Continuous centrifugation has been used for biological separations for many years. Implementation of centrifugation for animal cell harvest applications is more recent, as normal-flow depth filtration and tangential-flow microfiltration technologies become less feasible as culture volumes and cell population densities increase. A key benefit of cell harvesting via centrifugation compared with tangential-flow microfiltration is the avoidance of concentration and recycling of the cell mass, leading to membrane fouling and cell lysis.
Among the many different types of centrifuge, the best suited for animal cell separations are the tubular bowl, imperforate bowl and solids-ejecting disc stack centrifuges.
Tubular-bowl centrifuges have been employed in the biotechnology and pharmaceutical industry for many years and are an ideal choice for high value, low solids, cell cultures. The key design feature of the tubular-bowl machine is the high length-to-diameter ratio, which enables long retention times despite the small bowl diameter. Tubular-bowl machines offer lower-shear separation and typically yield drier solids than disc-stack centrifuges. In a tubular-bowl centrifuge the feed enters from the bottom, the solids are retained along the bowl wall and the clarified liquid travels out the top. Paring discs can be used to pump the clarified liquid out of the machine. Once the solids space is filled, the centrifuge must be stopped and taken apart to remove the separated solids. Industrial-scale tubular-bowl machines are capable of achieving g-forces from 13 000-62 000 but are limited to smaller batch sizes due to the manual removal of solids (Letki 1998). The largest tubular-bowl machines have diameters up to 130 mm and operate at flow rates less than 100 l/min with solids capacities less than 10 l (Axelsson 1999). As feed solids increase, tubular-bowl machines become less attractive than solids-ejecting discstack centrifuges.
Also called chamber-bowl centrifuges, the imperforate-bowl centrifuge typically has a cylindrical design in which the height and diameter are nearly equivalent. The classical design also requires manual removal of the solids; however a more recent design employs an automated scraping device to remove solids continually from the bowl during the harvest. As with the tubular-bowl machine, solids are collected at the bowl wall at high g-forces. Once the desired amount of solids has been captured, the bowl speed is reduced and a scraper arm is engaged to sweep the solids into a collection tank below the bowl. After the solids are discharged, the centrifuge returns to the operating speed and the separation cycle continues. This design is noted for its ability to produce extremely dry solids with minimal cell lysis (Betts et al. 2003).
Another adaptation of the imperforate-bowl machine has been tailored specifically for harvesting shear-sensitive animal cells. In this machine, shown in Figure 16.10, the feed tube is precisely centred on the axis of rotation so that the feed is introduced to the accelerator in a region of very low surface velocity, which minimizes shear forces as the cell culture enters the centrifuge bowl. Cells are then concentrated along a gentle path between the core and the bowl wall. The clarified supernatant is continuously discharged under atmospheric pressure, and the concentrated cells are periodically collected at 1 g. This machine is well suited for the collection of viable cells. Capable of operating at very high g-forces, this machine is available for bench to production (10 000 l) scale. Additional features include an automated control system, clean-in-place, sterilization and variable frequency drive for operation at lower g-forces.
Disc-stack centrifuges are among the more efficient separators used in the biotechnology industry, especially for separation of yeast and bacterial cells from the fermentation broth. Although, traditionally, disc-stack centrifuges have been found to cause a significant amount of cell lysis when processing sensitive cell cultures (Berthold & Kemken 1994), recent design changes have provided an opportunity to re-evaluate centrifugation of shear-sensitive cells (Tebbe et al. 1996). These design changes include modifications to the feed inlet zone to eliminate air-liquid interfaces, and incorporation of variable-frequency drives for operation at lower speeds. In addition,
hermetic machine designs in which air is completely removed from the internal centrifuge volume are also available. However, hermetic machines tend to be more complex and contain an additional mechanical seal, which must be well maintained for proper operation.
Regardless of the choice to use a hermetic or non-hermetic machine, cell separation via centrif-ugation can be accomplished with minimal cell lysis with current machine designs. Figure 16.11 shows a schematic of a solids-ejecting disc-stack centrifuge. The separation occurs within the narrow spaces of the disc stack as the solids are captured at the periphery of the bowl and the clarified liquid travels upward to the centre of the bowl. The collected solids are discharged intermittently at the operating bowl speed under extremely high pressures, while the clarified liquid exits the centrifuge through a paring disc. The clarified liquid can then be collected or subsequently filtered using depth-and sterilizing-grade filters depending on the separation requirements.
Disc-stack centrifuges are readily available in sizes ranging from 1000 m2 to greater than 200 000 m2 of settling area. Harvest volumes from a few litres to thousands of litres can be processed with the variety of available machine options. Most production-scale centrifuge systems, such as that shown in Figure 16.12, feature automated clean-in-place and operation modes enabling robust processing and data collection. Although widely used in many industrial applications,
CENTRIFUGATION THEORY AND PRINCIPLES
most centrifuges and accompanying process piping may be manufactured to conform to the specifications required for biopharmaceutical processing, especially highly polished surface finishes and capability for steam sanitization.
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