Concentrated feed medium M1 test in shake flasks. In the concentrated feed medium study, the original concentration is designated as 1X. The 1X medium was used as a control. Feed media with concentrations of 1X, 1.30X, 1.60X, 1.75X, 1.90X, and 2.50X were prepared by adding 10 N NaOH to increase the pH and dissolve all of the components into the solutions. The higher the feed concentration, the more the base needed for solubility, resulting in an increase of pH from pH 8.58 for 1X medium to pH 10.41 for 2.50X medium, as shown in Table I. The osmolality of the concentrated feed media was increased from 835 mOsm/kg of 1X medium to 2432 mOsm/kg for 2.50X medium. To test if a surfactant can increase the solubility of medium components without increasing the medium pH, 2.50X concentrated feed media with various percentages of PS80 or higher levels of P188 were made. The data show that these media required less NaOH compared to the 2.50X concentrated medium, resulting in a lower final medium pH and lower osmolality (see Table I).
The concentrated feed media described previously were used in the fed-batch culture of cell line A. The feed volumes are shown in Table I. Nutrients were added to the cell culture for the concentrated feed media at the same quantities for the 1X feed medium. The productivities on the last day of the process (day 14) showed that the 1.60X medium led to 12% and 20% increase of the titer and specific productivity respectively, which is significantly higher than the 1X medium control (p < 0.05). The 2.50X medium had the lowest productivity (see Figure 1a), suggesting that there was an upper concentration limit of feed media prepared by raising the pH, which was due to the lower cell viability at higher cell culture pH and osmolality (data not shown). Among the surfactants evaluated, the addition of 0.5% (w/v) P188 in 2.50X medium increased the titer and specific productivity by 15% and 25%, respectively, while the feed volume could be reduced by 2.5-fold. More favorable culture pH and osmolality profiles from the 2.50X + 0.5% P188 feed medium have been observed as these are closer to the 1X control condition (data not shown). The 2.50X + 0.5% P188 feed medium also led to 42% and 116% increase in the final titer and specific productivity of cell line B respectively (see Figure 1b).
