retained using a manual centrifugation step, followed by partial or total discarding

of supernatant and resuspension in fresh media [19]. Moreover, parallel experi-

ments can easily be performed. The lack of online probes and the limited working

volume usually lead to rigid exchange regimes considering predefined growth or

uptake rates for media exchanges. This rigid batch-wise regime often results in

profiles of (over-)feeding with abrupt metabolite gradients [11], although current

progress around shake flask cultures online monitoring instrumentation might

overcome these hurdles [80]. Nevertheless, semi-perfusion cultures have been

successfully used for perfusion process development for a variety of cell lines and

viruses [36,45,61,65,81]. Foremost, it enabled to verify in a simple manner if

medium and cells can reach higher cell concentrations and if then the CSVY can be

kept as compared to batch mode.

6.6.5

INCLINED SETTLER

Inclined settlers exploit the difference in density between the culture media and

cells by providing a quiescent liquid area in which the cells settle from suspension.

As no physical barrier is used for retention, clogging or mechanical shear stress

does not occur. This allows longer operation times and selective retention of viable

cells (non-viable cells have an approximately twofold lower sedimentation velocity)

[53]. However, the small size and low density of mammalian cells (with 1.03–1.08

g/cm³ around 5% higher than that of the medium) result in low gravitational settling

velocities (1–15 cm/h) [43,53]. This limitation is circumvented by inclined settlers,

which include several closely packed inclined flat plates (lamella), positioned at an

angle to the vertical. The effective sedimentation path and upward fluid velocity is

reduced (Boycott effect), thereby resulting in a more rapid sedimentation [52]. A

simplified overview and description of the inclined settler setup is shown in

Figure 6.8. The cells enter the inclined settler through the lower part of the device

and gravitational forces drive the cells toward the lower surface of each lamella.

Once settled, the cells slide down in a layer towards the plate periphery and are

transferred back into the bioreactor [52]. Supernatant containing small particles

(e.g., non-viable cells or virions) is removed from the top part of the settler. The

main disadvantages are the tendency of cells to adhere to the lamella, low perfusion

rates, and long RTs in un-oxygenated, unmixed environments [19,53]. Applying

special coatings to the lamella and vibrations are commonly used to reduce cell

adhesion [19]. Moreover, by cooling down the cells that exit the bioreactor in a heat

exchanger, sedimentation velocity is increased and oxygen limitations can be

overcome by reducing cell metabolism [82]. Large-scale GMP-compliant inclined

settlers were first developed by Bayer AG [63] in the 1980s and are successfully

used for the production of recombinant blood factors with operation times up to

3–5 months [83]. They also find application in the seed train of fed-batch processes

at scales up to 3,000 L/d [84].

Only few studies investigated the application of inclined settlers for the pro-

duction of viruses. Coronel et al. used an inclined settler-based perfusion process

for the production of IAV in suspension AGE1.CR.pIX cells up to 50E06 cells/mL

with cell retention efficiencies over 96%. No virus retention was observed and

Process intensification

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