(between 2–3 MHz), and the acoustic power is the only internal parameter that can

influence the retention efficiency. Frequencies below this range can lead to cavitation

resulting in cell damage and additional heating of the medium. External parameters

that can affect the separation efficiency are the duty cycle, flow rate, backflush fre-

quency, and recirculation rate [86]. As described before, duty cycles are the stop times

of the acoustic filter and harvest pump, allowing the transfer of aggregated cells back

to the bioreactor. Even though these duty cycles have little influence on the acoustic

settler performance, it was shown to be crucial to increase IAV yields [50]. To avoid

nutrient depletion at high cell concentrations during the cell growth phase and virus

production phase, high flow medium withdrawal rates through the acoustic filter need

to be achieved. However, this results in a lower separation efficiency as more cells

will be washed out. This decrease in separation efficiency could be mitigated by

increasing the acoustic power input. However, this leads to increased heat production

and increased temperature in the acoustic chamber. Alternatively, the backflush fre-

quency, which describes the number of times the acoustic chamber is cleared of

sedimented cells per hour, could be enhanced. But this would also lower the se-

paration efficiency. The last external parameter, the recirculation rate, describes the

flow rate with which sedimented cells in the acoustic chamber are recycled back into

the bioreactor [86].

Acoustic settlers have no physical barrier and no moving parts are required for cell

retention and are, therefore, less susceptible to mechanical failure and fouling [88].

Moreover, they are easily cleaned and sterilized-in-place further facilitating their

integration in perfusion processes. Maximum perfusion rates of 10, 50, 200, and

1,000 L d⁻¹ can be achieved over long periods at a high cell retention efficiency and

with high cell viabilities [63]. A fully integrated virus production process was recently

established by Gränicher et al., who used an acoustic settler to reach HCD and to pre-

clarify the virus containing harvest for subsequent in-line purification steps [41].

However, several issues limit the usability of this CRD: The power required to

generate the standing wave field is associated with a heating effect, which can

damage cells and virus particles. Solutions such as air cooling and water circulation

have been implemented for lab and pilot scales (200 L), but remain an issue for

larger scales [19]. Same as for other CRDs, acoustic filters are external devices,

where cells in the external loop are exposed to uncontrolled conditions and nutrient

gradients for short periods. Dalm et al. described significant oxygen concentration

gradients for recirculation pump rates smaller than 6 RV d⁻¹ [88]. If the acoustic

settler is used for HCD cultivations, more cells will be in the settling chamber. Due

to the compactness and aggregation of the cells at the nodal pressure planes, high

cell concentrations could lead to an attenuation of the acoustic wave, which could

weaken the standing 3D wave, resulting in a lower retention efficiency [86].

Compared to other CRDs, operation of acoustic filters seems slightly more complex

and less “plug-and-play,” requiring specially trained staff and constant maintenance.

6.7

USE OF DISPOSABLES

Single-use bioreactors (SUBs) have been adopted into several cell-culture−based

production systems including commercial vaccine production (also described in the

Process intensification

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