Figure 4
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Perfusion was initiated one day after the bioreactors were at full capacity. In perfusion mode, feed solutions were fed to
the bioreactor continuously, and spent media was constantly removed. Running a process in perfusion mode compared to in batch
or fed-batch mode can increase the longevity and cell density of the culture, which in turn increases the overall productivity
of the run. The perfusion rate (percent total culture volume per day) started at 5–12% and increased to >50% by the second
or third day of perfusion. The maximum cell density in the 25/50-L and 100/200-L bioreactor was approximately 9x106 and 5x106 cells/mL, respectively (Figure 4). These cell densities occurred 14 and 6 days after seeding and corresponded with a cell
viability of 65–77%.
Figure 5
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The concentration of glucose was generally 3.0–3.5 g/L. The use of perfusion kept the glucose metabolism product, lactate,
relatively low compared to cultures in shaker flasks. The maximum lactate for the 25/50-L and 100/200-L bioreactor was 1.15
and 1.25, respectively. The level of glutamine in the 25/50-L bioreactor was maintained between approximately 1–2 mM until
day 13 of culture, and the product from glutamine metabolism, ammonium, was generally 3–4 mM (Figure 5). Ammonia production
in the 100/200-L bioreactor was similar, but the glutamine was initially above 2 mM and fell to below 1 mM by day five of
culture.
Perfusate was collected for 11 and five days from the 25/50-L and 100/200-L bioreactors, respectively. The total collected
media volume of 151 L from the 25/50-L bioreactor contained an average IgG concentration of 117 mg/L. From the 100/200-L bioreactor,
320 L of media that was collected yielded an average IgG concentration of 73 mg/L, which correlates to the reduced viable
cell density in this bioreactor compared to the 25/50-L.
The controlled addition of nutrients and resultant lower levels of metabolites allowed for maximum product concentrations
of up to 124 mg/L. This is an improvement of approximately 80% compared to shake flask cultures, which typically reached a
maximum of 22 mg/L.
Chinese hamster ovary
Suspension cells were seeded from spinner flasks into the 25/50-L, 100/200-L, and 500/1,000-L bioreactors at 1x105 –4x105 cells/mL in serum free IS CHO-V (Irvine Scientific, Santa Ana, CA) with additives. Seeding cells at lower densities (e.g.,
6x104 cells/mL) is also possible with no deleterious effects on the cell growth or production. In addition, the cells can reach
much higher densities inside of the Cellbag than a spinner flask. This can most likely be attributed to the increased oxygenation
inside the Cellbag, as well as the ability to control pH. In a spinner flask, the density was maintained between 2x105 –6x105 cells/mL. This was increased to 8x105 –1x106 cells/mL in the Cellbag until perfusion was started, which allows for densities as high as 2x107 cells/mL to be maintained with this cell clone.
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