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Large-scale production is accomplished in large bioreactors, a term that is often used synonymously with fermentor. Industry convention dictates that the term "fermentation" be used for cultivation of single-celled organisms (bacteria and yeast), while "cell culture" is reserved for bioreactor batches of multi-cellular organisms (plants, insects, and mammals).
Mammalian cells are very delicate compared to bacteria. They are heat and shear sensitive, requiring the bioreactor to have strict temperature and agitation control. Low shear equipment must be employed when mixing or moving broth from once vessel to another as care must be taken to avoid destruction of the organisms. Many installations employ nitrogen overpressure when transferring live cells from seed to production reactors to avoid the potential destructive effects of pumps. However, peristaltic pumps are proven to be a mechanically effective means of fluid transport with little danger of contamination. Bacterial cells, in contrast, are very hardy and can withstand vigorous agitation and pumping forces. Rather than strict temperature control, heat removal is the problem, and must be properly accounted for when designing fermentor jackets and temperature control mechanisms.
The growth rate of mammalian cells is very slow compared to bacteria. Depending upon the cell line, it can take more than 10 hours to double the number of mammalian cells in culture compared to approximately 30 minutes for bacteria. Cell culture runs can be from 10 days up to several months. Because mammalian cells express their products outside the cell walls, it is possible to run cell culture in continuous perfusion mode. Cells are kept and regenerated inside the bioreactor while product is continuously withdrawn for several days, weeks, or months.
Specific measures must be taken in the bioreactor design to allow for use in this manner. For example, perfusion reactors usually withdraw product from the top of the bioreactor where the cell density is lowest. They often contain or utilize proprietary settling devices to keep the cells contained within the reactor as product is withdrawn. Continuous perfusion presents the most technically complicated design scenario, as the equipment must be maintained in sterile operation for many weeks, and the product harvest vessels must be kept within the sterile boundary. Many details must be considered when designing for continuous perfusion vs. batch mode, and Parsons and our staff has experience in both types of bioreactor designs, in sizes ranging from 10 liter to 15,000 liter.
Roller Bottles
Some cells do not function well in suspension, regardless, if they are free-floating or attached to microcarrier beads. Certain cells, known as attachment cells, must be attached to the wall of the culture vessel in order to function properly. In these cases, bioreactor cell culture is abandoned in favor of roller-bottle culture. Small 1 to 2 liter roller bottles, with serrated walls to maximize the wall surface area, are used as mini-bioreactors and function much the same way. Unique to roller-bottle culture though are the lack of controls for automated gas sparging and nutrient addition. Each bottle must be manipulated manually (or by automated robot) to change out the harvest supernatant for fresh media. Here, cells continue to grow and multiply, having their own "doubling time," and the number of roller bottles may increase 16-fold as the run progresses.
Facility design for a roller bottle operation differs from a large-scale bioreactor facility only in the layout of the fermentation area. Large-scale bioreactor suites are replaced by warm-rooms in which to keep the racks of bottles and robots (if desired) to manipulate the bottles during a campaign. Downstream purification operations can be treated similarly for each of the two designs; however, roller bottles tend to yield fermentation products requiring more purification steps. Parsons' staff has experience in the design of roller-bottle installations, including the associated robotics.
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