Controlling the Key Parameters of a Bioreactor

Temperature

The majority of mammalian cell lines have an optimal operation at the physiological temperature of 37 °C. A temperature of over 38 °C can quickly have a dramatic effect on the cell viability, while lower temperature can result in a slower cell metabolism. It is important to maintain a homogenous constant temperature in the bioreactor. This is controlled by temperate sensor, a water jacket on the bioreactor, and a temperature control unit (TCU).

The temperature sensor reads the actual process value of the culture medium, then sends a signal to the controller to drive a change to the TCU. The TCU heats or cools down water, or any heat transfer fluid recirculating in the jacket, around the bioreactor tank. Temperature of culture media in the biocontainer equilibrates by contact with the temperature of the jacket.

pH

Typically, mammalian cell lines have an optimal operation at the physiological pH in the range 7.0 - 7.4. Most of the culture medium contains bicarbonate buffer to naturally keep the pH in that range. However, due to cellular metabolisms, cells produce CO₂ and water as they convert glucose into lactate.

Culture medium becomes more acidic during the culture if no action is taken. In the beginning of the culture, pH is regulated in the range 7.0 - 7.4 by playing on the bicarbonate equilibrium. CO₂ is added in the sparger to increase dissolved CO₂ and decrease pH. Alternatively, air is added in the sparger to strip the dissolved CO₂ out and increase pH. When lactate accumulates in the culture medium, addition of air in the sparger is not sufficient enough to increase the pH.

A basic solution like NaOH or N_a2C_O3 0.5 – 1 M may be required to be pumped in the bioreactor. The addition of air, CO₂ or basic solution is automatically managed by a controller which compares signal measure by the pH probe inserted in the bioreactor with the defined setpoint for the process.

pO₂

O₂ is sparsely soluble in culture media and quickly consumed by cells. It is continuously added by sparging air, a mix of air and O₂, or pure O₂ into the bioreactor via the sparger, which is usually located below the impeller.

The level of dissolved oxygen (DO) is monitored by a sensor. Most mammalian cell cultures are performed with a DO of around 20 - 50% of the saturation with air. Automatic addition of air/O₂ is managed by a controller, based on the reading difference between a process value of the DO sensor directly submerged in the culture, against the desired setpoint that a specific process is set to. When the sensor reads a value above the setpoint, nitrogen can be added in the bioreactor through the sparger to strip the oxygen out of the culture medium.

An alternative and more common method is to let cells consume the oxygen until reaching the setpoint. When the process value is below a setpoint, air and O₂ blend are supplemented to increase the process value for DO back to the setpoint.

Agitation

The agitation system typically consists of an impeller, a drive mechanism (magnetic or direct) and a motor. The goal is to deliver a power input into the culture medium to generate efficient mixing, and to get a homogeneous distribution of the temperature, DO and pH inside the bioreactor.

The impeller type, size and location as well as the design of the sparger are critical factors to guarantee homogeneity while limiting shear stress effect due to hydrodynamics and aeration and their potential effect on the cells and the process.   

Pressure

As most single-use biocontainers are made of flexible plastic, they cannot resist overpressure of few hundred millibars for a long period of time. Pressure is monitored by using a single-use probe inserted inside the biocontainer.

A gas addition interlock strategy is put in place if pressure rises resulting from a misuse of the bioreactor, such as a closed clamp or blocked vent filters due to an excessive foaming, for example.