The technique of culturing mammalian cells on a large-scale is frequently performed in airlift reactors or stirred tank in batches. In order to achieve product concentrations and high cell densities fed-batch processes were introduced. Perfusion systems, where fresh medium enters the bioreactor and the spent medium uninterrupted, were used to extend culturing. Sometimes the medium containing the product is removed from the effluent stream. The important characteristic of a perfusion system is that it retains cell mass in the bioreactor. Compared to fed-batch systems or traditional-batch systems, perfusion systems with smaller bioreactor volumes, permit high cell densities for equivalent yields.
Further, the energy and labor costs are lower because of less frequent bioreactor cleaning and configuration. With this approach, high cell densities are maintained for extended periods of time thereby allowing extended collection secreted product (extracellular) through purge systems or harvest and for continual or occasional cell removal at high density via a purge system that is required for cell collection or cell production of a product (intracellular). Adapting cell lines, which are adherent, to suspension mode for large-scale production can reap additional benefits, e.g., introducing a potential heterogeneous culture conditions and eliminating porous carriers, which are the major cost factor.
However, the suspension-adapted cells that are present pose a challenge since it is tough to retain them in a perfusion system. Simple harvest screens are adequate enough to generate a potential cell-retention system for micro-carrier cultures, while more complex devices are required for creating single-cell suspension cultures.
The characteristic features of an optimal cell-retention device used in suspension systems are defined as follows:
• Cells are retained inside the bioreactor. Cell loss via a retention device should ne minimum in order to maintain cell density and reduce cell debris and lysate in the harvest.
• Cell culture viability should be maintained at optimum levels. Mechanical-sheer stress generated by certain retention devices can negatively affect the mammalian cells.
• The retention device should be minimally polluted (fouling). Cell cultures that are continuously perfused last for several months; the retention device should work through the entire period without any maintenance. In case fouling occurs, the retention device should be exchanged aseptically or cleared easily.
• The retention device should be easily sterilizable and cleanable. A reusable system is preferred to a device that is replaced after every run.
Majority of the cell retention devices, which are commonly used, have fall short in one or many of the properties mentioned above. The adaptability of the retention device is an important factor when it comes to supporting various reactor sizes and operational conditions. Scale-up includes increase in reactor volume and in the perfusion rate; i.e., the retention device should be able to accommodate varying and heavy flow rates. External and internal cell retention devices refer to the positioning of the device; whether inside or outside the bioreactor. The rotating filter, developed from static harvest screen, is an example of internal device.
Internal Retention Devices- Spin Filter
The rotating filter, also called spin filter, is an internal device. Its membrane, made from steel, sometimes materials such as porcelain and polyamide are also used, is attached to the impeller shaft. Since the filter setup in the system has limited use, its ability should be scaled up. Specially designed bioreactors are required by the filter and also, it does not permit membrane maintenance without terminating the fermentation process.
Further, the rotational speed is couples directly to the impeller speed thereby hindering the use of range of rotational rates. Frequent fouling of the membrane is another major problem associated with internal devices. To solve this problem, various approaches are being pursued; draft tubes provide good membrane clearance and pore sizes, which exceed the diameter of the cell.
External Retention Devices
External cell retention devices are designed primarily for acoustic cell filtration, sedimentation, hollow-filter filtration, di-electrophoresis and centrifugation. The simplest external retention devices are the settling tubes used on small-scale reactors. Scale-up is restricted because the settling tubes increase in dimension leading to high-residence times inside the retention device. This decreases viability of the cell because of longer residence times in a non-aerated environment. Cell attachment to the device in regions that have quiescent flow environment also poses a problem. Adherence problem can be prevented by vibrating the settling tubes.
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