Centrifugation

Similar to tangential-flow filtration, centrifugation is utilized for both perfusion and batch cultures. The focus of this section is those aspects of centrifugation that pertain to perfusion cultures. As described in more detail in the later section, centrifugation relies on the density differences between the cells and the medium to achieve cell retention. The key advantage of centrifugation technology for perfusion cultures is the rate at which the conditioned medium can be clarified and cells recycled. Efficient retention of viable cells should be feasible at scales in excess of 3000 l/day. Thus, centrifugation is really the only technology that can accommodate large perfusion rates (>500 l/day). If the centrifuge is oversized relative to the required perfusion rates, the centrifuge could be operated intermittently, allowing for sufficient downtime to perform maintenance between runs.

However centrifugation-mediated perfusion does have some challenges: centrifuges increase the operational complexity and risk of contamination. This can be a particular challenge to long-term perfusion cultures. Additionally, it is possible that the centrifugation process can negatively impact the cell culture. While properly designed centrifuges will minimize exposure to excessive shear and centrifugal forces, by their nature they subject the culture to periodic hyper-concentration. For example, the culture will spend some time in a very dense, probably oxygen-deprived, environment. While this periodic exposure may not affect the culture viability, a decrease in the specific productivity of the culture has been reported (Johnson et al. 1996). Detailed studies with representative cultures and operational conditions are recommended to ensure process robustness.

Kendro Laboratories manufactures two centrifuges designed for perfusion culture: the 300 l/day Lab II unit (Figure 16.4) and the larger 'Cell' unit for clarification up to 3000 l/day. Both centrifuges utilize the 'inverted J' or 'skip rope' technology in conjunction with disposable, aseptic plastic rotor inserts that help to simplify the technique and minimize the risk to aseptic processing. Centrifuge rotational speed, run time, cell concentrate discharge time, flow rates during centrifuge feeding and discharge can all be varied somewhat to tailor the operation. Viable cell retention efficiency, where reported, appears to be excellent (~99 %).

Various investigators have explored disc-stack and tubular bowl centrifuges modified specifically to enable perfusion culture (Tokashiki et al. 1990; Jager 1992: Bjorling et al. 1995; Takamatsu et al. 1996). Reports describing these changes demonstrate the feasibility of very large-scale

Figure 16.4 Centritech Lab II. Reproduced by permission of Kendro Laboratory Products perfusion; however, long-term, high perfusion rate, demonstration may not be reported until this technology and the ability to process such large volumes, is required.

In general, retention efficiency and device fouling are not significant issues for centrifugation-mediated perfusion. However, complications surrounding the need for long-term aseptic processing, the level of maintenance required, and possible effects of culture productivity have yet to be addressed. Appropriately sized and operated centrifugal devices have the potential to retain roughly 99 % of the viable cells, unless lower efficiencies (and lower bioreactor viable cell concentrations) are desired to maximize dead cell passage. Thus, for a standard perfusion culture at 10 X 106 cells/ml, 90 % viable, a conditioned medium with up to 1 X 105 cells/ml would be expected.

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