maximum virus yields [35]. To achieve high virus titers, cells should be

typically infected at the late exponential growth phase.

ii. Multiplicity of infection (MOI) at TOI: Infection with an optimal

amount of infectious virus particles per cell is necessary to optimize virus

production. This concerns in particular maximum titers and time of har-

vest. As before, ample supply of cells with a corresponding medium is

crucial [35].

iii. Residence time (RT) of the virions within the bioreactor and time of

harvest (TOH): The RT is defined as the time a virus particle (or a cell)

remains inside the bioreactor/CRD and is dependent on the cultivation

mode and CRD. Due to the lytic nature of most viruses, extracellular

contaminations (cell debris, inhibiting metabolites such as lactate and

ammonia, enzymes such as proteases, host cell DNA) continuously in-

crease during the infection phase, leading to variations of pH values and

possible virus inactivation, degradation, or aggregation [29,35]. As a re-

sult, an early TOH and a short RT is beneficial for viral vaccines (e.g.,

live attenuated vaccines, viral vectors), where potency is defined by in-

fectivity. Moreover, the higher the contamination level the more difficult

purification will become.

Perfusion processes involving a complete medium exchange prior to infection

[11,36–38] or a continuous medium renewal during cell proliferation and virus pro-

pagation constitute the foundation for an optimal metabolic state of the cells. In ad-

dition, medium renewal can prevent the accumulation of unwanted inhibitors of cell

growth and virus replication and with that reduce the so-called “cell-density effect”.

Many recent studies investigating HCD processes for viral vaccine production

have successfully demonstrated that the CSVY can be maintained or even in-

creased. Moreover, intensification strategies such as adaptation of various feeding

schemes after infection (e.g., “hybrid fed-batch/perfusion [37]) can further increase

both CSVY and VVP compared to perfusion-only strategies. To shorten seed train

timelines and maximize plant flexibility, inoculation procedures such as FASTEC

(frozen accelerated seed train for execution of a campaign) consisting of HCD

inoculation with disposable cryopreserved bags are increasingly finding application

[39]. The use of online capacitance probes for monitoring the cell concentration/

volume during cell growth and virus production allows for increased process ro-

bustness through improved control of substrate concentrations for example

[11,40,41]. In some cases, signals obtained can be correlated with the time of virus

particle release or maximum virus titers, which could be used to improve virus

harvesting strategies [41,42].

Besides the selection of appropriate process strategies, culture medium is the

most important factor in cell culture technology. The medium selected needs to

ensure all basic cell functions, proliferation, cell survival, and has a direct influence

on the product yield and quality. Compared to approaches requiring genetic en-

gineering of cell lines, media optimization is simple, delivers relatively fast results,

and is reasonably inexpensive at laboratory scale. Nevertheless, for host cell lines

used in virus production, there are currently only a few media commercially

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Bioprocessing of Viral Vaccines