towards a lower temperature) can be useful with a shift to 34 or

32

C

at a pre-defined timepoint being a widespread

approach [21].

2. The ability to inject gases in response to the demands of the

culture is one of the main advantages of using a bioreactor.

Injection of (a combination of) air, oxygen, and CO2 into the

headspace serves to adjust pH of the media, limit foam accu-

mulation and may fulfill the initial oxygen requirements of the

cells. It is typically not sufficient to do so once higher cell

densities have been reached. Instead, gas needs to be injected

directly into the culture medium. Spargers of different design

exist with open pipe-, macro- (such as a ring sparger) and

micro-spargers being the most popular ones. The choice of

sparger needs to be made based on the oxygen requirements

of the cells, sensitivity to bubble bursting, and potential equip-

ment limitations (capacity of the gas source). Micro-spargers

provide significantly better aeration due to the much higher

total surface volume of the large amount of small bubbles.

Conversely, this results in higher stress for the cells (see Note

18).

3. Stirring is important to ensure homogeneity of the culture and

to avoid the formation of gradients within the bioreactors.

Stirring also has an impact on shear stress. The appropriate

agitation rate needs to be determined based on the system.

Engineering parameters such as the maximum tip speed, mix-

ing time, and the power input per volume can support the

selection of these parameters [22].

4. Establishing a regulation cascade for dissolved oxygen ensures

that enough of this key substrate for growth and production is

available to the cells independent of their density and metabo-

lism. Typical setpoints are between 20 and 50% (compared to

concentration in the medium equilibrated with air under nor-

mal pressure). This parameter is typically controlled through

agitation rate and/or injection of air or oxygen through the

sparger. The cascade needs to be well defined to ensure the

setpoint can be met and to avoid spikes (see Note 19).

5. Active or passive pH regulation can be used. Cell culture pH is

commonly between 6.7 and 7.5. With a passive approach, pH

of the culture is usually dropping down during the first few days

due to the generation of acidic by-products. Shifts in metabo-

lism and addition of feeds then typically limit a further drop in

pH. Active pH control injecting CO2 in the headspace or in the

culture medium or removal of CO2 using sparging (in case of

small adjustments) or acid/base addition can be used to main-

tain pH at a desired setpoint. The impact of the latter on the

osmolality of the culture should not be overlooked.

8

Ange´ lique Schmid et al.