Large-scale transient gene expression (TGE) in mammalian cells is an attractive method to rapidly produce recombinant proteins for pre-clinical studies, with some processes reported to reach 100 L. However, the yield remains low, hardly over 20 mg protein/L, mainly because the current TGEs have been performed at low cell density (approximately 5 x 10(5) cells/mL). In this study, the strategy to improve TGE focuses on facilitating transfection at high cell density. A high-density perfusion culture of 293 EBNA1 cells was established in 2-L bioreactor using Freestyle 293 expression medium (Invitrogen, Singapore) to grow the cells for transfection. Transfection was then carried out at 1 x 10(7) cells/mL using polyethylenimine (PEI) as DNA carrier, at the optimized conditions of 6 microg DNA/10(7) cells and 1:3 DNA to PEI mass ratio. During the post-transfection phase, 80.8 mg/L of the model protein, EPO was obtained at day 5.5 post-transfection (130 mg total EPO production) using a fed-batch culture mode. In comparison, perfusion cultures using an enriched SFM II medium resulted in a longer post-transfection production phase (8 days), and 227 mg of EPO was produced in 10.7 L medium, showing that high-density TGE enables the production of several hundreds of milligrams of protein in a 2 L bioreactor. In addition, a protocol for economical plasmid preparation based on anion exchange was also established to satisfy TGE's demand in terms of quality and quantity. To the best of our knowledge, this is the first report of transient transfections at a high cell density of up to 1 x 10(7) cells/mL.
A high-concentration medium was developed for the high-density cultivation of Chinese hamster ovary (CHO) cells to produce tissue plasminogen activator (tPA). First, the effects of the concentrations of several medium components on the growth and metabolism of CHO cells were studied particularly in relation to the serum concentration, in order to develop a suitable concentrated medium. High concentrations of serum, glucose, lactate, and glutamine and a low ammonia concentration increased the specific growth rate and decreased the specific tPA production rate. Among these components, the effect of the serum concentration was the most significant. There was an inverse correlation between the specific rates of cell growth and tPA production. Using a medium containing glutamine at twice the concentration of the original medium (F-12) allowed the medium volume to be reduced to seven-ninths that of the original, without any reduction in tPA productivity. When the F-12 medium was supplemented five-fold with amino acids and vitamins, the total amount of tPA produced, specific rate of tPA production, and yield of tPA per unit volume of medium employed were respectively enhanced 1.7, 1.5, and 3.6 times. As a consequence, the frequency of medium exchange could be reduced. The results strongly indicate that the supplementation of nutrient amino acids and vitamins to a medium with a low concentration of serum are the most critical factors for cost-effective tPA production in high-density cell cultivation.
During the hydrolysis of an exopolysaccharide (EPS) produced by Citrobacter sp., the maximum liberation of hexosamine was obtained with 6 M HCl at 115 degrees C in an autoclave for 1 h. The glycosidic bond energy and degree of acetylation of the hexosamine in EPS were approximately 77 kJ mol(-1) and 61%, respectively. Thermal destruction of the hexosamines and the effect of salt on the hexosamine determination could be minimized under the optimized hydrolytic conditions. Using a modified Elson-Morgan method, maximum total hexosamine concentration was determined to be 3.2 g l(-1) (29% of crude EPS) after 96 h of fed-batch culture.
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