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  • 1
    Language: English
    In: BMJ Leader, 3 December 2018, Vol.2(4), p.144
    Description: The role of the general practitioner (GP) is central to the UK National Health Service, with the vast majority of healthcare being delivered in the community. Although a range of policy initiatives aim to address the immense pressures on GPs, the GP workforce in England is struggling to keep pace with demand. GP retention is therefore key.
    Keywords: General Practitioners ; Gps ; Coaching ; Workforce Retention
    ISSN: BMJ Leader
    E-ISSN: 2398-631X
    E-ISSN: 2398631X
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  • 2
    In: Wagstaff, Jane L. and Masterton, Rosalyn J. and Povey, Jane F. and Smales, Christopher Mark and Howard, Mark J. (2013) 1H NMR Spectroscopy Profiling of Metabolic Reprogramming of Chinese Hamster Ovary Cells upon a Temperature Shift during Culture. PLoS ONE, 8 (N/A). e77195-e77195.
    Description: We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero. We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and alanine with both cell lines responding in a similar manner and comparable to previously reported data. However, analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in response to temperature was cell line specific. The significance of our results is presented using principal component analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of metabolites in culture supernatant to improve growth or productivity.
    Keywords: QH301 Biology
    ISSN: 1932-6203
    Source: University of Kent
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  • 3
    Language: English
    In: PLoS ONE, 01 January 2013, Vol.8(10), p.e77195
    Description: We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero. We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and alanine with both cell lines responding in a similar manner and comparable to previously reported data. However, analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in response to temperature was cell line specific. The significance of our results is presented using principal component analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of metabolites in culture supernatant to improve growth or productivity.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 4
    Language: English
    In: Analytical and Bioanalytical Chemistry, 2013, Vol.405(25), pp.8251-8265
    Description: Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) has been exploited extensively in the field of microbiology for the characterisation of bacterial species, the detection of biomarkers for early disease diagnosis and bacterial identification. Here, the multivariate data analysis technique of partial least squares-discriminant analysis (PLS-DA) was applied to ‘intact cell’ MALDI-ToF MS data obtained from Escherichia coli cell samples to determine if such an approach could be used to distinguish between, and characterise, different growth phases. PLS-DA is a technique that has the potential to extract systematic variation from large and noisy data sets by identifying a lower-dimensional subspace that contains latent information. The application of PLS-DA to the MALDI-ToF data obtained from cells at different stages of growth resulted in the successful classification of the samples according to the growth phase of the bacteria cultures. A further outcome of the analysis was that it was possible to identify the mass-to-charge ( m / z ) ratio peaks or ion signals that contributed to the classification of the samples. The Swiss-Prot/TrEMBL database and primary literature were then used to provisionally assign a small number of these m / z ion signals to proteins, and these tentative assignments revealed that the major contributors from the exponential phase were ribosomal proteins. Additional assignments were possible for the stationary phase and the decline phase cultures where the proteins identified were consistent with previously observed biological interpretation. In summary, the results show that MALDI-ToF MS, PLS-DA and a protein database search can be used in combination to discriminate between ‘intact cell’ E. coli cell samples in different growth phases and thus could potentially be used as a tool in process development in the bioprocessing industry to enhance cell growth and cell engineering strategies.
    Keywords: Escherichia coli ; Growth phase ; ‘Intact cell’ ; MALDI-ToF mass spectrometry ; Partial least squares-discriminant analysis ; Biomarker profiling
    ISSN: 1618-2642
    E-ISSN: 1618-2650
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  • 5
    Language: English
    In: Nursing standard (Royal College of Nursing (Great Britain) : 1987), 2011, Vol.25(21), pp.42-6
    Description: In response to the growing challenge of obesity, South Essex Partnership University NHS Foundation Trust and South West Essex Primary Care Trust established a joint initiative to provide a psychological service for adults who were obese and had not been able to manage their weight through traditional methods. This article describes a pilot project providing psychological support for adults with chronic or morbid obesity. It presents the rationale for this approach and describes the service and preliminary outcome data. The implications for nurses working in this field are considered.
    Keywords: Behavior Therapy -- Methods ; Obesity -- Psychology
    ISSN: 0029-6570
    E-ISSN: 20479018
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  • 6
    Language: English
    In: Biotechnology Letters, 2016, Vol.38(4), pp.589-596
    Description: To access, purchase, authenticate, or subscribe to the full-text of this article, please visit this link: http://dx.doi.org/10.1007/s10529-015-2014-y Byline: Evdoxia Gourbatsi (1), Jane Povey (1), Shahid Uddin (2), C. Mark Smales (1) Keywords: Protein formulation; Mass spectrometry; Post-translational modification; Aggregation; Peptide model Abstract: Objectives The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic. Results Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme. Conclusions Peptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins. Author Affiliation: (1) School of Biosciences and Centre for Molecular Processing, University of Kent, Canterbury, CT2 7NJ, UK (2) Formulation Sciences, AKB Building, MedImmune, Granta Park, Cambridge, CB21 6GH, UK Article History: Registration Date: 07/12/2015 Received Date: 05/11/2015 Accepted Date: 02/12/2015 Online Date: 23/12/2015 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s10529-015-2014-y) contains supplementary material, which is available to authorized users.
