Process Development: Maximizing Plasmid Production in Shake Flasks and Bioreactors
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Date
2019-01-29
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Johns Hopkins University
Abstract
A living cell is a biochemical reactor that allows enzyme-catalyzed reactions to produce a remarkable spectrum of resoures in addition to generating more cells (Lim and Shin, 2013). Cell cultures, both–microbial and mammalian, are grown in laboratories and industrial settings to harness these biochemical reactions and collect the purified products. Culturing of microbes is referred to as fermentation in the biotechnology and bio-pharmaceutical industry. There are three broadly categorised modes of growing cells, namely – batch, semi-batch (fed-batch) and continuous. By optimizing the process of culturing cells, optimal growth conditions can be provided to enhance the production of highly valuable products such as recombinant DNA products, antibodies, amino acids, proteins, antibiotics and cells. Recombinant DNA products further offer means to synthesise proteins and hormones such as insulin, growth hormone and interferon (Bollon, 2017). This endows critical significance to devising processes that can be employed to manipulate the growing cells into producing these products. Therefore, optimizing fermentation processes by identifying relevant process parameters, understanding the complex biochemical assimilation of nutrients within the cells and establishing optimum parameter set-point ranges improves the overall yield and productivity. This thesis is a compilation of process development and optimization for batch and continuous fermentation using E.coli cells in the Cell Culture and Fermentation Sciences department at MedImmune (AstraZeneca). A platform process was developed using shake flasks with the aim of improving plasmid DNA yield from E.coli cells. Different process parameters, like pH, dissolved oxygen, carbon source concentration, that influence bacterial growth were tested to identify the limiting factor and determine the optimum growth conditions. The process was optimised through pH correction and confirmed by using statistical analysis. Comparison between the final new protocol and the existing protocol reflected that total production per flask (mg) increased by up to ~ 4.52 ± 0.17 times and total production in 2 days increased by up to ~ 2.26 ± 0.09 times by using half the number of shake flasks. This was followed by developing a pilot fermentation process to attain continuous production of cellular biomass for >20 hours in an agitated bioreactor. MedImmune had an optimised platform fed-batch fermentation protocol using these agitated bioreactors. Based on this fed-batch process, the steady state process was developed for continuous production of cellular biomass that could be used to harvest pDNA.
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Keywords
Fermentation, Plasmid, Process Development, E. coli, Shake Flasks, Bioreactors