Table 1. Combinations from the 16-component central composite design
Dilution series of selected feed components were performed to check for optimal concentration ranges and toxicity levels.
These experiments were performed in a shaker plate system. A serial dilution was performed from 2x through 0x. The plates
were incubated at 37°C, 5% CO2, and were run for 7 to 9 days. Cells were sampled on days 3, 5, 7, and 9 for cell viability assays. For one set of experiments,
it took one day to set up the experiment; after incubating the plates for 7 to 9 days, significant components were selected
based on cell viability and protein productivity assays. A schematic of the media development strategy is shown in Figure
Sixteen-Factor Central Composite Design
Figure 2. Cell viabilities of the control, run 258, and run 179 in triplicates are shown from day 0 to day 9. The controls
maintained good viability for 5 days and samples 258 and 179 maintained good viability for 7 to 8 days.
Approximately 16 components were selected for feed optimization based on the cell growth curves from feed formulation experiments.
A 16-component central composite design was set up in triplicates in a shaker plate system. Cells were seeded at a density
of 1.85 x 106/mL, and the plates were incubated at 37°C, 5% CO2, and were run for 7 to 9 days. Cells were sampled on days 3, 5, 7, and 9 for high-throughput cell viability and protein productivity
assays. Two of the top combinations from the shaker plate system were selected for shake flask studies based on protein concentration.
The factorial design of some of the combinations is shown in Table 1.
Shake Flask Studies
Figure 3. Viable cell counts of the control, run 258, and run 179 in triplicates are shown from day 0 to day 9. The maximum
viable cell count for the controls was on day 5 and for runs 179 and 258 was on day 6 and day 7, respectively.
Shake flask studies were performed with combinations 179 and 258, as shown in Table 1, to confirm the results from the shaker
plate system. These studies were performed in triplicates along with the controls. The feed components were added according
to the combinations, and the controls were added with the same amount of media but no additional components. This process
was followed to ensure the same volumes in all the experimental shake flasks. The initial seeding density after adding the
components was 1.72 x 106/mL. Cells were sampled on each day up to 9 days. Cell viability and cell counts are shown in Figures 2 and 3. The controls
reached a maximum viable cell density of 4 x 106/mL on day 5 and dropped below 2 x 106/mL with 45% viability on day 6.