available that support all process needs. In particular, medium development should

consider specific requirements of cells to achieve high cell concentrations versus

those relevant to accomplish high virus yields. Furthermore, there is certainly a

need for medium formulations, which can support high cell concentrations at low

volumetric perfusion rates [18].

Regarding DSP, adverse consequences cannot be excluded for intensified viral

HCD processes. This concerns in particular, potential problems with subsequent

unit operations such as filtering or chromatography, i.e., filter blocking or losses in

purification steps, and is directly related to the incremental increase in con-

taminating host cell DNA and proteins with increasing cell concentrations.

However, for processes operated in perfusion mode, high medium exchange rates

after virus infection can mitigate the accumulation of such contaminants in the virus

harvest. For example, Gränicher et al. showed that process intensification in MVA

production had no negative impact regarding cell clarification, host cell DNA re-

moval, and purification compared to a batch process [41]. Furthermore, the ratio of

viral genome copy numbers to infectious virions and antigen glycosylation were not

affected by HCD cultivation [37]. Nevertheless, small-scale studies addressing the

impact of process changes on the performance of unit operations in DSP are re-

quired to avoid virus losses and guarantee that an intensified process will con-

sistently result in a product that meets its predetermined specifications and quality

attributes are to ensure safety and efficacy of vaccines.

6.6

CELL RETENTION DEVICES

The retention of cells inside of the bioreactor is critical to reach high cell con-

centrations. Therefore, the selection of the appropriate retention device including the

corresponding parameters for its operation is of utmost importance. A detailed

overview of various CRDs is given in Table 6.3. For industrial processes, CRDs need

to comply to GMP requirements, should be commercially available at various sizes

(preferably in single-use), combine a high perfusion capacity (at least 1,000 L d⁻¹)

with a high retention efficiency while not damaging the cells, allow high-yield pro-

duction, and operate over a complete run without maintenance [43]. Nowadays, a

large variety of CRDs are available (Figure 6.3). Membrane-based systems such as

spin-filters [32], tangential flow filtration (TFF) [5,44], or alternating tangential flow

filtration (ATF) [37,38,45] are currently the most commonly considered systems for

virus vaccine production. Hollow-fiber bioreactors (HFBRs) [46] follow the same

idea; however, here the bioreactor itself is responsible for the cell retention within the

extracapillary space. Due to the lytic nature of most viruses, their size (up to 350 nm

for MVA), and their surface properties, the usage of membrane-based retention de-

vices has been shown to be challenging [37,38]. Particularly, membrane clogging and

unwanted virus accumulation inside the bioreactor or the modules are well known

drawbacks of most membrane-based retention systems. Alternatively, retention

technologies that make use of density differences for separation can be used. Such

systems do not use a physical barrier and can allow sustainable long-term operation

[43]. Examples for such devices are acoustic settlers [47–50], centrifuges, hydro-

cyclones [51], and inclined settlers [52,53] (Figure 6.3). Moreover, those devices

Process intensification

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