2. HPLC (high performance liquid chromatography)-grade elu-

ents (i.e., purified water).

3. Anion exchange chromatography column (e.g., Supelcogel-

column (Agilent, Vienna, Austria)).

4. All fed carbon sources and all residual metabolites in solid/

liquid form for quantification.

(a)

In this case, feeding is conducted with lactose and glyc-

erol. In addition, glucose and galactose, resulting from

hydrolyzed lactose, need to be quantified. Furthermore,

any metabolite potentially accumulating (e.g., acetate)

needs to be prepared as a standard reference.

5. Syringes and 0.2-μm syringe filters.

2.4.4

Product

Determination

This section is highly dependent on the expressed target protein

and must be conducted according to established analytics. In case

of intracellular product formation, a high-pressure homogenizer is

required (e.g., PANDA+ 2000, GEA, Biberach, Germany).

3

Methods

3.1

Cultivation Setup

1. All bioreactor and preculture cultivations are carried out using

the same medium composition to avoid deviations. We recom-

mend a defined chemical medium, to reduce process distribu-

tions (e.g., DeLisa et al., 1999, [42]).

2. Cultivation setup depicted in Fig. 1.

3. For the adjustment of the correct dilution rate, dip-pipes need

to be adjusted to the required height in the bioreactors:

(a)

Calculate the desired residence time (or dilution rate)

given the applied volumetric flow in the reactor. Dilution

rate can then be adjusted either (i) via volumetric flow or

(ii) via volume in the reactor; e.g., 100 mL/h is the

volumetric flow in the reactor and the residence time is

desired at 10 h. Hence, 1 L operating volume is required

for continuous cultivation.

(b)

Fill reactor with desired volume prior to sterilization and

apply process parameters (e.g., 1,400 rpm and 2 vvm).

(c)

Make sure reactor contains all probes and pipes at this

stage, which could influence the liquid level in the reactor.

(d)

Set immersion pipe to adequate height and mark at the

reactor side in case deviations might occur during

sterilization.

4. For continuous cultivation, constant volume can be adjusted in

two ways:

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Julian Kopp and Oliver Spadiut