Ali, S; (2012) Establishment and evaluation of high cell density fermentation processes using a miniature bioreactor in conjunction with ultra-scale cell recovery tools. Doctoral thesis , UCL (University College London).

Abstract

The use of small scale bioreactors that are mechanically and functionally similar to large scale reactors is highly desirable to accelerate bioprocess development because they enable well defined scale translations. In this study a 25 mL miniaturised stirred tank bioreactor (MSBR) has been characterised in terms of its power input, hydrodynamics and volumetric oxygen transfer coefficient (kLa) to assess its potential to grow high cell density (HCD) cultures using adequate scale-down criteria. Engineering characterisation results showed scale-down, based on matched specific power input (PG/V) and kLa was feasible from the 20 L and 75 L pilot scale stirred tank bioreactors used in this study. In addition, kLa in the MSBR was found to be highest among all three bioreactors suggesting that it was feasible to perform high cell density fermentation in the MSBR. Improvement of mechanical and operational design and optimisation of fed-batch operation resulted in high cell density establishment in the MSBR. Scale-down was performed from 20 L STR to the 25 mL MSBR at matched PG/V, matched kLa and based on dissolved oxygen (DOT>30%) using Fab’ producing E. coli W3110. Comparison of results from the three scaledown strategies shows that matched PG/V was the best strategy and 25 mL MSBR accurately scaled-down the 20 L fermentation performance in terms of growth, Fab’ production and harvest material characteristics at matched PG/V. Scale-down based on dissolved oxygen did not produce reproducible results. Successful scale-down at matched PG/V in the MSBR resulted in maximum cell density of OD600nm~ 114 and total Fab’ concentration of 940 μg/mL compared to OD600nm~118 and 990 μg/mL in 20 L STR. Furthermore, the use of the MSBR in conjunction with primary recovery scale-down tools to assess the harvest material of both reactors showed comparable extracellular viscosity, shear sensitivity and centrifugation performance at both scales when scale-down was performed at matched PG/V. The conjoint use of the MSBR with ultra scale-down centrifugation mimics can provide a cost-efficient manner in which to design and develop bioprocesses that account for good upstream performance as well as their manufacturability downstream. To assess the feasibility of the MSBR to be used for bioprocess development purpose an in-house variant strain of E. coli W3110 co-expressing Fab’ and Staphylococcus aureus nuclease was characterised in 25ml, 20 L and 75 L bioreactor for growth and productivity. Growth and productivity profiles in the pilot scale reactors were successfully scaled-down by 25 mL MSBR when Fab’ fermentation was performed at matched PG/V. Results highlighted in this thesis for successful scale-down high cell density cultivation, use of scale-down recovery tools to predict downstream processibility of the 25 mL harvest, and application of the MSBR for upstream process development of a new strain, suggest that the MSBR can be used for bioprocess development in parallel with pilot scale reactors.

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