Incorporation of Developability into Cell Line Selection

The pharmaceutical industry is under increasing pressure to deliver new medicines quickly and cost effectively; traditional small molecule product pipelines have dried up and companies are increasingly investing into biopharmaceuticals. To date, the most successful biopharmaceuticals have been monoclonal antibodies. The ability to construct common manufacturing platforms for a range of antibody products has underpinned this interest. Antibodies are most often produced as heterologous proteins at large scale in stirred tank reactors. However, at manufacturing scale there is limited opportunity to undertake process development and optimisation. If a manufacturing process can be ‘scaled down’ experiments could be carried out at much greater throughput and occur in parallel throughout the entire product lifecycle. In creating a small scale model, the fundamental challenge lies in accurately recreating the engineering environment experienced at large scale in order to yield process relevant data. In this thesis a miniature, single use, 24-well shaken bioreactor platform was investigated as a small scale cell culture device. This plate format can operate either using direct (REG plate) or headspace sparging (PERC plate) i.e. with either the presence or absence of a dispersed gas phase. Initial work involved the experimental and theoretical characterisation of the novel, miniature bioreactor (7 mL) and the conventional stirred bioreactors (1.5 L), themselves mimics of pilot scale GSK cell culture processes. Under typical operating conditions in the miniature bioreactor, measured mixing times were 0.8 – 13 s and apparent kLa values in the range 5 – 50 hr-1. Based on these findings, cell culture kinetics were investigated. A methodology for consistent, parallel cell cultures was first established and then used to determine the impact of the dispersed gas phase on culture kinetics of a model CHO cell line. Cultures performed with head space aeration showed the highest viable cell density (15.2 × 106 cells mL-1) and antibody titre (1.58 g L-1). Final cell density in the PERC plate was nearly 40 % greater than shake flask cultures due to the improved control of process conditions. In contrast, cultures performed with direct gas sparging showed a 25 – 45% reduction in cell growth and 40 – 70 % reduction in antibody titre. The platform nature of the system was confirmed with similar findings obtained using a second antibody and cell line cultured under different conditions. The miniature bioreactor was then investigated for use as an early stage, cell line selection tool. A strong positive correlation between PERC and shake flask data was found (0.88), indicating the suitability of the platform for this application. In contrast, selection results in the REG plate format differed notably, highlighting the fact that the presence of a dispersed gas phase can significantly alter cell culture kinetics; and potentially cell line selection. A panel of four CHO clones was then investigated alongside bench scale bioreactors, operating at matched mixing times; the REG plate format provided the most comparable match in terms of cell growth and product titre. Primary recovery studies investigated use of a small scale depth filtration tool to analyse material generated previously with regards to ease of processing. Data showed that cells cultures in the presence of a dispersed gas phase yielded the most accurate prediction of primary recovery data. Subsequently, detailed product quality analysis confirmed consistent product quality attributes across the different cell culture formats. In summary, this work shows the utility of miniature bioreactor systems for high throughput strain selection under process relevant conditions

High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017

Impact of aeration strategies on fed-batch cell culture kinetics in a single-use 24-well miniature bioreactor

The need to bring new biopharmaceutical products to market more quickly and to reduce final manufacturing costs is driving early stage, small scale bioprocess development. This work describes a comprehensive engineering characterisation of a novel, single-use 24-well parallel miniature bioreactor system. Cell culture performance is also investigated, with particular focus on the aeration strategies adopted at this small scale (7 mL) either by headspace sparging alone or by direct gas sparging into the culture medium. Apparent volumetric oxygen mass transfer coefficient (kLa) values ranged between 3–22 h−1 and 4–53 h−1 for headspace aeration and direct gas sparging respectively. The higher kLa values with direct gas sparging correlated directly with the increase in gas–liquid interfacial area per unit volume. Mixing times (tm) over a range of conditions were generally in the range 1–13 s and decreased with increasing shaking frequency (500–800 rpm). Direct gas sparging also served to reduce tm values by a factor of up to 19 fold. The impact of aeration strategies on cell culture kinetics of a model CHO cell line was also determined. Cultures performed with head space aeration alone showed the highest viable cell density (VCD) (15.2 × 106 cells mL−1), viability retention and antibody titre (1.58 g L−1). These were greater than in conventional shake flask cultures due to the improved control of the μ24 bioreactor system. In all cases the miniature bioreactor managed good control of process parameters such as pH 6.95 ± 0.4, temperature T°C 37 ± 0.4 and DO% 57 ± 32. Cultures performed with direct gas sparging showed a 25–45% reduction in VCD (depending on the aeration strategy used) and a similar reduction in antibody titre. Overall this work shows the successful application of the miniature bioreactor system for industrially relevant fed-batch cultures and highlights the impact of the dispersed gas phase on cell culture performance at the small scale

Bacterial adaptation is constrained in complex communities

© 2020, The Author(s). A major unresolved question is how bacteria living in complex communities respond to environmental changes. In communities, biotic interactions may either facilitate or constrain evolution depending on whether the interactions expand or contract the range of ecological opportunities. A fundamental challenge is to understand how the surrounding biotic community modifies evolutionary trajectories as species adapt to novel environmental conditions. Here we show that community context can dramatically alter evolutionary dynamics using a novel approach that ‘cages’ individual focal strains within complex communities. We find that evolution of focal bacterial strains depends on properties both of the focal strain and of the surrounding community. In particular, there is a stronger evolutionary response in low-diversity communities, and when the focal species have a larger genome and are initially poorly adapted. We see how community context affects resource usage and detect genetic changes involved in carbon metabolism and inter-specific interaction. The findings demonstrate that adaptation to new environmental conditions should be investigated in the context of interspecific interactions

A simple method to determine evaporation and compensate for liquid losses in small-scale cell culture systems

Objectives: Establish a method to indirectly measure evaporation in microwell-based cell culture systems and show that the proposed method allows compensating for liquid losses in fed-batch processes. Results: A correlation between evaporation and the concentration of Na + was found (R 2 = 0.95) when using the 24-well-based miniature bioreactor system (micro-Matrix) for a batch culture with GS-CHO. Based on these results, a method was developed to counteract evaporation with periodic water additions based on measurements of the Na + concentration. Implementation of this method resulted in a reduction of the relative liquid loss after 15 days of a fed-batch cultivation from 36.7 ± 6.7% without volume corrections to 6.9 ± 6.5% with volume corrections. Conclusion: A procedure was established to indirectly measure evaporation through a correlation with the level of Na + ions in solution and deriving a simple formula to account for liquid losses