Due to wide range of interest in use of bio-economic models to gain insight into the scientific management of renewable resources like fisheries and forestry,variational iteration method (VIM) is employed to approximate the solution of the ratio-dependent predator-prey system with constant effort prey harvesting.The results are compared with the results obtained by Adomian decomposition method and reveal that VIM is very effective and convenient for solving nonlinear differential equations.

A potentially scalable method for the harvesting of hMSCs from microcarriers

The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on microcarriers in bioreactors. Whilst much research investigating hMSC culture on microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill microcarriers in a 5L bioreactor [1]. Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell 'detachment' from the microcarriers' surface followed by the 'separation' of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip® vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too. © 2014 The Authors.

Economy of the island of eco-tourism: the role of shark-diving and shark harvesting

Beyond its importance in maintaining ecosystems, sharks provide services that play important socioeconomic roles. The rise in their exploitation as a tourism resource in recent years has highlighted economic potential of non-destructive uses of sharks and the extent of economic losses associated to declines in their population. In this work, we present estimates for use value of sharks in Fernando de Noronha Island - the only ecotouristic site offering shark diving experience in the Atlantic coast of South America. Through the Travel Cost Method we estimate the total touristic use value aggregated to Noronha Island by the travel cost was up to USD 312 million annually, of which USD 91.1 million are transferred to the local economy. Interviewing people from five different economic sectors, we show shark-diving contribute with USD 2.5 million per year to Noronha’s economy, representing 19% of the island’s GDP. Shark-diving provides USD 128.5 thousand of income to employed islanders, USD 72.6 thousand to government in taxes and USD 5.3 thousand to fishers due to the increase in fish consumption demanded by shark divers. We discover, though, that fishers who actually are still involved in shark fishing earn more by catching sharks than selling other fish for consumption by shark divers. We conclude, however, that the non-consumptive use of sharks is most likely to benefit large number of people by generating and money flow if compared to the shark fishing, providing economic arguments to promote the conservation of these species.

Electrically tunable piezoelectric bimorph cantilever for energy harvesting

Con la presente tesi viene esaminato un metodo per modificare la frequenza di risonanza di trasduttori piezoelettrici mediante applicazione di carichi elettrici esterni. L'elaborato inizia con la presentazione dei cristalli utilizzati nel lavoro di tesi, concentrandosi sul processo di fabbricazione di un bimorph cantilever impiegato come convertitore elettromeccanico di energia, la cui frequenza di risonanza è modellizzata analiticamente mediante la legge di Newton e il modello di Euler-Bernoulli. Su tale struttura vengono condotte misure mediante shaker elettrodinamico e analizzatore d'impedenza, ai fini di giusticare il modello analitico presentato. Con lo scopo di sincronizzare la frequenza di risonanza del cantilever con la vibrazione dell'ambiente per massimizzare la potenza disponibile, viene proposto un algoritmo MPPT secondo l'approccio Perturba e Osserva (P&O), al quale è fornita in ingresso la tensione efficace di un layer di materiale piezoelettrico. Valutare la sua risposta in tensione, presenta dei limiti applicativi che hanno portato a prendere in considerazione un approccio totalmente diff�erente, basato sullo sfasamento tra la tensione di un trasduttore piezoelettrico e il segnale di accelerazione impiegato come eccitazione. Misure sperimentali sono state condotte con l'obiettivo di validare l'efficacia di quest'ultimo approccio qualora si voglia sincronizzare la frequenza di risonanza dei piezo con segnali di vibrazione reali.

Piezoelectric energy harvesting using blood-flow-like excitation for implantable devices

The goal of this research is to produce a system for powering medical implants to increase the lifetime of the implanted devices and reduce the battery size. The system consists of a number of elements – the piezoelectric material for generating power, the device design, the circuit for rectification and energy storage. The piezoelectric material is analysed and a process for producing a repeatable high quality piezoelectric material is described. A full width half maximum (FWHM) of the rocking curve X-Ray diffraction (XRD) scan of between ~1.5° to ~1.7° for test wafers was achieved. This is state of the art for AlN on silicon and means devices with good piezoelectric constants can be fabricated. Finite element modelling FEM) was used to design the structures for energy harvesting. The models developed in this work were established to have an accuracy better than 5% in terms of the difference between measured and modelled results. Devices made from this material were analysed for power harvesting ability as well as the effect that they have on the flow of liquid which is an important consideration for implantable devices. The FEM results are compared to experimental results from laser Doppler vibrometry (LDV), magnetic shaker and perfusion machine tests. The rectifying circuitry for the energy harvester was also investigated. The final solution uses multiple devices to provide the power to augment the battery and so this was a key feature to be considered. Many circuits were examined and a solution based on a fully autonomous circuit was advanced. This circuit was analysed for use with multiple low power inputs similar to the results from previous investigations into the energy harvesting devices. Polymer materials were also studied for use as a substitute for the piezoelectric material as well as the substrate because silicon is more brittle.

