Additional ports allow sterile additions and sample withdrawal without the need to place the bioreactor inside a laminar flow
cabinet. A sterile tubing welder is used to attach media, buffer, and glucose stock solutions to the system.
A hemacytometer was used for cell counts, and viability was established by trypan blue dye exclusion. Optical density (OD600) was measured with a spectrophotometer (Bio-Rad SmartSpec 3000). Cell culture biochemistry analysis was conducted on a Bioprofile
300A and 400 (Nova Biomedical, Waltham, MA). The pH was determined online using the Cellbag probe and offline using a PHM220
pH meter (Meter Lab, Lyon, France). Oxygen, CO2, temperature, rocker speed, and angle were determined and controlled through the Wave Bioreactor controllers.
GROWTH AND PRODUCTIVITY
E. coli
The 20/50-L bioreactor was seeded from an overnight E. coli culture in 20 L of Terrific Broth with additives. The culture was maintained for 10 hours at 40–45 rocks per minute (rpm)
and a rocking angle of 11.9–12.0°. A foam trap was attached in place of the outlet filter to avoid clogging from the high
rpm and angle used for bacterial fermentation.
Figure 1
|
The starting OD600 was 0.2, and a maximum OD600 of 19.8 was reached after 9.5 hours (Figure 1) versus shake flasks cultures with maximum OD600 of 2.0 after overnight culture. This strong growth was supported by maintaining the dissolved oxygen at >75% (Figure 2).
Initial glutamine and glutamate concentrations were approximately 6 and 8 mM, respectively, and were reduced to 4 and 3 mM
(Figure 3). Variability (2–10 g/L) in the glucose concentration indicates spiking of the media followed by consumption. Even
with the rapid growth of the bacteria, pH was maintained at an average of 6.75.
Figure 2
|
Therefore, by using the Wave platform, an improvement in bacterial growth compared to shake flask culture of almost 10-fold
was achieved without any optimization of growth media or feeds.
Hybridoma
Figure 3
|
Wave bioreactors were seeded at a cell density of 1–2x106 cells/mL in HyQ ADCF-MAb media (HyClone, Logan, UT). The 25/50-L bioreactor ran at 22.5–24.4 rpm and an angle of 8.0°. The
100/200-L bioreactor ran at 20.1–21.1 rpm and 8.0° angle. The level of CO2 was adjusted to maintain the pH of the cultures at 6.9–7.1. The 25/50-L run required 6% CO2 with the exception of the last day at 4% CO2, whereas the 100/200-L run occurred in the presence of 7–8% CO2. Additional O2 was added to the 100/200-L run on the fourth day after perfusion was initiated.
|
