Guy, HM; (2015) Microscale characterisation of a manufacturing route for lentiviral vectors. Doctoral thesis , UCL (University College London).

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Abstract

Lentiviral vectors used in clinical trials are currently produced by transient transfection of adherent human embryonic kidney (HEK)293(T) cells. However, this approach is not scalable and for commercialisation the development of alternative strategies based on suspension-adapted producer cell lines, that have all genes for vector production stably integrated, is desired. To assist progress in this area, the aim of this thesis was to establish a microscale cell culture platform that enables key bioprocess design data to be acquired rapidly and cost-effectively. ProSavin®, an equine infectious anaemia virus (EIAV)-derived lentiviral vector developed for the treatment of Parkinson’s disease (Palfi et al., 2014) was used as a model system. First, the suitability of a shaken 24-well plate system for the suspension culture of HEK293T-derived producer cells was established. This system was shown to support equivalent cell growth and ProSavin® titres to conventional shake flasks while providing substantially greater opportunity for parallelisation. Second, the utility of the microscale platform when combined with statistical Design of Experiments (DoE) techniques for optimising titres and informing the design of a scale-up strategy was demonstrated. An initial screening experiment identified three parameters as having a critical influence on ProSavin® titres, which were post-induction period, liquid fill volume and concentration of doxycycline (inducer compound). Subsequent optimisation experiments defined operating ranges for these parameters. Third, the insights obtained during the microwell investigations were shown to aid successful scale-up of the ProSavin® process to a single-use 2 L WAVE bioreactor. Fourth, with a view to informing further improvements in process design, the half-life of ProSavin® and other EIAV-based lentiviral vectors was determined, and approaches to moderate the rate of decay during upstream processing trialled. Overall, it was concluded that the microwell platform may be viewed as an effective tool for future use in the development of lentiviral vector bioprocesses.

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