    Keywords: Protein formulation ; Mass spectrometry ; Post-translational modification ; Aggregation ; Peptide model
    ISSN: 0141-5492
    E-ISSN: 1573-6776
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  • 7
    In: Gourbatsi, Evdoxia and Povey, Jane F. and Smales, Christopher Mark (2018) The effect of formulation variables on protein stability and integrity of a model IgG4 monoclonal antibody and translation to formulation of a model ScFv. Biotechnology Letters, 40 (1). pp. 33-46.
    Description: Objectives: There are a number of blockbuster monoclonal antibodies on the market used for the treatment of a variety of diseases. Although the formulation of many antibodies is achieved in ‘platform’ formulations, some are so difficult to formulate that it can result in an inability to develop a finished drug product. Further, a large number of antibody-inspired or-based molecules are now being developed and assessed for biotherapeutic purposes and less is understood around the required active protein drug concentrations, excipients and additives required in final product formulations. Results: We investigated the effect of formulation variables (pH, buffer composition, glycine and NaCl concentration, time and temperature of accelerated stability studies) on antibody solubility/aggregation and activity using a Plackett–Burman Experimental Design approach. We then used the findings from this study and applied these to the formulation of a single chain variable fragment (ScFv) molecule. Our data shows that prediction of ScFc stability from a model monoclonal antibody could be achieved although further formulation optimization was required. Mass spectrometry analysis confirmed changes to the mass and hence authenticity of both the model antibody and ScFv under formulation conditions that did not provide appropriate conditions for protection of the molecules. Conclusions: The role of the different formulation conditions on maintaining protein integrity is described and using mass spectrometry shows that protein integrity is compromised under particular conditions. The implications for predicting successful formulations for protein molecules is discussed and how antibody formulations could be used to predict formulation components for novel antibody based molecules.
    Keywords: Q Science
    ISSN: 0141-5492
    Source: University of Kent
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  • 8
    Language: English
    In: Biotechnology Letters, 2018, Vol.40(1), pp.33-46
    Description: To access, purchase, authenticate, or subscribe to the full-text of this article, please visit this link: http://dx.doi.org/10.1007/s10529-017-2443-x Byline: Evdoxia Gourbatsi (1), Jane F. Povey (1), C. Mark Smales (1) Keywords: Aggregation; Formulation; Monoclonal antibody; Recombinant biotherapeutic protein; ScFc; Stability Abstract: Objectives There are a number of blockbuster monoclonal antibodies on the market used for the treatment of a variety of diseases. Although the formulation of many antibodies is achieved in 'platform' formulations, some are so difficult to formulate that it can result in an inability to develop a finished drug product. Further, a large number of antibody-inspired or-based molecules are now being developed and assessed for biotherapeutic purposes and less is understood around the required active protein drug concentrations, excipients and additives required in final product formulations. Results We investigated the effect of formulation variables (pH, buffer composition, glycine and NaCl concentration, time and temperature of accelerated stability studies) on antibody solubility/aggregation and activity using a Plackett--Burman Experimental Design approach. We then used the findings from this study and applied these to the formulation of a single chain variable fragment (ScFv) molecule. Our data shows that prediction of ScFc stability from a model monoclonal antibody could be achieved although further formulation optimization was required. Mass spectrometry analysis confirmed changes to the mass and hence authenticity of both the model antibody and ScFv under formulation conditions that did not provide appropriate conditions for protection of the molecules. Conclusions The role of the different formulation conditions on maintaining protein integrity is described and using mass spectrometry shows that protein integrity is compromised under particular conditions. The implications for predicting successful formulations for protein molecules is discussed and how antibody formulations could be used to predict formulation components for novel antibody based molecules. Author Affiliation: (1) 0000 0001 2232 2818, grid.9759.2, School of Biosciences and Industrial Biotechnology Centre, University of Kent, Canterbury, CT2 7NJ, UK Article History: Registration Date: 16/09/2017 Received Date: 01/08/2017 Accepted Date: 14/09/2017 Online Date: 23/09/2017 Article note: Electronic supplementary material The online version of this article (doi:10.1007/s10529-017-2443-x) contains supplementary material, which is available to authorized users.
    Keywords: Aggregation ; Formulation ; Monoclonal antibody ; Recombinant biotherapeutic protein ; ScFc ; Stability
    ISSN: 0141-5492
    E-ISSN: 1573-6776
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  • 9
    Language: English
    In: Journal of Biotechnology, 20 August 2014, Vol.184, pp.84-93
    Description: Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10 L bioreactor model of Lonza's large-scale (up to 20,000 L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10 L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10 L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines’ performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements.
    Keywords: Cell Line Development ; Chinese Hamster Ovary Cells ; Whole Cell Maldi-Tof Mass Spectrometry ; Pls-Da Modelling ; Cell Line Prediction ; Engineering
    ISSN: 0168-1656
    E-ISSN: 1873-4863
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  • 10
    Language: English
    In: International Journal for Parasitology, March 2018, Vol.48(3-4), pp.197-201
    Description: parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on and the development of anti- drugs.
    Keywords: Cryptosporidium ; Cell Culture ; Colo-680n ; Lipidomics ; Proteomics ; Atomic Force Microscopy ; Immunofluorescence Microscopy ; Electron Microscopy ; Biology ; Zoology
    ISSN: 0020-7519
    E-ISSN: 1879-0135
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