At F Diaphragm Pump Cycle

PRESSURIZATION CYCLE

EXHAUST CYCLE

AIR FLOW OUT

AIR FLOW OUT

HOLLOW FIBER MODULE

FLUID FLOW TO PROCESS VESSEL

FLUID FLOW FROM PROCESS VESSEL

Figure 16.2 Schematic of the Alternating Tangential-Flow Device from Refine Technology (reproduced from Furey (2002) with permission of Genetic Engineering News Inc).

HOLLOW FIBER MODULE

FLUID FLOW TO PROCESS VESSEL

FLUID FLOW FROM PROCESS VESSEL

Figure 16.2 Schematic of the Alternating Tangential-Flow Device from Refine Technology (reproduced from Furey (2002) with permission of Genetic Engineering News Inc).

Figure 16.3 AppliSense 50L acoustic filter. (Reproduced with permission from Applisense product literature.) NB: Note the vertical lines of trapped cells. Liquid flow is vertically out of the resonance chamber.

on the tops of the plates; as with the acoustic filter, cells are then returned to the bioreactor via periodic flow reversals or via an air purge of the system.

These technologies offer the advantage of being able to operate indefinitely without fouling, which is a significant benefit for long-term cultures at high perfusion rates and/or high cell population densities. However, viable cell retention of these devices is highly dependent on the culture conditions and is generally less than 100 %. Perfusion rate is generally inversely related to retention efficiency for both units. Additionally, since the cells settle on the inclined plate and may not be returned to the bioreactor for several hours, it is possible that this suboptimal environment may negatively impact the culture viability or specific productivity. More frequent settler back flushes with air or conditioned medium can help mitigate this effect, but at the expense of a higher forward flow rate, which may reduce retention efficiency. Some investigators have successfully utilized chilling of the withdrawn culture in external settlers to slow cellular metabolism and reduce this environmental effect (Searles et al. 1994).

CELL RETENTION IN PERFUSION CULTURE

For acoustic filters, cavitation and temperature control (particularly at large scale) are concerns. Cavitation is more likely to be an issue at low cell population density, with serum-free cultures, and at high power settings. Larger-scale units such as the BioSep 200L are equipped with jackets to facilitate temperature control. The quality of the particulate removal for both devices will be influenced by the bioreactor conditions, the desired perfusion rate and sizing of the device, and the operational settings (for the acoustic filter). With gravitational settlers, careful control of temperature throughout the settler is critical for avoiding convective currents, which can lead to separation problems and reduced retention efficiency. The retention efficiency of both technologies will suffer if there is any gas bubble entrainment. For the acoustic filter, separation efficiency can be optimized to some extent by adjusting the power applied to the resonance chamber, the forward and stop/reverse run times, the recirculation rate in the loop, and the temperature of the chamber.

Acoustic filters capable of approximately 200 l /day are currently available from Applisense (Applikon). A 1000 l/day device, which is essentially a bundling of five 200 l/day units, is currently under development. Gravitational settlers have been used successfully at the 100-500 l/day scale with moderate retention efficiencies, but are typically custom-made and are difficult to scale further. For both acoustic filters and gravitational settlers, appropriately sized and operated devices may be expected to retain ~90 % of the viable cells. Considering a typical 90 % viable culture with 10 X 106 cells/ml, this would yield a conditioned medium with up to approximately 1 X 106 cells/ml.

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