their high metabolic demand. Resulting heterogeneous cell populations (due to e.g.,

changes in cell size, cell aggregates, syncytia) can cause problems that require

specific process control strategies to maintain optimal conditions for consistent

virus production [20]. In combination with long operation times, technical failures

are more likely to occur. As a consequence, operating staff needs to be specially

trained. Another important point to consider is the use of media. Compared to

recombinant protein production, there are only a handful of commercially available

media for virus production. Even fewer media are optimized for cultivations per-

formed at low perfusion rates to minimize media consumption. Furthermore, the

very high virus titers that can be achieved in intensified processes might need ad-

ditional biosafety measures.

Alternatively, viral yields can be increased utilizing true continuous production

systems such as chemostats [21] or two-stage systems [22]. In a chemostat, new

medium is supplied continuously, while consumed medium, virus, and cells are

harvested. Here, the feed and harvest have the same flow rates to enable a constant

working volume. In this system, the applied dilution rate (D) determines the specific

cell growth rate (µ); hence, the cells are maintained in a steady-state condition (D is

equal to µ). However, if the applied D is too high and approaches the maximum

specific cell growth rate (µmax), cell washout occurs, which has to be avoided.

Chemostats can only be used for the production of non-lytic viruses as lytic viruses

do not allow continuous cell growth. For lytic viruses, cell growth and virus pro-

pagation have to occur in separated vessels, which can be realized with a two-stage

continuous stirred tank reactor (CSTR) cultivation system. Here, cells are grown in

the first stirred tank bioreactor (STR) (operated as a chemostat) under steady-state

conditions and continuously transferred into a second STR, where virus propagation

takes place. The application of a two-stage CSTR was already reported for the

production of baculovirus [23–25], poliovirus [26], influenza A virus (IAV) [22],

and modified Vaccinia virus Ankara (MVA) [27]. For the genetically stable virus

MVA, the production in a two-stage CSTR showed a higher concentration of vir-

ions produced compared to batch mode starting at 25 days of process time [28].

However, for IAV, the production led to periodic oscillations in virus titers, de-

creasing the VVP significantly compared to batch cultivations [22]. This was

mainly due to the accumulation of defective interfering particles (DIPs, see previous

chapter). This limits the usability of continuous processes for the production of IAV

and other viruses spontaneously generating DIPs. Further limitations comprise the

required high qualification level for technical staff and the increasing risk of con-

taminations due to the complexity of the setup and the prolonged process time.

A simplified overview of the most important parameters of all operation modes

is given in Table 6.2. In summary, perfusion systems seem to be the most promising

option to satisfy the ever-growing demand for viruses. As higher cell concentrations

compared to batch and fed-batch processes are reached, higher VVP can be at-

tained. As a result, smaller-sized equipment might be used, thereby reducing fixed

costs and correlated peripheral costs e.g., facility, clean in-place operation, down-

times. Combined with today’s trend to utilize single-use technology, the small

footprint of perfusion systems is seen as an enabler for this development [18]. For

viruses that only have very low CSVY such as HCV or flaviviruses, bioreactor

Process intensification

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