Betts, JPJ; (2015) Incorporation of Developability into Cell Line Selection. Doctoral thesis , UCL (University College London).

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Abstract

The pharmaceutical industry is under increasing pressure to deliver new medicines quickly and cost effectively; traditional small molecule product pipelines have dried up and companies are increasingly investing into biopharmaceuticals. To date, the most successful biopharmaceuticals have been monoclonal antibodies. The ability to construct common manufacturing platforms for a range of antibody products has underpinned this interest. Antibodies are most often produced as heterologous proteins at large scale in stirred tank reactors. However, at manufacturing scale there is limited opportunity to undertake process development and optimisation. If a manufacturing process can be ‘scaled down’ experiments could be carried out at much greater throughput and occur in parallel throughout the entire product lifecycle. In creating a small scale model, the fundamental challenge lies in accurately recreating the engineering environment experienced at large scale in order to yield process relevant data. In this thesis a miniature, single use, 24-well shaken bioreactor platform was investigated as a small scale cell culture device. This plate format can operate either using direct (REG plate) or headspace sparging (PERC plate) i.e. with either the presence or absence of a dispersed gas phase. Initial work involved the experimental and theoretical characterisation of the novel, miniature bioreactor (7 mL) and the conventional stirred bioreactors (1.5 L), themselves mimics of pilot scale GSK cell culture processes. Under typical operating conditions in the miniature bioreactor, measured mixing times were 0.8 – 13 s and apparent kLa values in the range 5 – 50 hr-1. Based on these findings, cell culture kinetics were investigated. A methodology for consistent, parallel cell cultures was first established and then used to determine the impact of the dispersed gas phase on culture kinetics of a model CHO cell line. Cultures performed with head space aeration showed the highest viable cell density (15.2 × 106 cells mL-1) and antibody titre (1.58 g L-1). Final cell density in the PERC plate was nearly 40 % greater than shake flask cultures due to the improved control of process conditions. In contrast, cultures performed with direct gas sparging showed a 25 – 45% reduction in cell growth and 40 – 70 % reduction in antibody titre. The platform nature of the system was confirmed with similar findings obtained using a second antibody and cell line cultured under different conditions. The miniature bioreactor was then investigated for use as an early stage, cell line selection tool. A strong positive correlation between PERC and shake flask data was found (0.88), indicating the suitability of the platform for this application. In contrast, selection results in the REG plate format differed notably, highlighting the fact that the presence of a dispersed gas phase can significantly alter cell culture kinetics; and potentially cell line selection. A panel of four CHO clones was then investigated alongside bench scale bioreactors, operating at matched mixing times; the REG plate format provided the most comparable match in terms of cell growth and product titre. Primary recovery studies investigated use of a small scale depth filtration tool to analyse material generated previously with regards to ease of processing. Data showed that cells cultures in the presence of a dispersed gas phase yielded the most accurate prediction of primary recovery data. Subsequently, detailed product quality analysis confirmed consistent product quality attributes across the different cell culture formats. In summary, this work shows the utility of miniature bioreactor systems for high throughput strain selection under process relevant conditions.

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