Versatility of a Single-Use Bioreactor Platform for Culture of Diverse Cell Types - Disposable bioreactors can support growth of bacteria and different mammalian cell types, as well as allow efficient

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Versatility of a Single-Use Bioreactor Platform for Culture of Diverse Cell Types
Disposable bioreactors can support growth of bacteria and different mammalian cell types, as well as allow efficient scale-up.


BioPharm International
Volume 22, Issue 2

The 25/50-L and 100/200-L bioreactors ran at 24 and 20 rpm, respectively, at an angle of 8.0. The 500/1,000-L bioreactor ran at 6.2 rpm and 2.0 angle. The level of CO2 was adjusted to maintain the pH of the culture at 6.9–7.1. Each run began with a mixture containing 10% CO2, which eventually was reduced to less than 1% CO2 added. Additional O2 was added to each run after perfusion was initiated.

Once the cells reached 1x106–2x106 cells/mL, perfusion was started and the volume exchange per day was approximately 75%. Perfusion was started for 25/50-L on day 13. Perfusion was started for 100/200-L and 500/1,000-L on day 8. The reactor was perfused for as long as cell viability was greater than 50%. Once the cells reached approximately 2x107 cells/mL in the 25/50-L bioreactor run, it became more difficult to control the accumulation of lactate and other toxic subproducts, which in turn rendered pH control very difficult. For the 100/200-L and 500/1,000-L runs, cells were removed to maintain a density of 1x107–2x107 cells/mL and lactate levels of 2.2 g/L or less. The volume exchange per day was increased to approximately 100%.


Figure 6
Figure 6 shows that the cell growth and cell densities for each bioreactor were comparable. The viability and cell growth in the 25/50-L bioreactor dropped precipitously and was not recoverable after the cell density reached 3x107 cells/mL, presumably caused by a lack of nutrients and build up of toxic substances, including high levels of lactate in the culture (Figure 7). Hypoxia was not a likely cause for the decreased viability because dissolved oxygen was maintained at ≥73% for each reactor run.


Figure 7.
The glucose and lactate concentrations for each of the runs are similar, with the exception of the glucose spike for 100/200-L on day 9, which resulted from a high concentration added on day 8 (Figure 7). As each bioreactor run progressed and the cell density increased, glucose was consumed and lactate increased. The spikes in glucose concentration in the latter part of each run were caused by additions of 50% glucose stock when the bioreactor glucose level fell below 2.0 g/L.

The 25/50-L bioreactor perfused for 13 days at an average of 12.5 mg/L of protein harvested, while the 100/200-L bioreactor perfused for 18 days at an average of 8.3 mg/L of protein harvested. This was five days longer than the 25/50-L reactor, which resulted in a gain of 6.7 g of harvested protein over those days. The 500/1,000-L bioreactor perfused for 16 days at an average of 12.2 mg/L of protein harvested, which was three days longer than the 25/50-L bioreactor and which resulted in a gain of 16.4 g of harvested protein over those days. Any increase in the viability and longevity of a bioreactor run resulted in a higher yield. The overall protein produced for each reactor was as follows: 25/50-L – 7.8 g, 100/200-L – 21.4 g, and 500/1,000-L – 149.2 g.

This study demonstrated the scaling of a process from 25- to 500-L in less than five months. The flexibility and ease of use of this single-use disposable platform enabled rapid scale-up without any loss in product quality.

PLATFORM VERSATILITY

A disposable bioreactor offers advantages over reusable bioreactors in the areas of cleaning, sterilization, validation, set-up, and turn-around time between runs. We have demonstrated that these disposable bioreactors can support the growth of bacteria as well as different mammalian cell types. Further, systems run with a 25-, 100-, or 500-L working volume are comparable for cell growth, protein production, glucose consumption, and lactate production.

In conclusion, as the bioprocess industry continues its push to fully implement single-use, disposable systems, it is advantageous for facilities to select the simplest, most cost-effective components.

Kristin DeFife is vice president of operations and Leigh Pierce is president at PacificGMP, San Diego, CA, 858.550.4094,


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