Lunson, Mary Alice Gallaway; (2021) Establishment of High Cell Density Fed-Batch Microbial Cultures at the Microwell Scale. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

The rate limiting steps of biopharmaceutical process development are clone evaluation and process optimisation. To improve the efficiency of this step, miniature bioreactors are increasingly being used as a tool for high throughput experimentation. At industrial scale, microbial cultivations are usually performed in fed-batch mode to allow for high cell density cost-effective processes; however, many commercially available miniature bioreactors do not have an inbuilt feeding capacity. There are several challenges that need to be addressed to establish high cell density fed-batch cultivation at microscale: attaining high oxygen mass transfer rates, achieving good mixing for the duration of the culture and implementation of an industrially relevant feeding strategy requiring low volume additions. The overall aim of this project was to develop a scale-down fermentation platform suitable for the study and optimisation of high cell density cultures. The first objective of this work was to evaluate options for fed-batch cultures in a commercially available 24-well shaken microbioreactor. To achieve this, two feeding strategies were evaluated using an E. coli strain expressing a domain antibody: in situ feeding by the enzymatic release of glucose from polymeric starch, and direct feeding using a bespoke feed delivery system. In situ feeding was investigated as it is a simple option that does not require a physical method of feed delivery; cellular productivity was enhanced in comparison to batch cultures, however the glucose release was insufficient to sustain high cell density cultures representative of laboratory and pilot scale processes. To enable direct and continuous feed delivery to the microbioreactor a bespoke 3D-printed feeding system was developed that can operate at flow rates of 20μL h-1 and above, and enables up to twelve fed-batch cultures to be run in parallel. E. coli fermentations were performed on complex medium containing glycerol with direct feeding of a 23% w/v glycerol solution initiated at around 18 hours. The second objective of this project was to establish an industrially relevant feeding strategy in the microbioreactor, comparable to a laboratory scale fed-batch process. To this end, the direct feeding strategy was refined in terms of cell growth and product expression; the feed rate and concentration were modified, the DO set point was increased, and a pre-feeding hold period was implemented to allow for consumption of the inhibitory by-products generated in the batch phase. It was found that direct feeding enhanced biomass production by ~70% and product expression by ~2.4 fold in comparison to non-fed cultures. The third objective of this work was to demonstrate the applicability of the new feeding system as a tool for process optimisation experiments. The effect of IPTG concentration and post-induction temperature on product expression was performed using the both the microbioreactor feeding system and the 1L laboratory scale process. The data trends were consistent between scales; product expression was enhanced at a higher post-induction temperature, and IPTG concentration did not affect product expression over the concentration range tested. This demonstrates that the microbioreactor, is predicative of the 1L laboratory scale process terms of sensitivity to change in process conditions The fourth objective of this work was to characterise the microbioreactor in terms of oxygen transfer capability and fluid mixing. To achieve this aim, the volumetric oxygen mass transfer coefficient (kLa) and liquid phase mixing time (tm) of the microbioreactor were determined. The impact of shaking frequency, total gas flow rate and fill volume on oxygen transfer and fluid mixing were investigated and the optimum operating conditions were determined. Within the operating ranges of the miniature bioreactor system, it was found that oxygen transfer was dependant on both shaking frequency and gas flow rate, but was independent of fill volume. The oxygen mass transfer coefficient, kLa increased with both increasing shaking frequency (500-800rpm) and gas flow rate (0.1-20 mL min-1) over the range 3-101h-1; this is at the lower end of the range for conventional stirred tank reactors. It was demonstrated that the miniature bioreactor system is well mixed under the range of operating conditions evaluated. The liquid phase mixing time, tm under non-aerated conditions increased with shaking frequency and decreased with fill volume over the range 0.5-15s. The final objective this project was to demonstrate suitability of the microbioreactor as a scale-down model of an industrial fermentation process. 50L pilot scale, 1L laboratory scale, and 4mL microbioreactor fed-batch fermentations were performed under optimum conditions. The 4mL microbioreactor fed-batch process was shown to better predict the 50L pilot-scale process than the 1L laboratory-scale process based on cell growth, product expression and product quality. This could be explained by mixing and oxygen mass transfer phenomena. At 1L scale, oxygen mass transfer and fluid mixing are most efficient, meaning cell growth and productivity were the highest of the three processes. It appears that the limitations in oxygen mass transfer in the microbioreactor and fluid mixing in the 50L scale vessel, results in a comparable cellular environment, and therefore cell growth, productivity and product quality. In summary, this work has demonstrated the ability to conduct high cell density, fed-batch microbial cultures in parallel, using a shaken miniature bioreactor system. A bespoke, 3D-printed feed delivery system was developed allowing for twelve industrially-relevant microbial fed-batch cultures to be run in parallel. The microbioreactor fed-batch cultures were shown to be predictive of, a 50L pilot scale process in terms of cell growth, productivity and product quality.

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