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.

The ecology of paper mill by-product and its evaluation as a casing medium in the culture of Agaricus bisporus (Lange) Pilat

Effluent from pulp and paper production at the Kemsley mill of Bowaters U.K. Paper Company Limited passes through two treatment stages before its discharge into the Swale estuary. Suspended material removed during treatment is deposited on wasteground as a thin sludge. The solids it contains are mainly wood components lost during pulp production, whilst it also has a high salt content, derived from chemicals used in pulping processes. After deposition the sludge undergoes an ageing process during which it dries out and its salt content is reduced. This ageing can be reproduced and accelerated by improved drainage under controlled conditions. The paper mill sludge was investigated as a casing medium in the culture of Agaricus bisporus (Lange) Pilat, the cultivated mushroom. It was unsuitable up to one year from deposition due largely to the inhibitory effect of its salt content on fruiting. Material eighteen months or more in age gave yields comparable to standard peat casing. Before use as a casing the material must be shredded to a satisfactory structure, neutralised with chalk, and pasteurised to eliminate organisms harmful to the crop. The prepared medium has a high water holding capacity and a structure resilient to management procedures, important requirements of a good casing. A passive movement of salts from the compost to the casing was shown to occur during culture, capable of enhancing the natural decline in cropping if sufficiently great. The ions chloride, potassium, sodium and sulphate were shown to be responsible, their damaging effects being due to high conductivity created in the casing. Studies of elements available during culture suggested phosphate availability in the compost could limit crop potential, whilst iron released by mycelium of A.bisporus in the casing may be utilised by associated micro-organisms.

Analysing organisational culture : a critical ethnography of public relations and personnel specialists in a state bureaucracy

Following an introductory chapter, I examine (i) typologies which have differentiated the literature on organisational culture and symbolism (Chapter 2), (ii) the contribution of organisation theory to organisation culture (Chapter 3), and (iii) recent literature on organisational culture and symbolism (Chapter 4). Within these chapters, I adopt Habermas' (1972) notion of knowledge-constitutive interests, assessing the contrubutions to understanding organisational culture made by literature guided by technical, practical and emancipatory cognitive interests. In doing so, I suggest that more critical studies on organisational culture and symbolism have been comparatively neglected. Lamenting this neglect, I suggest that Giddens' theory of structuration can be employed to advance the development of a critical, emancipatory conceptualisation of organisational culture. In particular, I argue that this Giddensian analysis, by penetrating the existential, poltical and material processes of cultural reproduction (Chapter 5), is able to disclose some of the more contradictory features of organisation culture. The remainder of the thesis comprises of a critical ethnography of the work cultures of public relations and personnel specialists located in a state bureaucracy. I begin the ethnography with a dicussion of my research methods (Chapter 6) and an overview of the departments studied (Chapter 7): I then examine (i) the work cultures of the specialists (Chapter 8), (ii) the specialists' management of the relationships with the hosts bureaucracy (Chapter 9); and, (iii) opportunities the specialists had for developing an emancipatory praxis (Chapter 10). Finally, in a concluding section, I offer some critical reflections on the contributions of the thesis and suggest areas for future research.

Development of a co-culture model of the human lungs for toxicity testing and identification of biomarkers of inhalation toxicity

