Page 398 - Academic Press Encyclopedia of Physical Science and Technology 3rd Chemical Engineering

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Encyclopedia of Physical Science and Technology EN009K-419 July 19, 2001 20:57

Membranes, Synthetic, Applications 333

FIGURE 44 Closed-loop cascade HPTFF system operating in diafiltration mode with buffer regeneration by a con-
ventional ultrafiltration unit. [Adapted from Zydney, A. L. and van Reis, R. (2001). In “Membrane Separation in Biotech-
nology” (W. H. Wong, ed.), Marcel Dekker, New York.]

operated in diafiltration mode, using a second UF stage to a wash step to remove indigenous residues from the col-
regenerate buffer solution from the permeate, as shown in umn, the protein is detached from the ligand by means
the figure. of an appropriate buffer solution and recovered free of
Studies show that HPTFF can be used to purify bov- contaminants. While this method is highly specific, the
ine serum albumin (BSA; MW = 68,000 daltons) by re- large pressure drop typical of packed beds also limits the
moving hemoglobin (Hb; MW = 67,000 daltons) as an throughput rates achievable. It is also difficult to project
impurity. Despite their almost identical molecular weight, large column performance based on the behavior of small
hemoglobin exhibits a strong negative charge at pH 7 systems. Another problem with scale-up is the extraordi-
while BSA is more neutral. By using a negatively charged nary costs associated with populating a large-scale column
membrane and adjusting the feed solution to neutral pH, with affinity ligands, and the correspondingly high risk of
electrostatic repulsion rejects the hemoglobin almost com- loss associated with process upsets. A production-scale
pletely. A purification factor of 100 for BSA was achieved. batchwise protein separation by column chromatography
Even more remarkably, BSA may be separated from an can require hours or days.
antigen-binding fragment (Fab; MW = 45,000) by a pu- A membrane analog of the affinity column is shown
rification factor of over 800. in Fig. 45. Ligand is attached to the internal surfaces of
a microporous membrane, which can be thought of as
a very wide but very thin (on the order of 10–100 µm)
C. Membrane-Based Affinity Separation
column. Pressure drop is kept low by using a micro-
Biospecific recognition is among nature’s most selective porous membrane of sufficiently large pore size not to
mechanisms. It is the basis of immune responses and the cause separation by physical retention. Target protein is
myriad interactions in living organisms. In certain pro- captured when the feed solution flows through the mem-
teins, the combination of amino acid sequence and spatial brane, quickly occupying the limited ligand capacity of-
configuration permits stable binding only with a unique fered by the membrane matrix. Following a rinse cycle
species of complementary functionality and shape. This to remove extraneous species held in the membrane, the
ligand–ligate recognition and attachment, such as that be- target protein is released by dissociative elution. Finally,
tween an antigen and a monoclonal antibody, or that be- the membrane is regenerated to prepare it for the next cy-
tween specific chemical dyes and proteins, is the principle cle of capture/release. Since the hold-up volume is very
underlying affinity separations (“Improved tools,” 2000). small, all flow cycles can be quite short; the entire purifi-
Commercial exploitation of affinity separation occurred cation sequence can be completed on the order of minutes.
first in the form of column chromatography. Ligands are By repeating the cycle many times automatically, a small
attached to various passive matrices such as crosslinked quantity of ligand has the cumulative capacity to harvest
cellulosic gel particles. When a solution containing a tar- the product from a large volume of feed material.
get protein flows through a packed bed of these gel par- A key feature of affinity membrane separations is
ticles, the target protein attaches to the ligand. Following the combination of sieving and selective adsorption