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Title: Characterisation of a biosensor surface for use with scale-down technologies in the process monitoring of Escherichia coli antibody fragment production
Author: Perez Pardo, M. A.
ISNI:       0000 0004 5362 8643
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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This thesis illustrates the need to consider overall process performance through the use of biosensors, scale-down and low-volume analytical technologies to achieve processes that account for cell engineering, upstream processing and its effects on downstream processing performance; the upstream/downstream interface. An Escherichia coli fed-batch fermentation expressing antibody fragment (Fab’) was used as a system representative of current manufacturing challenges. Sensor surface characterisation using dual polarisation interferometry (DPI) was explored and a step-wise fabrication and characterisation of a multi-layer DPI based biosensor was carried out. A chemical characterisation of the sensor surface was performed ex-situ using x-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS) to investigate the chemical identity and homogeneity of the chemical layers. The studies uncovered UV/ozone cleaning as the method of choice in comparison with surface acid washing and the use of hydrophilic surfaces as optimal for antibody recognition in comparison to hydrophobic surfaces. The studies highlighted the use of DPI as a sensor characterisation tool. Biological characterisation assessed the potential of the sensor as a monitoring tool. Following characterisation, method development ensued, to quantify antibody fragments via interaction with protein G. The surfaces were assessed through non-specific binding studies and challenged with complex fermentation materials. The characterised biosensors were utilised to assess Fab’ product loss and Fab’ quality during bioprocessing. The biosensor was shown to be sensitive down to 1.7 µg/mL of Fab’ with low interference due to non-specific binding (NSB) and good percentage recovery in 4-fold diluted extracellular broth. The sensor surfaces were then used in conjunction with scale-down tools for primary recovery and low-volume analytical technologies to obtain insights into the interaction between cells from the upstream process and downstream processing operations. For the process studied, the net yield at the end of the primary recovery, which takes into account both product losses during fermentation and centrifugation, was the critical parameter due to the intercellular nature of the product. Studies showed that 30 h was the optimal harvest time with regard to product loss. To benefit from increased product titres at 40 h and above requires that cell strength and integrity be improved. The effect of cell engineering on the growth and productivity of the upstream process as well as its effect on cell integrity at harvest was conducted. This was conducted through the assessment of the growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain. This modification allows the removal of DNA upon cell disruption. The Co-expression of staphylococcal nuclease and antibody product in the same sub-cellular compartment showed no impact on the growth characteristics and Fab’ yield of the host strain. However, introduction of the periplasmic nuclease did perturb kinetics of Fab’ production; increasing the rate of intracellular Fab’ accumulation and concomitantly increasing in the rate of appearance of Fab’ in the external medium. This thesis highlights the need to consider the effects of upstream processing on primary recovery and shows how harvest point determination is a critical process parameter to assess during the design of a bioprocess.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available