cell growth phase followed by a virus replication phase that is initiated with ad-

dition of a seed virus (see Figure 6.2A). Following intracellular virus replication,

progeny virions are assembled and released. In the case of lytic viruses, this process

is coupled with cell lysis resulting in release of contaminants including host cell

proteins and cellular DNA [29]. For the establishment of HCD cultures with the

respective high cell concentration (>1E07 cells/mL) and subsequent virus produc-

tion phase, the cell growth phase will be extended depending on cell inoculation

concentration and the respective maximum cell concentration envisaged. The most

important parameter is the CSVY, which should not decrease by increasing cell

concentration at TOI. However, the so-called “cell-density effect,” a reduction of

the CSVY at high cell concentrations, has been reported repeatedly for previous

virus production processes [29–34]. While the exact cause for this decrease still

often remained unclear, it can often be related to the accumulation of inhibitory

factors such as ammonia and lactate or a limitation of nutrients [33]. In addition, it

was often speculated that still unknown inhibitors of virus replication would ac-

cumulate [31,33]. Chapter 5 outlined three key factors that are critical to intensify

the production process and maximize the virus yields [29]:

i. Metabolic state of the cells and cell concentration at TOI: Well-

nourished cells with an appropriate growth and metabolic state as well as

a sufficient supply of extracellular substrates is necessary to achieve

TFF

ATF

Spin filter

Centrifuge

Inclined settler

Hydrocyclone

Acoustic filter

HFBR

FIGURE 6.3 Available cell retention devices/options and their connections to a bioreactor

vessel. Gray arrows indicate the direction of flow, blue arrows indicate cooling systems or

other flows (e.g., air for ATF diaphragm pump) with respective flow directions. The symbol

(V) enclosed by a circle represents a pump. Orange dots represent cells. Abbreviations: ATF:

alternating tangential flow filtration; TFF: tangential flow filtration; HFBR: hollow fiber

bioreactor, here the bioreactor itself is responsible for the cell retention, therefore not ex-

plicitly further discussed as cell retention device.

Process intensification

147