demands for sensors and few operative interventions are the main reasons for the

wide use of batch processes in industry. Moreover, high VVP and high nutrient use

can be achieved, thereby reducing the amount of required media. One major

drawback is the large downtime between runs (cleaning, setup of next run, ster-

ilization), which renders such processes often less economical. In addition, to plan,

build, validate, and commission plants for conventional large-scale bioreactor op-

eration in batch mode is costly and time consuming [18]. For this reason, there is a

growing interest in realizing the benefits of fed-batch, perfusion, and continuous

biomanufacturing. In addition, single-use production receives increasing attention.

A first step towards process intensification can be achieved by applying a fed-

batch strategy to increase cell concentrations and virus yields. Here, an initial

growth phase of the cells under batch conditions is followed by a (typically step-

wise) addition of fresh medium or concentrated substrates. This leads to a corre-

sponding increase in working volume and an extension of the cell growth phase as

feeding provides additional nutrients to the cells (Figure 6.2B). This strategy has

advantages over conventional batch production as higher cell concentrations before

infection can be reached, which can result in higher virus titers as well as increased

STY. However, as higher cell concentrations are achieved, accumulation of sec-

ondary metabolites like lactate and ammonia can inhibit cell growth and virus re-

plication. Moreover, medium addition to bioreactors is limited due to restrictions in

the total volume of vessels, aeration, and mixing.

One strategy to overcome most of the limitations of fed-batch processes is op-

eration in perfusion mode. Cell retention devices are used to retain cells in the

bioreactor throughout the run, a continuous renewal of medium without or with low

cell losses can be achieved. After an initial batch phase, the perfusion mode is

initiated where fresh medium is added to the bioreactor at a controlled rate, while

spent medium is simultaneously removed at the same flow rate to keep the bior-

eactor volume constant (Figure 6.2C). As nutrient limitations and accumulation of

unwanted toxic-by products are prevented, high cell concentrations can be reached.

Moreover, longer cultivation times, higher viability, better control of the cell en-

vironment, and higher volumetric yields can be achieved [19]. Depending on the

cell retention device, either co-accumulation of virus together with the cells in the

bioreactor or direct harvesting of the virus material and subsequent cooling in

harvest tanks is possible. Direct harvesting can impact virus stability and with that

virus titer. All in all, compared to fed-batch processes, smaller bioreactors can

be used for perfusion cultivations. This reduces the initial capital costs [19].

Nevertheless, there are some drawbacks to perfusion processes. First, perfusion rate

and cell retention need to be controlled tightly. In addition, efficient aeration sys-

tems have to be used as surface aeration is not sufficient to supply oxygen in high

cell density (HCD) cultures. While microspargers can ensure a high mass transfer

coefficient (kLa) even for low gas flow rates, the generated microbubbles may lead

to foaming problems. On the other hand, macrospargers resulting in larger bubble

sizes might reduce foaming and sparging-related cell death, but require higher gas

flow rates, to supply similar cell concentrations [20]. More on aeration and CO2

control can be found in the previous chapter. Problems related to aeration but also

mixing and pH control are particularly challenging at high cell concentrations with

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Bioprocessing of Viral Vaccines