Page 260 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

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Encyclopedia of Physical Science and Technology EN014J-683 July 30, 2001 20:3

668 Separation and Purification of Biochemicals

and the desired final product quality are altered. The ef-
fect of the particle diameter on the efficiency of the column
tends to be less pronounced under overloading conditions.
However, an optimal ratio of column length to the square
of the particle diameter can be found so that in the most
common applications, the optimum particle diameter is in
the range from 10 to 30 µm. Broad particle size distribu-
tion often leads to an inferior separation efficiency with
concomitantly higher backpressure.

C. Processes for Preparative Chromatography
FIGURE 11 Schematic representation of a fluidized bed system.
The use of a typical—but large—batch column packed
with porous particles is possible and common practice in
preparative chromatography; and columns of more than a upward through the porous plate at such a flow rate that
cubicmeterhavebeenused.Someoftheproblemsencoun- the particles become “fluidized” within the confinements
tered with biochromatography on preparative columns of the container, as illustrated in Fig. 11. There are several
have been discussed in Section IIB (Scale-Up Considera- ways of using adsorptive separations with fluidized beds
tions). However, several alternatives have been suggested, in biotechnology. A major advantage of the approach is
which either circumvent some of the typical problems that particles (e.g., also residual cells) pose no problem.
encountered when using columns early on in the down- As long as they do not stick to the particles, solids move
stream process or during scale-up (fluidized bed, radial with the fluidizing liquid through the expanded bed and
chromatography), or which allow to operate a chromato- are thus removed without any risk of column clogging,
graphic separation in a continuous manner (continuous an- while the product is bound to the adsorbent. The product
nular chromatography, simulated moving bed chromatog- is simultaneously concentrated and, to a certain extent,
raphy). On the production scale, a continuous separation purified in a single-step operation. Since solids can be ex-
process is often preferable, as such processes are usually changed and recycled in fluidized beds, and clogging is
more economical, are automated more easily, and provide not a serious problem, such beds are putatively interest-
a more homogeneous product quality. Furthermore, they ing systems for applications involving applying the crude
enable a better use of the adsorbent and mobile phase and cell culture suspension directly into the expanded bed sys-
may facilitate recycling. tem, thus bypassing the tedious step of cell and cell debris
The advantages of continuous chromatographic separa- removal.
tions are fully realized only in large-scale processes; there- Since the 1970s, the stabilization of a fluidized bed of
fore such chromatographic processes are found mainly magnetic particles by means of applying a magnetic field
in the petrochemical and sugar industries. However, as has been investigated. This was shown to suppress particle
industrial biotechnology advances, continuous chromato- circulation to a large extent and solids seem to move in
graphic systems are expected to play an increasing role nearly plug-flow manner in such fluidized beds. As both
also for large-scale purification and isolation of biochem- the solid and the fluid movement are controllable, magnet-
icals, e.g., in the food or (bio)pharmaceutical industry. ically stabilized fluidized beds are interesting albeit me-
Such systems can work at countercurrent or crosscurrent chanically complex continuous chromatographic separa-
flow. The moving bed and the simulated moving bed are tors. Both crossflow fluidized beds, in which the solids
examples of the countercurrent approach, since the ad- move perpendicularly to the direction of the fluid, and
sorbent and the eluent move in opposite directions. Con- countercurrent fluidized beds have been described. An-
tinuous annular chromatography, on the other hand, is an other way to stabilize the fluidized bed is to use adsorbent
example for a crosscurrent flow system. While counter- particles with different densities (establishment of a den-
current methods are restricted to the separation of binary sity gradient). In this case, the heavier particles will stay
mixtures, crosscurrent methods are able to also separate close to the bottom of the column, while the lighter ones
multicomponent mixtures. raise preferably to the top. The result is a putative multi-
stage separation system (expanded bed) as opposed to an
indiscriminately fluidized bed, which can only achieve a
1. Expanded (Fluidized) Bed Chromatography
single stage separation.
In an expanded or fluidized bed, the adsorbent particles are One can mention that affinity beads are also being used
placed in a vessel with a porous bottom plate. A fluid flows in batch affinity purification by suspending the beads in