Laminar shear is the primary mechanism of cell damage, limiting flow rate (and hence flux) in crossflow microfiltration of animal cells. Sensitivity to hydrodynamic and interfacial stress is reduced by the addition of 0.1% Pluronic polyol. A critical average wall shear rate of 3000 s(-1) (above which damage occurs) is found for several cell types, including mammalian and insect cells. Hydrodynamic stress also limits the maximum tip speed in a rotary lobe pump to less than 350 cm/s. Turbulent flow in the recirculation loop piping at Reynolds numbers of up to 71,000 does not cause cell damage. Maximum sustainable flux decreases with cell concentration and increases with cell size (in qualitative agreement with the hydrodynamic lift model). A flux of 30 to 75 L/m(2) h (depending on cell size) can be sustained during 20-fold concentration from 2.5 x 10(6) cells/ml, while maintaining high cell viability.
Recombinant tumor necrosis factor (TNF) is expressed in Escherichia coli as a soluble intracellular protein. A purification process is described that incorporates a hydrophilic membrane (cellulosic) separation followed by a hydrophobic one (PTFE). The hydrophilic step is a traditional one in that species are separated primarily on the basis of size. The hydrophobic step separates species on the basis of parameters apparently not related to size. By combining these two steps, an increase in TNF purity of 7-10-fold can be achieved with a yield of 50%. The effects of cellular debris and pH on selectivity and recovery of the hydrophobic filtration step are discussed.
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