Vibration based health monitoring of civil infrastructure using energy harvesting techniques

The use of structural health monitoring of civil structures is ever expanding and by assessing the dynamical condition of structures, informed maintenance management can be conducted at both individual and network levels. With the continued growth of information age technology, the potential arises for smart monitoring systems to be integrated with civil infrastructure to provide efficient information on the condition of a structure. The focus of this thesis is the integration of smart technology with civil infrastructure for the purposes of structural health monitoring. The technology considered in this regard are devices based on energy harvesting materials. While there has been considerable focus on the development and optimisation of such devices using steady state loading conditions, their applications for civil infrastructure are less known. Although research is still in initial stages, studies into the uses associated with such applications are very promising. Through the use of the dynamical response of structures to a variety of loading conditions, the energy harvesting outputs from such devices is established and the potential power output determined. Through a power variance output approach, damage detection of deteriorating structures using the energy harvesting devices is investigated. Further applications of the integration of energy harvesting devices with civil infrastructure investigated by this research includes the use of the power output as a indicator for control. Four approaches are undertaken to determine the potential applications arising from integrating smart technology with civil infrastructure, namely • Theoretical analysis to determine the applications of energy harvesting devices for vibration based health monitoring of civil infrastructure. • Laboratory experimentation to verify the performance of different energy harvesting configurations for civil infrastructure applications. • Scaled model testing as a method to experimentally validate the integration of the energy harvesting devices with civil infrastructure. • Full scale deployment of energy harvesting device with a bridge structure. These four approaches validate the application of energy harvesting technology with civil infrastructure from a theoretical, experimental and practical perspective.

Wireless Networks with Energy Harvesting and Power Transfer: Joint Power and Time Allocation

In this letter, we consider wireless powered communication networks which could operate perpetually, as the base station (BS) broadcasts energy to the multiple energy harvesting (EH) information transmitters. These employ “harvest then transmit” mechanism, as they spend all of their energy harvested during the previous BS energy broadcast to transmit the information towards the BS. Assuming time division multiple access (TDMA), we propose a novel transmission scheme for jointly optimal allocation of the BS broadcasting power and time sharing among the wireless nodes, which maximizes the overall network throughput, under the constraint of average transmit power and maximum transmit power at the BS. The proposed scheme significantly outperforms “state of the art” schemes that employ only the optimal time allocation. If a single EH transmitter is considered, we generalize the optimal solutions for the case of fixed circuit power consumption, which refers to a much more practical scenario.

Rate Maximization of Decode-and-Forward Relaying Systems with RF Energy Harvesting

We consider a three-node decode-and-forward (DF) half-duplex relaying system, where the source first harvests RF energy from the relay, and then uses this energy to transmit information to the destination via the relay. We assume that the information transfer and wireless power transfer phases alternate over time in the same frequency band, and their time fraction (TF) may change or be fixed from one transmission epoch (fading state) to the next. For this system, we maximize the achievable average data rate. Thereby, we propose two schemes: (1) jointly optimal power and TF allocation, and (2) optimal power allocation with fixed TF. Due to the small amounts of harvested power at the source, the two schemes achieve similar information rates, but yield significant performance gains compared to a benchmark system with fixed power and fixed TF allocation.

RF energy harvesting two-way cognitive DF relaying with transceiver impairments

This paper analyzes the impact of transceiver impairments on outage probability (OP) and throughput of decode-and-forward two-way cognitive relay (TWCR) networks, where the relay is self-powered by harvesting energy from the transmitted signals. We consider two bidirectional relaying protocols namely, multiple access broadcast (MABC) protocol and time division broadcast (TDBC) protocol, as well as, two power transfer policies namely, dual-source (DS) energy transfer and single-fixed-source (SFS) energy transfer. Closed-form expressions for OP and throughput of the network are derived in the context of delay-limited transmission. Numerical results corroborate our analysis, thereby we can quantify the degradation of OP and throughput of TWCR networks due to transceiver hardware impairments. Under the specific parameters, our results indicate that the MABC protocol achieves asymptotically a higher throughput by 0.65 [bits/s/Hz] than the TDBC protocol, while the DS energy transfer scheme offers better performance than the SFS policy for both relaying protocols.

Impact of primary networks on the performance of energy harvesting cognitive radio networks

In this paper, we investigate the effect of of the primary network on the secondary network when harvesting energy in cognitive radio in the presence of multiple power beacons and multiple secondary transmitters. In particular, the influence of the primary transmitter's transmit power on the energy harvesting secondary network is examined by studying two scenarios of primary transmitter's location, i.e., the primary transmitter's location is near to the secondary network and the primary transmitter's location is far from the secondary network. In the scenario where the primary transmitter locates near to the secondary network, although secondary transmitter can be benefit from the harvested energy from the primary transmitter, the interference caused by the primary transmitter suppresses the secondary network performance. Meanwhile, in both scenarios, despite the fact that the transmit power of the secondary transmitter can be improved by the support of powerful power beacons, the peak interference constraint at the primary receiver limits this advantage. In addition, the deployment of multiple power beacons and multiple secondary transmitters can improve the performance of the secondary network. The analytical expressions of the outage probability of the secondary network in the two scenarios are also provided and verified by numerical simulations.