Silk, NJ; (2014) High throughput approaches to mammalian cell culture process development. Doctoral thesis , UCL (University College London).

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Abstract

Commercial pressures to reduce the costs and timelines involved in bringing new medicines to market are driving investment in methods for high throughput bioprocess development. The establishment of mammalian cell culture processes for therapeutic antibody production is an area of particular interest. This is an experimentally intensive procedure initially involving evaluation of a large number of clones followed by optimisation and scale-up of cell culture conditions. In this thesis a high throughput microwell platform is established for use in early stage cell line selection and cell culture process development. It was first demonstrated that shaken 24 SRW microwells were suitable for batch suspension culture of a commercially available CHO cell line, provided that appropriate culture conditions were selected. However, a high rate of evaporation from the microwells was identified as a potential limiting factor. Further work with an industrial GS-CHO cell line led to the development of a fed-batch method. This used a combination of diluted liquid feeds and a ‘sandwich lid’ to counteract microwell liquid losses by water replacement and reduction of evaporation to negligible levels. This led to comparable cell growth and product formation kinetics as well as similar metabolite utilization kinetics in 24 SRW plates and conventional shake flasks. Engineering characterisation of 24 SRW, shake flask and laboratory scale (5L) stirred tank systems was subsequently performed, encompassing the oxygen mass transfer coefficient (kLa), mean energy dissipation rate (P/V), and liquid phase mixing time (tm). Mixing times in particular showed a strong dependence on the speed and diameter of orbital shaking while in general kLa values were sufficient (>1h-1) for oxygen transfer not to be rate limiting. Consequently it was suggested that matched liquid phase mixing times could be a suitable scale translation parameter for the types of small scale bioreactor formats used in early stage cell culture process development. It was subsequently demonstrated that at mixing times that promoted a homogeneous culture environment at each scale (tm = 5s) similar culture performance in all three bioreactor formats could be achieved using this engineering basis for two distinct cell culture processes involving different GS-CHO cell lines and alternative feeding methodologies. Peak viable cell densities achieved in stirred tank, shake flask and 24 SRW formats were 5.9, 6.7 and 6.4 x 106 cells mL-1 respectively, while antibody titres were 1.23, 0.81 and 0.88 g L-1. The higher IgG concentration in the stirred tank was attributed to tighter on-line pH control. The utilization rates for key metabolites were also closely matched. Finally the industrial relevance of this methodology was demonstrated through the successful parallel fed-batch cultivation of more than 50 GS-CHO cell lines. The rank order of cells based on product titre agreed closely with existing development procedures using shake flasks and static microwell plates. The microwell cell culture process presented here thus offers the potential for a considerable increase in throughput during the early stages of biopharmaceutical product development.

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