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Dissertation
Influence of cultivation conditions on intracellular properties and recombinant protein production
Doctor of Philosophy (Ph.D.), Drexel University
2000
DOI:
https://doi.org/10.17918/00002482
Abstract
Traditional methods of process optimization for animal cells are based on controlling certain changes in the cell environment and not the cellular state itself. For biological systems, basing process optimization on "metabolic state" instead of environmental variables in the bioreactor would be a more solid foundation. Intracellular pH (pHi) is a metabolic parameter that affects many important cellular functions. At present there is no method available to measure pHi under cultivation conditions. In this thesis, an instant and accurate pHi measurement method based on rate of increase in initial fluorescence ratio of BCECF during dye loading is developed. Experimental results of ammonium chloride challenge response of Spodoptera frugiperda (Sf9) insect and Chinese hamster ovary (CHO) cells successfully compared with the previous work. Intracellular pH of growing cultures was measured at bioreactor conditions. The results indicate that pHi is dictated mainly by extracellular pH under cultivation conditions. Cells growing at a higher growth rate maintain a slightly more acidic intracellular pH. Growing cultures took as long as 180 minutes to adjust their pHi in response to external pH changes. This is in contrast to current understanding that pHi adjustments occur in time scales of 1 to 3 minutes. Metabolic studies of CHO cells in pseudo-continuous culture showed that at pHe 7.5, CHO cells utilized glutamine, whereas at pHe 6.8 and 7.1, the growth was mainly on glucose. Fraction of glucose utilized via aerobic pathway decreased form 90% to 40% as dilution rate increased from 0.015 hr-1 to 0.040 hr-1. Specific rate of alkaline phosphatase (SEAP) production increased with increasing growth rate. Skewness of the cell size correlated successfully with specific rate of SEAP production. A fed-batch feeding strategy at maximum growth rate is recommended. A successful sample preparation for matrix-assisted laser-desorption ionization/time-of flight (MALDI-TOF) mass spectroscopy analysis of carbohydrates from glycoproteins was achieved with model glycoprotein ovalbumin. Application of this approach yielded preliminary glycosylation data for reactor samples, which suggests significant further development is needed to link cultivation conditions to glycosylation pattern.
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Details
- Title
- Influence of cultivation conditions on intracellular properties and recombinant protein production
- Creators
- Pinar Ozkan
- Contributors
- Rajakkannu Mutharasan (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xvi, 233 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Engineering (1970-2026); Drexel University
- Other Identifier
- 991021888950104721