Page 21 - Subyek Encyclopedia - Encyclopedia of Separation Science

16 I / AFFINITY SEPARATION/ Derivatization

are high and the recovery of bound proteins is Af\nity Membranes
easier, created by the process operating with fewer
theoretical plates than those generated by chromatog- UltraRltration membranes are commonly employed
raphy columns. This technique has also been com- as a ‘polishing’ stage of multistage separation pro-
bined with afTnity precipitation, where a homo- cesses for several commercially important proteins.
bifunctional ligand composed of two ligand entities Consequently attaching standard afRnity ligands
connected by a spacer (for example a bis-dye) is used. to create afRnity membranes has become an ac-
However, even in combination this approach suf- tively researched area. The most obvious advantage
fers from considerable nonspeciRc binding and rela- of a membrane structure is the high rate of transport
tively low puriRcation factors. A review of this com- of the medium through the porous structure by Rltra-
bination suggests that it is more suited to large scale, tion, thus minimizing the normally encountered dif-
low purity products. In contrast, perSuorocarbon fusion limitations of mass transfer. High adsorption
emulsion chemistry utilizing mixer-settlers may of- rates are achieved, especially if long distance electro-
fer more promise. By using a series of mixer-settlers static interactions are involved in the binding mech-
connected in a loop a continuous process has been anism. However, in contrast to ion exchange
developed. A ligand (usually a reactive dye) is membranes, similar high transport effects are not
covalently bonded to a high density perSuorocarbon observed when used in the afRnity mode, elimin-
emulsion and contacted with the crude protein solu- ating much of the initial attraction of this form of
tion. After settling in the Rrst tank the emulsion is device. Other theoretically attractive features in-
pumped to a second settler and washed before passing cluded: an inherent ability to control pore size across
to the third settler for elution. The emulsion is regen- a very wide range, offering an opportunity to
erated in the fourth settler. The supernatants from increase capacity of a given system; and ability to
each settler, still containing some unbound target operate in either batch mode or Rltration mode. In
protein, are normally discarded. Although reasonable both cases experimental data have not conRrmed these
recoveries and yields are obtained, signiRcant devel- assumed advantages; a 10-fold change in the pore size
opment is needed for this type of system to become resulted in only a two-fold capacity increase and when
competitive with conventional chromatography col- in Rltration mode, although adsorption is fast, severe
umn methods. peak broadening on elution is experienced.
Another favoured research approach to improving The chemistry relevant to particulate media is iden-
efRciency is to use expanded beds. Various tech- tical to that required for membranes, in effect
niques have been tried, with the primary objective of making the systems compatible and allowing an easy
eliminating the ‘solid bed’ effect, where the bed technology interchange. The covalent bonding of af-
acts as a Rlter, trapping insolubles and creating signif- Rnity ligands to the surface of a membrane follows
icant back-pressure. By partially removing the normal exactly the same chemistry as that applied to partic-
constraints of upper and lower retaining frits, which ulate media, and the same adsorption/desorption
pack the particles tightly in the bed, the particles can principles apply to both. Consequently the only dif-
expand, thereby releasing trapped solid impurities. ference between membrane systems and those of con-
Consequently longer operational cycles and higher ventional chromatography is the exploitation of the
Sows result. One limitation of the expanded bed characteristics of the membrane matrix compared
system is that adsorption can only be carried out in with a particulate bed. Although the high mechanical
one stage, resulting in a less efRcient process. strength of membranes is one major advantage, plus
Expanded beds are only an intermediate stage to- the scale-up is claimed to be very easy by stacking
wards Uuidized beds. Several variations of Suidized membranes (although scaling afRnity columns is
bed technology have been adopted to evaluate them also very straightforward), it has been discovered that
for afRnity processing. One example is the use of if the pressure drop across the membrane is too high
perSuorocarbon emulsions in a countercurrent con- sealing problems occur; the mobile phase then Sows
tactor. The afRnity perSuorocarbon emulsion is beyond the edges and past the membranes. Further-
loaded with crude source material into the base of more afRnity membranes should be capable of
a column in a similar manner to that of an expanded use with unclariRed extracts, but is has been generally
bed. The adsorbent is then removed from the base of observed that membrane capacity and lifetime are
the bed and carried forward through four identical progressively reduced with time of use. Even with
contactors where washing, elution and regeneration clariRed broths, membrane fouling regularly occurs.
are carried out successively. This process is claimed to This is almost certainly the reason why afRnity
improve signiRcantly removal of target proteins com- membranes have not found favour in large scale
pared to an expanded bed system. processing.