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Titel
Studying the effects of frequency and amplitude of periodic oxygen-related stress on recombinant protein production in Pichia Pastoris / Gülbahar Cekici
VerfasserCekici, Gülbahar
Begutachter / BegutachterinHerwig, Christoph
Erschienen2014
Umfang72 Bl. : Ill, graph. Darst.
HochschulschriftWien, Techn. Univ., Dipl.-Arb., 2014
SpracheEnglisch
DokumenttypDiplomarbeit
Schlagwörter (DE)recombinant protein production / two compartment bioreactor / cell stress
Schlagwörter (EN)recombinant protein production / two compartment bioreactor / cell stress
URNurn:nbn:at:at-ubtuw:1-67767 Persistent Identifier (URN)
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 Das Werk ist frei verfügbar
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Studying the effects of frequency and amplitude of periodic oxygen-related stress on recombinant protein production in Pichia Pastoris [17.81 mb]
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Dynamic variations of operating conditions in which microorganisms are living and growing influence the performance of the culture. These variations impose stress on cells and can render positive or negative metabolic actions. The aim of this work was to apply oxygen-related stress on the cells of Pichia pastoris periodically by means of a two-compartment system (a single reactor coupled with a Plug Flow Reactor) in order to observe the effects on protein quality and quantity. Specifically we aimed at investigating different amplitude (the dissolved oxygen level in the reactor) and frequency (residence time of broth in Plug Flow Reactor) of stress on cells to understand how the specific productivity, titer and cell physiology change. The results were compared with the reference run that is a production strategy executed in normal single reactor (one-compartment system). The influence of different dissolved oxygen levels in reactor was going to be explored. There were no major differences in the specific productivity and titer (total activity) observed between the experiments, which were made at different dissolved oxygen (DO) levels in the one-compartment system. The analysis showed, that the DO level in one-compartment system does not play an important role as long as the culture is not oxygen limited. However, the results of the two-compartment experiments demonstrated that the protein production can be increased to several times without influencing the physiology of the cells. In the two-compartment system, the dissolved oxygen in the Plug Flow Reactor (PFR) was zero. A multivariable data analysis was performed to check the accuracy of the results, and the analysis reflected that the oxygen-related stress increase the protein quality and quantity. The frequency of the stress (residence time), however, has no influence on these variables. It is assumed that it does not matter how long the cells stay in this equilibrium state however it is important to only experience the oxygen-related stress. Environmental conditions such as temperature, pH, or oxygen content in reactor, play an important role in the cellular production. Therefore, the changes in these parameters can lead to the enormous changes in the cell metabolism. To compare the different setups, such as one-compartment system and two-compartment system, it is necessary to ensure that the cell physiology is the same in both construction. To that end, there are physiological parameters such as biomass yield and carbon dioxide yield (YX/s and YCO2/s), which are determined off-line. The results of Multivariate Data Analysis were also demonstrated that the YX/s and YCO2/s are the same in all experiments, and thus the physiology of the cells remains unchanged. In two-compartment system, the circulation of the fermentation broth between two different zones, the main reactor as homogenously aerated zone and the PFR as oxygen-free zone, caused an oxygen-related stress on the cells. The changes in environmental conditions caused by the periodic circulation led to remain the cells in their active state over the process time and thus to more protein production. The increase in the production of horseradish peroxidase (HRP) in Pichia pastoris due to the circulation was remarkable, and that meant the two-compartment setup should be further developed. Since the DO in PFR is always kept to zero, the critical factors in the decision of an optimal two-compartment system are the DO level in the main reactor (amplitude) and the residence time, which the cells in the PFR spend (frequency). The analysis showed that the high and low level of DO (70% and 5%) influences negatively and the intermediate level of DO (25%) results in two-fold increase in the specific productivity and titer (quadratic effect). The two-compartment system as a novel process technology is a proper tool to apply oxygen-related stress and successfully operates in laboratory-scale bioreactors. Hence, the analysis showed that this new proposed methodology could be implemented in the large-scale systems with less investment costs and more income, which represent crucial factors for biopharmaceuticals.