3.7

Fed-Batch

(Sampling and

Feeding)

1. During the culture, which usually lasts around 2 weeks, sam-

pling is done every day to acquire process understanding and to

allow comparison with other bioreactors (see Note 24).

2. After a few days, some of the nutrients contained in the basal

media are completely consumed by the cells, raising the need to

add new ones. Thus, feed addition typically starts around day 2

to day 4. The appropriate addition strategy should be selected

to supply enough nutrients to cells and allow them to both

grow and produce the product of interest. Overfeeding should

be avoided as it would lead to critical sub-optimal perfor-

mances. Thus, an appropriate balance is required. The starting

time for feed addition, the type and number of feeds as well as

the daily quantities are typically the parameters that need to be

studied to develop an appropriate feeding strategy (see Note

25).

3. Multiple process parameters can be critical to ensure a robust

and efficient production process. Among them, the glucose

concentration is paramount. Indeed, a shortage of glucose is

likely to quickly lead to cell death. This means that specific

control over those parameters might be required.

4. During the culture, foam is likely to accumulate due to the

sparging. Controlling the level of foam is of great importance

to avoid clogging of the exhaust filter (see Note 26).

5. The fed-batch run is terminated either at a particular time-

point, or when a minimal cell viability (typically between

50 and 75%) is reached.

3.8

Harvest and

Clarification

1. When the aim of the bioreactor run is to obtain the (product

containing) culture media, a final step to separate cells and

culture liquid is required. Depending on the scale and equip-

ment availability, this is usually done by centrifugation or depth

filtration or a combination of these. In addition, proper inacti-

vation and disposal of biological material in accordance with

local regulations is essential.

4

Notes

1. Considerations for the choice and optimal operation of a bio-

reactor are numerous and complex and well beyond the scope

of this publication. The aim is to provide a starting point for

testing and optimization. Detailed description of operation

parameters can be found in publications dedicated to this sub-

ject [10] as well as textbooks.

2. Metabolite analyzers of different complexity are available from

several suppliers. As a minimum requirement, a method for the

10

Ange´ lique Schmid et al.