were introduced. Thus, additional air exchange with the outside of the vessel is

realized and with CO2 incubators the composition of this air can be controlled. These

vessels often require accessory equipment such as incubators or roller bottle racks to

accomplish e.g., temperature and CO2 control.

Multiwell-plates and dishes are used mainly for cell screening and discovery

stages, but also for cell-based assays such as virus titrations. Dishes consist of a flat

bottom and a cap commonly round with surface areas from 8.8 cm² up to 145 cm².

T-flasks are the workhorse for cell culture expansion in laboratories. They come in

different sizes from 25 cm² up to 175 cm² surface. Roller bottles and stacked

multitrays achieve further expansion at low scale (>850 cm²; Table 5.6). Although

some systems, like roller bottles, can sustain medium mixing and gas diffusion, the

lack of control options for pH and pO2 can limit their use. Systems with perfusion

options circumvent these limitations. Beyond tens to hundreds m² scales, adherent

cell culture is carried out in packed-bed reactors, either in fixed or flow mode. In

CelCradle and TideXcell (Esco VacciXell), aeration takes place by the direct in-

termittent exposition of the carriers in the vessel´s headspace that is replenished

with fresh air, created by the alternating up and down media flow. In iCellis (Pall),

CelliGen/BioBLU (Eppendorf), and scale-X (Univercells technologies) vessels the

medium is moved and distributed through the packed bed or sheets. Clearly, at

larger size and volume, manual handling of these vessels becomes difficult and

automation is often available, like harvesting systems that supply reagents and

shake vigorously the vessel for in-situ cell detachment.

For suspension cells, preculture is mainly done in shake flasks. Thus, passaging

and scale-up is promoting the growth of cells that are perfectly adapted to shaken

mode and head space aeration. These cells are then transferred to a STR where they

need to adapt to stirring and (conventionally) additional aeration through spargers.

The gas supplied is either air, a mixture of nitrogen, oxygen and carbon dioxide, or

pure oxygen. In addition, the pH is controlled by adjustment of gases (in particular

CO2) and addition of buffers, acids (HCl) or bases (NaOH or Na2CO3). Thus, for

the cells many parameters change and often this results in a decreased growth rate

after transfer into STRs. Since many years, the impact of this switch is widely

discussed. As alternatives, small-scale Ambr15 cultivation system (15 mL working

volume (wv)) could be used for cell line development and screening under stirred

conditions. Likewise, orbital shaken bioreactors (OSBs) allow to continue culti-

vation in shaken mode up to a 2,500 L scale.

For adherent cells, e.g., growing on Cytodex 1 microcarriers, very low stirrer

speeds are recommended. Especially, if serum-free medium is used that does not

support cell attachment as good as serum containing medium, selection of the

proper stirring speed might be crucial for process performance (see also Figure 5.2).

Typically, 60 to 100 rpm are used to achieve a balance of shear stress resulting in

detachment of cells from the carriers and a high enough agitation to keep the mi-

crocarriers in motion and prevent settling to the bottom. During the initial phase of

cultivations (first 1−3 h), intermittent stirring (stirrer 5 min on, 30 min off) might be

useful to support cell attachment. For suspension cultures, stirring speeds can be

higher depending on the cell line and the cell concentration. Here, 100−200 rpm

are often described. For all cells, together with the aeration mode (head space,

Upstream processing for viral vaccines

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