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Currently, a large number of gene therapy applications make use of retroviruses as vehicles for gene delivery [1], since retroviruses provide permanent integration of foreign genes in cellular DNA [2] and, in contrast to other viral delivery systems, stable helper cell lines for their production are available [3]. However, several factors are limiting the commercial production procedures and wider clinical applications of retrovirus mediated gene delivery. The most relevant are the low titers obtained in culture, which seldom rise above 106 infective particles per milliliter and the low retrovirus stability not only during culture but also during downstream processing and storage [4]. This stresses the need for integrated optimization since degradation will occur in all steps of the production process. Nonetheless, few protocols and systematic optimization methods have been published. For this purpose in the present work the production of retroviruses was mathematically modeled in order to optimize the controllable intermediary steps between helper cell growth and final retrovirus titer. [ABSTRACT FROM AUTHOR]

BHK-21 cells expressing a rhIgG-IL2 fusion protein were grown under different nutrient conditions in a continuoussystem. At very low glucose (<0.5 mM) or glutamine (<02 mM) concentrations, a shift towards an energetically more efficient metabolism was observed. Cell specific productivity was maintained under metabolically shifted growth conditions and at the same time a constant energy state was observed. No significant differences in the oligosaccharide structures were observed from the rhIgG-IL2 obtained under the different metabolic states. Only neutral diantennary oligosaccharides with or without core α1-6-linked fucose were detected that carried no, one or two Galβ1-4 linked galactose. The data obtained point to the presence of the classical NeuAcα2-3Galβ1-3GalNAc structure for O-linked oligosaccharides that are present in the IL-2 moiety of the protein. [ABSTRACT FROM AUTHOR]

Activation of the constitutively expressed interferon-regulatoiy-factor-1/estrogen receptor fusionprotein (IRF-1-hER) in BHK cells was accomplished through the addition of estradiol to the culture medium, which enabled IRF-1 to gain its transcriptional activator function and inhibit cell growth. With the addition of 100 nM of estradiol at the beginning of the exponential phase, IRF-1 activation leads to a rapid cell growth inhibition but also to a significant decrease in cell viability. To apply this concept in industry, strategies to extend the stationary phase are required. Cycles of estradiol addition/removal were performed in 2 L stirred tank bioreactor operated under perfusion in order to reduce the time span of estradiol exposure by slow dilution after a step addition of 100 nM estradiol (perfusion rate between 0.7 and 1.4 day−1). Cell growth inhibition of the regulated/non-producer clone was achieved for three consecutive times, showing that the cells were able to respond to estradiol addition independently of the cycle. Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM lead to cell growth and viability recovery, independently of the perfusion rate used. These observations led to the definition of strategies to operate IRF-1 regulated cells by pulse estradiol addition followed by the longest possible period withestradiol and by the fast perfusion to low estradiol concentration. The response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with a regulated/Factor VII producer clone, where the time of estradiol exposure and perfusion rate were varied. This clone presents a stronger response to IRF-1 activation without increase on factor VII specific productivity after cell growth inhibition; clearly indicating that the stationary phase obtained is clone dependent. The final conclusion is that it is possible to modulate the IRF-1 effect by the manipulation of cycles of addition/removal of estradiol. [ABSTRACT FROM AUTHOR]

A genetic approach based on the activation of interferon-regulated-factor-1 (IRF-1) has been applied to regulate BHK cell growth. The presence of 100 nM estradiol in the culture medium in agitated flasks leads to the activation of the constitutively expressed IRF-1/estrogen receptor fusion protein (IRF-1-hER) and inhibits cell growth. Two days after estradiol addition cell concentration was still maintained but a significant decrease in cell viability was observed. The IRF-1 activation clearly interferes with the cell energetic metabolism, since there is an extra metabolic activity, with higher glucose, glutamine and oxygen consumption rates, while the yield lactate/glucose is maintained. Although there is a higher proteolytic activity, the protein content per cell increases significantly, suggesting an overall increase in the protein synthesis. A significant increase in the lactate dehydrogenase enzyme activity and a higher reduction of the MTT were also observed, indicating the increase in cell activity after cell growth inhibition. ATP, ADP and AMP contents were evaluated, and the cells were able to maintain the energy charge (EC) higher than 0.8. The significant increase in the total content on protein per cell can be the reason for the higher energetic needs. Thus, in order to maintain the EC the cells need to increase the ATP production, i.e., increase the catabolic metabolism. Nevertheless, 2 to 3 days after estradiol addition this equilibrium is no longer stable, leading to an increase in the ADP and AMP contents, and at that time the cell viability decreases and the other alterations previously referred start to be significant. The IRF-1 activation leads to a significant increase of the cell activity until the cell is no longer able to maintain its metabolic equilibrium and starts to die. [ABSTRACT FROM AUTHOR]