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Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications - Production and Downstream Processing of Biotech Compounds

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Filtration/Centrifugation - Downstream Processing of Biotech Compounds

Products from biotechnological industry must be separated from biological systems that contain suspended particulate material, including whole cells, lysed cell material, and fragments of broken cells generated when cell breakage has been necessary to release intracellular products.

Filtration/Centrifugation

Products from biotechnological industry must be separated from biological systems that contain suspended particulate material, including whole cells, lysed cell material, and fragments of broken cells generated when cell breakage has been necessary to release intracellular products. Most downstream processing flow sheets will, therefore, include at least one unit-operation for the removal (“clarification”) or concentration, just the opposite, of particulates. Most frequently used methods are centrifugation and filtration techniques (e.g., ultrafiltration, diafiltration and microfiltration). However, the expense and effectiveness of such methods is highly dependent on the physical nature of the particulate material and of the product.


Filtration

Filtration can concentrate the biomass prior to further purification. Several filtration systems have been developed for separation of cells from media, the most successful being tangential flow systems (also referred to as “cross flow”) where high shear across the membrane surface limits fouling, gel layer forma-tion and concentration polarization. In ultrafiltration, mixtures of molecules of different molecular dimen-sions are separated by passage of a dispersion under pressure across a membrane with a defined pore size (Minton, 1990). In general, ultrafiltration achieves little purification, because of the relatively large pore size distribution of the membranes. However, this technique is widely used to concentrate macromole-cules, and also to change the aqueous phase in which the particles are dispersed or in which molecules are dissolved (diafiltration) to one required for the subsequent purification steps.

 

 

Centrifugation

Subcellular particles and organelles, suspended in a viscous liquid (for example the particles produced when cells are disrupted by mechanical procedures) are difficult to separate either by using one fixed centrifugation step or by filtration. But, they can be isolated efficiently by centrifugation at different speeds. For instance, nuclei can be obtained by centrifugation at 400xg for 20 minutes, while plasma membrane vesicles are pelleted at higher centrifugation rates and longer centrifugation times (fractional centrifugation). In many cases, however, total biomass can easily be separated from the medium by centri-fugation (e.g., continuous disc-stack centrifuge). Buoyant density centrifugation can be useful for separation of particles as well. This technique uses a viscous fluid with a continuous gradient of density in a centrifuge tube. Particles and molecules of various densities within the density range in the tube will cease to move when the isopycnic region has been reached. Both techniques of continuous (fluid densi-ties within a range) and discontinuous (blocks of fluidwith different density) density gradient centrifugation are used in buoyant density centrifugation on a laboratory scale. For application on the industrial scale, however, continuous centrifuges (e.g., tubular bowl centrifuges) are only used for discontinuous buoyant density centrifugation. This type of industrial centrifuge is mainly applied to recover precipitated proteins or contaminants.

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