protein production use ATF systems [28,73,74]. Vázquez-Ramírez et al. used an

ATF-based perfusion process for the production of MVA using suspension

AGE1.CR.pIX cells, reaching up to 50E06 cells/mL with high infectious MVA

titers around 1E10 TCID50/mL, thus demonstrating the potential of ATF systems

also in virus production [37]. However, several drawbacks limit the usability of

ATF systems, especially for the production of viruses. Due to the lytic nature of the

virus production process and larger sizes of some viruses (100–400 nm), mem-

branes tend to clog. Larger pore sizes in the membrane (currently available pore

sizes: 45 kDa-650 nm) could allow continuous harvesting of virus particles, facil-

itating higher CSVY and virus stability due to shortened RT in the bioreactor.

This was further investigated by Genzel et al. by comparing three different ATF

membrane cut-offs in the range of 45 kDa-650 nm for IAV production at

25E06 cells/mL. Pore sizes of 0.65 μm still not allowed to harvest IAV virus

particles of about 0.2 μm size through the membrane. This clearly illustrates the

need for larger cut-offs or other membrane materials [38]. A recent study performed

by Hein et al. described a novel tubular membrane (Artemis Biosystems), which

allowed 100% of produced virus particles (IAV) to pass through [40]. Finally,

compared to TFF systems, the shear stress generated by ATF systems is lower by a

Feed pump

HF Membrane

Diaphragm

pump

Vacuum and

pressure line

ATF controller

Balance

Weight control

Permeate pump

Spent medium

Feed

medium

1

2

FIGURE 6.6 Schematic illustration of an ATF setup and a hollow-fiber unit. The dia-

phragm pump pushes the liquid in a bidirectional flow direction: 1) Exhaust cycle:

Pressurized air is pressing the diaphragm into the liquid chamber, resulting in a backflush of

the cells and medium into the bioreactor. 2) Pressure cycle: A vacuum pulls the diaphragm

into the liquid chamber, resulting in an inflow of cell broth into the filter unit. Cell-free

supernatant is removed through the membrane pores during the pressure cycle, allowing fresh

medium to be added to the bioreactor. Figure adapted from [ 65].

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Bioprocessing of Viral Vaccines