Wiegmann, Vincent; (2021) Characterisation of a parallel microbioreactor system and its application to accelerate cell culture process development. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Recent advancements in the field of microbioreactor technologies have transformed early- and mid-stage process development. Microbioreactor systems benefit from monitoring and control capabilities akin to larger scale bioreactors that have been realised at a much smaller working volume, thereby promoting the quick accumulation of process knowledge early on in the development timeline. Furthermore, the control of key process parameters at the small scale led to improved scalability when compared to traditionally used systems such as shake flasks and microtitre plates. Ultimately, the successful implementation of microbioreactor systems in a bioprocess development workflow can reduce the time to market and decrease the price of biotechnology products. This thesis sought to investigate whether the micro-Matrix microbioreactor (Applikon-Biotechnology BV) is a suitable instrument for cell culture process development. The system is based on a shaken 24 deep square well microtitre plate format with a working volume between 2 - 5 mL in which each well can be individually controlled for temperature, pH, and dissolved oxygen (DO) concentration. The system was first characterised to gain a better understanding of the cultivation environment within each well and to identify a suitable scaling criterion to a reference benchtop-scale bioreactor system. Mixing times were found to range between 1 – 42 s, while the volumetric mass transfer coefficient (kLa) was 2.4 – 240.8 h-1. Computational fluid dynamics was used to derive the power input, which was found to range between 4 W m-3 – 765 W m-3. Initial cell cultivations revealed considerable evaporation rates and well-to-well variabilities, which were successfully addressed by establishing a method for the periodic replacement of evaporated liquid. Mixing time was identified as suitable scaling criterion and a GS-CHO fed-batch process was scaled down from a reference stirred tank reactor (STR) with a working volume of 5 L to the micro-Matrix system. A problem specific to pH controlled small- and benchtop-scale bioreactors was highlighted, where the removal of CO2 from the cultivation broth is very efficient and eventually CO2 is stripped out completely. A low CO2 concentration was shown to negatively affect the maximum viable cell concentration in either system. A combination of matched mixing time and matched minimum CO2 fraction in the inflowing gas was therefore proposed as suitable scaling criterion. Scalability of growth and production kinetics as well as antibody glycosylation were demonstrated between micro-Matrix and the 5 L reference STR system. Subsequently, the micro-Matrix was used for the rapid optimisation of a GS-CHO feeding strategy. First, several bolus, continuous, and dynamic feed addition strategies were compared and bolus feeding was shown to be sufficient for the cell line under investigation. With the help of response surface methodology, the bolus feeding regime was optimised, which led to a 25.4% increase of the space-time yield and a 25 % increase of the final titre. Following a highly replicated validation of the results in the micro-Matrix, the optimised feeding strategy was scaled up to the 5 L STR system and shown to yield equivalent results. In the final chapter of the thesis, the focus was shifted to an emerging application for small-scale process development systems by investigating the effect of the cultivation environment on the growth and differentiation of the primary T cells. Initially, a perfusion-mimic process was identified as suitable mode of operation, necessitated by the need to repeatedly replenish nutrients and remove waste metabolites. Using this perfusion-mimic process, pH, DO, and shaking speed setpoints were then investigated as part of a full-factorial design and explored for their effect on growth kinetics and differentiation of primary T cells. The numerical optimisation established a locally optimal final cell concentration and differentiation profile for a shaking speed of 200 rpm, a pH of 7.4, and a DO of 25%. In summary, this work demonstrates the utility of the micro-Matrix microbioreactor in two bioprocess development applications and provides a toolbox and framework to aide with the implementation of the micro-Matrix in further bioprocessing applications.

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