The airway epithelium is the first point of contact in the lung for inhaled material, including infectious pathogens and particulate matter, and protects against toxicity from these substances by trapping and clearance via the mucociliary escalator, presence of a protective barrier with tight junctions and initiation of a local inflammatory response. The inflammatory response involves recruitment of phagocytic cells to neutralise and remove and invading materials and is oftern modelled using rodents. However, development of valid in vitro airway epithelial models is of great importance due to the restrictions on animal studies for cosmetic compound testing implicit in the 7th amendment to the European Union Cosmetics Directive. Further, rodent innate immune responses have fundamental differences to human. Pulmonary endothelial cells and leukocytes are also involved in the innate response initiated during pulmonary inflammation. Co-culture models of the airways, in particular where epithelial cells are cultured at air liquid interface with the presence of tight junctions and differentiated mucociliary cells, offer a solution to this problem. Ideally validated models will allow for detection of early biomarkers of response to exposure and investigation into inflammatory response during exposure. This thesis describes the approaches taken towards developing an in vitro epithelial/endothelial cell model of the human airways and identification biomarkers of response to exposure to xenobiotics. The model comprised normal human primary microvascular endothelial cells and the bronchial epithelial cell line BEAS-2B or normal human bronchial epithelial cells. BEAS-2B were chosen as their characterisation at air liquid interface is limited but they are robust in culture, thereby predicted to provide a more reliable test system. Proteomics analysis was undertaken on challenged cells to investigate biomarkers of exposure. BEAS-2B morphology was characterised at air liquid interface compared with normal human bronchial epithelial cells. The results indicate that BEAS-2B cells at an air liquid interface form tight junctions as shown by expression of the tight junction protein zonula occludens-1. To this author’s knowledge this is the first time this result has been reported. The inflammatory response of BEAS-2B (measured as secretion of the inflammatory mediators interleukin-8 and -6) air liquid interface mono-cultures to Escherichia coli lipopolysaccharide or particulate matter (fine and ultrafine titanium dioxide) was comparable to published data for epithelial cells. Cells were also exposed to polymers of “commercial interest” which were in the nanoparticle range (and referred to particles hereafter). BEAS-2B mono-cultures showed an increased secretion of inflammatory mediators after challenge. Inclusion of microvascular endothelial cells resulted in protection against LPS- and particle- induced epithelial toxicity, measured as cell viability and inflammatory response, indicating the importance of co-cultures for investigations into toxicity. Two-dimensional proteomic analysis of lysates from particle-challenged cells failed to identify biomarkers of toxicity due to assay interference and experimental variability. Separately, decreased plasma concentrations of serine protease inhibitors, and the negative acute phase proteins transthyretin, histidine-rich glycoprotein and alpha2-HS glycoprotein were identified as potential biomarkers of methyl methacrylate/ethyl methacrylate/butylacrylate treatment in rats.

An initial evaluation of gellan gum as a material for tissue engineering applications

Alpha-modified minimum essential medium (αMEM) has been found to cross-link a 1% gellan gum solution, resulting in the formation of a self-supporting hydrogel in 1:1 and 5:1 ratios of polysaccharide: αMEM. Rheological data from temperature sweeps confirm that in addition to orders of magnitude differences in G' between 1% gellan and 1% gellan with αMEM, there is also a 20°C increase in the temperature at which the onset of gelation takes place when αMEM is present. Frequency sweeps confirm the formation of a true gel; mechanical spectra for mixtures of gellan and αMEM clearly demonstrate G' to be independent of frequency. It is possible to immobilize cells within a three-dimensional (3D) gellan matrix that remain viable for up to 21 days in culture by adding a suspension of rat bone marrow cells (rBMC) in αMEM to 1% gellan solution. This extremely simple approach to cell immobilization within 3D constructs, made possible by the fact that gellan solutions cross-link in the presence of millimolar concentrations of cations, poses a very low risk to a cell population immobilized within a gellan matrix and thus indicates the potential of gellan for use as a tissue engineering scaffold. © 2007 Sage Publications.

Special issue:key debates in the translation of advertising material

Much has been written about the marketing aspects of promotional material in general, and several scholars (particularly in linguistics) have addressed questions relating to the structure and function of advertisements, focusing on images, rhetorical structure, semiotic functions, discourse features and audio-visual media, amongst other aspects of the genre. Not much, on the other hand, has been written within translation studies about the complexities involved in the transfer of an advertising message. Contributors to this volume explore various interdependent aspects of the interlingual and intercultural transfer of an advertising message. They emphasize features of culture specificity, of multi-medial semiotic interaction, of values and stereotypes, and most importantly, they recommend strategies and approaches to assist translators. Topics covered include a critique of the Western-based approach to advertising in the context of the Far East; different perceptions of the concept of cleanliness in advertising texts in Italy, Russia and the UK; the Walls Cornetto strategy of internationalization of product appeal, followed by localization; the role of the translator in recreating appeal in different lingua-cultural contexts; what constitutes 'Italianness' in advertisements for British consumers; and strategies for repackaging France as a tourist destination.