1.3 Bioreactor Engineering Issues

It is necessary to monitor and control culture parameters such as dissolved oxygen concentration, pH, temperature, and mixing regardless of the process that is used to grow cells. Changes in these parameters can significantly affect the process yield and the stability of product protein.

Optimal growth of E. coli cells and many other microorganisms that are used as hosts (see section on Molecular Biology) for recombinant genes usually require large amounts of dissolved oxygen. Because oxygen is sparingly soluble in water (8.4 mg/L at 25°C), it must be supplied continuously -- generally in the form of sterilized air -- to a growing culture. The air produces bubbles and the stirrer is used to break up the bubbles and mix the content of the reactor. If air flow is inadequate or the air bubbles are too large, the rate of transfer of oxygen to the cells is low and is not sufficient to meet cellular oxygen demand. Thus the fermentors are equipped to monitor dissolved oxygen level of the medium, to transfer oxygen efficiently to the culture medium, and to mix the broth to provide a uniform culture environment.

Temperature is another physiological parameter that is be monitored and controlled. Microorganisms have optimal temperature for growth. If grown at a temperature below the optimum, growth occurs slowly resulting in a reduced rate of cellular production. On the other hand, if the growth temperature is too high, not only will death occur, but in situations where the target protein may be under the control of temperature sensitive promoter, it may be expressed prematurely, lowering product yield.

Most microorganisms grow optimally between pH 5 and 7. As the cells grow, metabolites are released into the medium, a process that can change medium pH. Therefore, the pH of the medium must be monitored and be adjusted by base or acid addition to maintain a constant pH.

Adequate mixing of a microbial culture is essential for ensuring adequate supply of nutrients and prevention of the accumulation of any toxic metabolites within the bioreactor. Although good mixing is easy to achieve at small scales, it is one of the major problems in increasing the scale of bioreactors. Agitation of the broth also affects the rate of transfer of oxygen and heat transfer removal via cooling coils. Excessive agitation can cause mechanical damage to microbial or mammalian cells. Hence a balance must be reached between the need to provide good mixing and the need to avoid cell damage..

The process design should also include factors that make it easy to implement Good Manufacturing Practices. Although most recombinant microorganisms are not hazardous, it is important to design processes that ensure that they are not inadvertently released into the environment. Hence, fail-safe systems should be considered in equipment design and operation to prevent accidental spills of live recombinant organisms and to contain them if a spill does occur. Furthermore, all recombinant microorganisms must be treated by a verified procedure to render them nonviable before they are discharged from the production facility, and the spent culture medium must also be treated to ensure that it does not contain viable organisms and that its disposal does not create an environmental hazard.