5.6.2.1

Temperature

Typically, cultivation temperature is set to 37°C (<±1°C)). Decreasing to 32−34°C

at TOI can help to increase virus yield as enzyme activities of cellular proteases are

reduced and the produced virus can be more stable. Furthermore, oxygen supply

will change.

5.6.2.2

pH and CO2

The pH set point is equally important. There will always be an optimal pH for cell

growth, one for virus production and one for enzyme activities such as trypsin or

other important enzymes for virus binding for example. Thus, different pH regimes

during the different process phases might be accepted. Typically, the pH value is

controlled in the interval of 7.2−7.4. Lactate and ammonia produced by the cells

exert the major changes in cell culture supernatant, which are buffered preferably by

a CO2/bicarbonate system. For this buffer system, there is a very fragile balance

between aeration rate, CO2 addition, choice of sparger, stirring speed and head

space aeration that needs to be fine-tuned for each filling height of the STRs.

However, also for static bioreactors in CO2 incubators, the CO2 and NaHCO3

buffering has to be considered, as pH decreases and increases occur when a T-flask

is taken in or out of the incubator, respectively. Equilibration of fresh medium in the

incubator might take as long as 1 hour Increasing CO2 set point of the incubator

from e.g., 5 to 10% can reduce the pH by up to 0.5.

To increase the pH value often either CO2 supply is decreased or a base is added.

Typically, this base is NaHCO3. Addition of more NaHCO3 to a medium that already

contains up to 12 mM will increase the pH sharply (up to 1 log unit). In contrast, with

NaHCO3 concentrations exceeding 12 mM, further addition of NaHCO3 will not

increase pH significantly anymore. Often, NaOH is used as an alternative for pH

control, but it is not well tolerated by many cell lines. Thus, both options should be

evaluated at small scale. Intracellular pH is often overlooked but of great relevance for

cell physiology. As long as the extracellular pH is above 7.2, the intracellular pH

value is typically lower than the extracellular value [54].

5.6.2.3

Shear Stress

Cells, but equally virus particles, are sensitive to shear stress. Shear stress can be

understood as a force applied tangentially on a fluid element at rest over a static

surface that deforms the fluid element parallelly through planes slippage. Newton’s

first law relates the velocity gradient and the shear stress as follows [55]:

u

y

=

x

(5.1)

where

[Pa] is the shear tress,

[Pa.s] the dynamic viscosity and ^u

y

x [s−1] the

velocity gradient. The resulting flow has a characteristic velocity profile. Two

general flow regimes are distinguished depending on the hydrodynamic properties

of the fluid and the flow conditions. Low shear stress conditions are found in la-

minar flow regimes, where the fluid flows in layers in one direction. Increasing

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Bioprocessing of Viral Vaccines