Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/15531
Title: Optimization of 293-HEK suspension cultures for adenovirus production
Authors: LEE YIH YEAN
Keywords: 293-HEK; adenovirus; fed-batch; protein-free, chemically-defined media; microarray; metabolic engineering
Issue Date: 19-Sep-2006
Source: LEE YIH YEAN (2006-09-19). Optimization of 293-HEK suspension cultures for adenovirus production. ScholarBank@NUS Repository.
Abstract: 293-HEK has traditionally been the packaging cell line of choice for the production of adenoviral vectors for gene therapy protocols. An increase in demand for these vectors for clinical trials necessitates the need for development of robust and efficient cell culture process for vector production.A low glutamine fed-batch platform was developed for 293-HEK cells with the aim to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. This fed-batch system demonstrated reduction in glutamine and glucose consumption, as well as waste metabolites production, resulting in improvement of maximum cell concentrations attainable up to 6 million cells/ml and enhancement of virus titers up to 3 X 1011 pfu/ml in a protein-free chemically defined media (PF-CDM).Transcriptional profiling was conducted to decipher the cellular regulation underlying the changes observed in the fed-batch process and results revealed a reduction in glutaminolysis, increased electron transport chain activity in the fed-batch, indicative of a more efficient metabolic state, and differences in the cell cycle regulation.Genetic intervention, via an antisense approach, was conducted with the aim to modulate glutamine metabolism without the employment of complicated online fed-batch instrumentations. The compensatory up-regulation of gamma-glutamyltransferase (I?-GT), an alternate glutamine catabolic pathway novel to mammalian cell bioprocessing, when glutaminase was down-regulated was discovered and serves to highlight its importance and emphasize the need for its consideration in future metabolic engineering work.
URI: http://scholarbank.nus.edu.sg/handle/10635/15531
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