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Abstract(s)
Current developments in gene therapy and DNA vaccination, plasmid DNA vectors are
becoming increasingly appealing as therapeutics towards a large number of diseases such as cancer, infectious and cardiovascular diseases. This popularity is creating the demand for high quantities of highly purified plasmid DNA which in turn requires the design of high pDNA yield
bioprocesses. However, opposed to recombinant protein production, research on pDNA production is still needed, in order to have a clear comprehension of all the challenges and bottlenecks faced during the production of plasmid DNA (pDNA).
The design of a plasmid DNA production process usually begins with the choice of a suitable culture medium to cultivate the expression system containing the therapeutic plasmid. After defining all medium components, the influence of culture conditions, such as pH, temperature and dissolved oxygen, on biomass and plasmid yields is generally studied. Since
the appropriate conditions for maximizing biomass production and plasmid replication are not usually the same, a compromise solution is usually considered in these cases. When designing a large scale plasmid DNA production process, the employment of a correct fermentation strategy is also necessary in order to improve yields while reducing production costs.
So far, reports on plasmid DNA production in E. coli are focused essentially on the influence of different medium composition, fermentation conditions and feeding strategies on overall biomass and pDNA mass and volumetric titres. Nevertheless, due to the complex nature of microbial growth and the application of several modes of operation such as batch, fed-batch
and continuous cultivations, the constant monitoring and control of pDNA bioprocesses
represents an engineering challenge that should not be disregarded. As a highest yield process may not correspond necessarily to the best fermentation design, the improvement in off-line, at-line and online monitoring techniques should be seen as a crucial task in the design of the
fermentation. Two of the most relevant factors for fermentation performance are the
existence of host cell metabolic stress and plasmid instability; hence, the characterization of cell physiology and plasmid segregational stability has to be considered and monitored during the process.
With this thesis, we attempted to improve plasmid DNA yields while gaining new insights on plasmid DNA fermentation processes through the use of novel monitoring techniques such as flow cytometry and real-time quantitative PCR.
We started this work studying the influence of growth temperature and tryptone
concentration on plasmid DNA production in a previously developed semi-defined medium.
The analysis of pDNA yields and E. coli morphology revealed that higher pDNA specific yields were obtained at higher temperatures (37 and 40 ºC). Also, at these temperatures, E. coli filamentation was observed, possibly indicating a higher metabolic stress due to higher plasmid replication and higher culture temperatures. When analyzing the influence of tryptone concentration on plasmid yield, the best results were achieved with the lowest
tryptone concentration used. The use of limiting tryptone concentrations at a temperature of 37 ºC was shown to be a powerful tool to promote plasmid amplification, keeping the desirable plasmid structure (supercoiled isoform); thus favouring the attainment of product
quality. Our results suggest that by using tryptone alone as an amino acid source, pDNA
amplification was improved, proving that this strategy is able to increase pDNA yield even at small scale.
Since this first study revealed some evidence of E. coli metabolic stress during cultivation, the next task consisted in the development of new flow cytometric methodologies that allowed single cell physiology monitoring during cultivation for a better understanding of cultivation conditions influence in the host metabolic activity. Because one of the parameters
that enable a better characterization of cell metabolic cell as well as population
heterogeneity is cell cycle progression, in the second part of the work, we developed a flow cytometric method to evaluate cell cycle progression in E. coli cultivation using a newly developed far-red dye, DRAQ5. In this study we demonstrated that the use of DRAQ5 as a DNA-specific labelling stain provided an easy assessment of intracellular DNA content and cell-cycle phases in the Gram-negative bacteria E. coli.
Besides the previously reported method for cell cycle analysis, another method for assessing cell viability was implemented using flow cytometry. In this method, a propidium iodide/bis- (1,3-dibutylbarbituric acid)trimethine oxonol (BOX) dual staining was used to distinguish between three different populations: healthy cells (no staining), cells with depolarized
membranes (stained with BOX) and cells with permeabilized membranes (stained with
propidium iodide and BOX). In order to evaluate plasmid copy number (PCN) throughout the fermentation, a real-time quantitative PCR method was developed for absolute plasmid copy number quantification in whole E. coli cells. After developing and implementing these methods, we evaluated the impact of several plasmid DNA induction strategies, such as amino acid limitation, AMP addition and temperature up-shift, on cell physiology and plasmid segregational stability. This study showed that all induction strategies caused cell
filamentation, due to an increase in forward scatter values, and decreased viability at the end of fermentation, as was seen by an increase in the percentage of depolarized and
permeabilized cells. The results also suggest that an amino acid limitation with AMP addition induction strategy resulted in the highest specific yields and, concomitantly, highest PCN values. In conclusion, amino acid limitation-based amplification strategies seemed to be suitable approaches to be implemented at a large scale level since they do not require any additional energy and also had proved to be efficient in plasmid amplification, without causing any detrimental effects in plasmid stability and cellular viability.
The last step of this work aimed at improving plasmid DNA yield through the study of different batch and fed-batch fermentations in bioreactors. Also, the influence of different glycerol and tryptone concentrations and different non-feedback feeding profiles, namely exponential and constant feed rates, on cell physiology and plasmid stability was evaluated
by means of flow cytometry and real-time qPCR, respectively; investigating the potential of these two techniques as valuable tools for bioprocess monitoring and design. The results
showed that all fermentation strategies caused a slight decrease of cell viability at the end of fermentation, being this decrease more pronounced in fed-batch fermentations than in batch fermentations. The time-course assessment of plasmid copy number revealed that PCN values suffered an increase at the end of batch fermentations, which is in agreement with our previous results obtained in batch fermentations performed in shake flasks. However, in fedbatch fermentations, there were pronounced fluctuations in PCN values throughout the fermentations, indicating some plasmid segregational instability. As supposed, fed-batch fermentations with exponential or constant feeding profiles yielded higher biomass and plasmid DNA than batch fermentations with the highest biomass and plasmid yields being obtained with a fed-batch strategy with an exponential feed rate of 0.2 h-1. Notwithstanding
the high biomass (95.64 OD600) and plasmid yields (344.30 mg pDNA/L) obtained, this
fermentation also exhibited higher plasmid instability and lower percentage of viable cells.
This work showed that the fermentation strategy used, not only influences product yield, but also cell physiology and pDNA segregational stability. Furthermore, the new findings described herein draw attention towards the relevance of monitoring bioprocess performance and not just overall biomass and product yields.
In conclusion, in this thesis we evaluated and improved pVAX1-LacZ plasmid production in
Escherichia coli DH5 alpha taking into account not only the overall biomass and plasmid yields, but also considering that cell physiology and plasmid segregational stability, that are two pivotal features to the design and development of these production bioprocesses. In order to study these two factors, several techniques were implemented and were later used to evaluate the influence of several fermentation parameters such as induction strategies and
fermentation strategies on overall process performance.
Description
Keywords
DNA plasmídico Escherichia coli