Low‐Glutamine Fed‐Batch Cultures of 293‐HEK Serum‐Free Suspension Cells for Adenovirus Production

Abstract

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase

“…At the process level, the apparent deregulation of glucose and glutamine uptake rates when these nutrients are present in excess has led to the implementation of controlled feeding strategies to maintain low but non-limiting levels of the main nutrient concentrations (Europa et al, 2000;Gambhir et al, 1999;Kurokawa et al, 1994;Lee et al, 2003;Haggstrom, 1992, 1994;Omasa et al, 1992aOmasa et al, , 1992bSiegwart et al, 1999;Zhou et al, 1995). Such strategies have proven effective at reducing the overall yield of lactate on glucose, but require on-line monitoring of nutrient concentrations and automatic feed rate adjustment during the culture.…”

Dynamic Modeling of Apoptosis and its Interaction with Cell Growth in Mammalian Cell Culture

“…In standard cell culture media cell densities in the range 1-4 x 10 6 cells/mL have reached (Lee et al, 2003;Merten, 1987;Ozturk and Palsson, 1990a). In other studies, significantly higher cell densities of >10 7 cells/mL have been reported to be achieved for hybridoma cells (Jo et al, 1993).…”

Revisiting Verhulst and Monod models: analysis of batch and fed-batch cultures

“…The cells were cultured in 5L bioreactor (B. Braun, Melsungen, Germany) with working volume of 4 l culture medium (HyQ CHO MPS medium from Hyclone, Logan, UT, USA). Fed batch operation was performed using a modified online dynamic feeding strategy as described by Lee et al (2003). Details of fed batch and set-point control operations can be found in the published work of Chee et al (2005).…”