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Measurement of bioprocess containment by quantitative polymerase chain reaction

Noble, Michael; (1997) Measurement of bioprocess containment by quantitative polymerase chain reaction. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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This thesis describes the development and application of a method for the measurement of the release of genetically modified micro-organisms from large scale bioprocesses. Polymerase chain reaction (PCR) assays for two E. coli K-12 strains have been shown to be specific for the target strain and have sufficiently low limits of detection (less than 50 cells per PCR) for monitoring of bioprocess release. A quantitative PCR assay, using a competitive internal standard, for one E. coli strain allows measurement of the concentration of the bacteria over a range of up to 6 orders of magnitude with a measurement error of ±0.11 logs. This method has been applied to samples taken from an Aerojet General Cyclone air sampling device allowing the determination of the number of whole cells of the target organism in a sampled aerosol. Using this method, good correlation has been observed between the number of cells released by atomisation into a fixed, contained volume and the number of cells captured and enumerated. Aspects of large scale fermentation, homogenisation and centrifugation unit operations have been studied to determine the effectiveness of their containment. Airborne release of process micro-organisms has been detected in some instances, but the scale of the release was generally found to be small considering the total biomass involved in the bioprocess. Implications of the methodology and the findings from model and case studies on current engineering practice and bioprocess risk assessment are discussed. Areas for further improvement of the method and applications outside of bioprocess containment validation are identified.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Measurement of bioprocess containment by quantitative polymerase chain reaction
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Applied sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10098312
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