(determined by daily measurement) and a pre-defined setpoint

in the range of the glucose concentration in the basal media

(typically between 3 and 10.5 g/L). However, high glucose

concentration could negatively impact culture performances

and appropriate setpoints should be defined based on culture

needs [24].

7. Since many of the agents are based on an emulsion of silicone

beads, sterile filtration is generally not possible. Some can be

sterilized by autoclavation, while others are only applicable for

bioreactor use when purchased in a pre-sterilized (gamma-

irradiated) format.

8. Depending on available equipment and process understanding,

possibilities for automatization and detailed control are almost

endless. Where limitations exist, many functions of a controller

can be substituted by using external equipment like pumps and

scales or placing the bioreactor inside of an incubator. Control

of the DO levels by defined sparging of gas however should be

considered

as

an

essential

functionality

of

any

control

equipment.

9. While often used, control of the culture pH by base addition is

not always required and omitting it reduces stress the cells

experience from increased osmolality [25].

10. During all handling steps involving suspension cells, great care

needs to be taken to properly homogenize the liquid culture so

that the counted and transferred materials are aligned.

11. It is advisable to passage the cells several times after thawing,

not only to obtain sufficient material, but also to give them

time to fully recover. Furthermore, it is essential to maintain

cells in logarithmic growth as both too high and too low cell

densities can negatively influence stability of the cell line and

performance in subsequent steps.

12. At least a two-point calibration of the pH probes should be

performed using reference solutions with values similar to what

would be expected in the bioreactors (usually 7.0  1.0). As in

most systems the calibration is specific to a particular combina-

tion of pH probe and controller, care should be taken to

perform this step for the equipment pair being used during

the run and to label devices accordingly.

13. A two-point calibration of the DO probe will need to be

performed in the bioreactor when the media is equilibrated.

14. When using paper autoclaving bags, double bagging is advised

and probes should quickly be moved to a sterile zone after

removal from the autoclave as wet paper is permeable and

might promote contamination.

12

Ange´ lique Schmid et al.