Once the optimal impeller speed was established, a series of experiments examined the effect of pH (pH 6.8, 7.0, 7.2, and 7.6) on growth and antibody production (Figures 2C and 2D). The optimum pH was in the region of 7.0 to 7.2 where similar growth patterns and antibody titers were achieved. The higher (pH 7.6) and lower (pH 6.8) values resulted in decreased growth rates and viable cell numbers as well as lower antibody titres. Therefore, the pH used for subsequent experiments was pH 7.0.
The penultimate set of experimental conditions examined the effects of temperature (30, 34, 37, and 39ºC) (Figures 2E and 2F). At 34ºC there was a long lag phase and a decreased maximum viable cell number compared to cells grown at 37ºC. However, at 34ºC the cells exhibited an increased stationary phase (from one to three days), extending the duration of the culture by two days.
This data was also examined in relation to antibody production, which exhibited an initial lag period before reaching values similar to that observed with cells grown at 37ºC by day 12. By contrast, cells grown at 39ºC initially grew more quickly than cells grown at 37ºC; however, cells at 39ºC had a decreased maximum viable cell number and decreased antibody production. At 30ºC the cells did not grow, and they simply entered a slow decline phase with a small amount of antibody production. The temperature used for the last experiment was 37ºC.
The final set of experiments tested the effect of maintaining a minimum dissolved oxygen (DO) level (no control or minimum set points of 30%, 50%, 70%) in the bioreactors (Figures 2G and 2H). Following overnight equilibration by sparging of air, initial DO levels in the medium were set at 100%. As the cells grew, the DO concentration in the medium decreased.
The DO concentration was prevented from falling below the minimum DO set point (e.g., 30%) by automatically increasing the oxygen ratio in the gas mixture using the mass flow controllers. In the conditions identified as “no control”, the cells were grown without DO control and, consequently, under these conditions the DO level was allowed to fall without any intervention at a minimum set point.
Increasing the minimum DO concentration (from NC to 70% DO) resulted in increased maximum viable cell numbers (from 1 x 107 to 1.4 x 107) and overall length of culture (from 12 to 16 days). The increased growth and viability with increased DO concentration was not mirrored for antibody production. While the 30% set point improved both growth and productivity, the two highest DO levels (50% and 70%) resulted in decreased antibody titers.
The findings from these experiments led us to define our standard conditions for growth of CHO cell lines as: temperature 37ºC, pH 7.0, impeller speed 80 rpm, minimum dissolved oxygen 30%. (These conditions correspond to the 30% minimum DO set point data [30%] from Figures 2G and 2H).
The data presented in this article defines a standard set of conditions that can be used for the growth of CHO cell lines using the Dasgip Parallel Bioreactor System. These conditions were determined in nine weeks and provide a starting point for the further optimization of culture conditions for specific cell lines expressing different recombinant proteins (e.g., mAb variants).