Newton, Joesph Matthew; (2018) Understanding cell lysis in fermentation and its impact on primary recovery using viscosity monitoring. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

The high level of innovation in drug discovery in recent years has presented a significant challenge for drug manufacturing process development, which must constantly evolve to meet this increasingly diverse demand. As a result, novel process monitoring technologies to rapidly optimise these processes, reduce development costs and improve time to market are in high demand in the biopharmaceutical industry. Within the context of bioprocess development and manufacturing, the main focus of this work is on fermentation and its impact on, and interaction with, primary recovery. Although E. coli is the most widely researched host organism for recombinant protein production and cell death during fermentation has been observed for decades, very little is understood about how to quantify and detect cell lysis in late stage fermentation, which leads to a number of problems in the downstream process such as product loss and poor operational performance. The complex nature of the cell broth means that it is difficult to observe lysis directly, and current analytical technologies are unable to rapidly and accurately monitor the shift between optimum intracellular product concentration and leakage to the cell broth. This thesis proposes that by monitoring the physical properties of the cell broth, i.e. by monitoring the viscosity, it may be possible to indirectly infer cell lysis, as the release of intracellular content, such as host cell protein and nucleic acids, to the cell broth at the point of lysis are known to cause an increase in the broth viscosity. In this thesis, cell lysis was first characterised in an industrially relevant E. coli fermentation producing antibody fragments (Fab'), using a range of common analytical techniques. Following this, a method has been developed to rapidly detect cell lysis and product loss using at-line viscosity monitoring, and a strong correlation was shown to exist between DNA release, product leakage, cytotoxicity and viscosity. Viscosity monitoring to detect cell lysis was shown to perform better than optical density measurements and online capacitance probes, and could detect lysis faster than HPLC, flow cytometry, cytotoxicity assays and DNA quantification. Subsequently, a model has been developed to quantify cell lysis using rapid viscosity monitoring. Viscoelasticity studies have also been performed to provide novel insight into changes in cell strength during fermentation. Finally, a case study has been carried out to demonstrate an application of viscosity monitoring in process development, and to enable insight into the impact of upstream processing conditions on the efficiency of downstream unit operations. A novel process design using crossflow filtration and flocculation achieved a 2.53-fold improvement in total product recovered, a 3-fold improvement in solids removal and a 3.6-fold improvement in product purity, in comparison to the existing Fab' primary recovery process. This work presents the novel use of viscosity monitoring in biopharmaceutical fermentation to rapidly detect cell lysis and product loss. In doing so, a deeper understanding of changes in the physical properties of cell broths during fermentation has been obtained, as well as insight into the impact of lysis on various primary recovery unit operations. The use of viscosity monitoring to rapidly detect lysis and product loss has been shown to be a promising analytical tool to enable optimisation in process development and facilitate harvest decision making for large scale operation.

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