factor of 0.64 which can be advantageous for shear stress sensitive cell lines such as

suspension HEK293 cells [75]. Furthermore, often the fact that only one tube is

connected instead of two ports for a TFF system is considered as an advantage.

The biggest challenge for the use of ATF systems are cultivations at very high cell

concentrations. With increasing concentrations, the viscosity of the cell suspension

increases and the pull or compression capacity of the diaphragm pump decreases [76].

Moreover, long-term cultivation can increase the risk for potential adsorption of

media components to the membranes, leading to changes in the medium composition.

However, cell densities used in virus production processes are not too high, yet. As

most of the studies in virus production so far were done in small-scale systems, it is

still to be seen, how both ATF and TFF systems will perform at larger scales.

6.6.4

DISC CENTRIFUGE

Centrifuges use centripetal acceleration to separate particles of greater and lesser

density. Therefore, they can be very efficient for separating cells from culture

media. Centrifuges are commonly applied for harvesting animal cells or as a CRD

in long-term perfusion cultivations [52]. As separation is based on the sedimenta-

tion of cells in a centrifugal field, the separation efficiency is greatly improved

compared to gravitational settling. In the early 1990s, Westfalia Separator AG [77]

and Alfa Laval [63] developed special centrifuges (disk-stack centrifuges and

Centritech®) for continuous cultivations with animal cells. Regardless of the cen-

trifuge type, the main streams of the separator can be divided into three parts, as

shown in Figure 6.7: The feed stream containing cells is pumped from the bior-

eactor into the centrifuge. Centrifugal forces push the cells outward and separate

them as the underflow, which can be pumped back into the bioreactor. The cell-free

supernatant can be harvested via the overflow. The main advantages of using

centrifuges as a CRD for perfusion cultivations are the lack of fouling or clogging, a

tightly controllable separation rate by g-force and feed flow rate, and a separation of

viable and dead cells by adjusting the g-force [17]. As separation occurs under a

centrifugal field, cells are subjected to relatively high shear stress. Another draw-

back, particularly for stainless steel centrifuges, is a certain sterility risk due to the

necessity of an additional waste line for permeate withdrawal. The combination of

fast moving parts with the risk of sealing breakages or gaps further complicate

sterilization procedures.

The disk-stack centrifuges by Westfalia (now GEA Westfalia Separator Group

GmbH) were investigated in two studies for the perfusion cultivation of hybridoma,

HeLa, and CHO cells [77,78]. No negative effects on cell viability and cell growth

were detected despite high angular velocities and flow rates for either study. However,

decreased retention efficiencies were observed by Björling et al. due to clogging in the

concentrate channel [78]. Centritech® centrifuges were originally developed by Alfa

Laval, but are available today from CARR/Pneumatic Scale Corporation (USA) after

several rounds of acquisitions and purchases [19]. They were developed to minimize

shear stress and consist of a disposable insert bag, which is mechanically fixed on a

conical rotor in continuous rotation [19,52]. The cell broth enters the insert bag at one

top end and the overflow is harvested at the other top end. Concentrated cells are

Process intensification

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