Thesis

Study of transient expression using new technology: Free Style Max system

Advances in molecular biology using different types of cells, vectors, and novel methods of DNA delivery, are paving the way for the development of reproducible bioprocesses that yield large amounts of recombinant proteins. A new mammalian cell expression technology has been applied in this study. The FreeStyle TM MAX Expression System offers the advantages of a completely animal origin-free, large-scale transient transfection system that produces high quantity recombinant proteins. Different combinations of plasmid DNAs and transfection reagents with FreeStyle TM CHO-S and FreeStyleTM 293-F cells were tested in order to identify the combination yielding optimal transfection efficiencies. The use of different culture systems, such as shake flasks, Wave Bioreactors, and Stir Tank Bioreactors, to analyze and compare the cell viability, transfection efficiencies, and expression levels using cultures at 1 liter, 1 0 liter, and 30 liter scales for FreeStyleTM CHO or FreeStyleTM 293-F cells. The results demonstrated that the system was able to achieve over 60% transfection efficiency for GFP expression and over 14 mg/L of human lgG production from 10 liter FreeStyleTM CHO-S cell cultures. In terms of yield, functionality, and fully modified proteins, these results suggest optimal production of proteins in FreeStyleTM CHO requires the use of the FreeStyle TM MAX transfection reagent.. Keywords: Transient expression, mammalian cells, liposome, transfection efficiency, recombinant protein, bioreactor, ELISA

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