Page 253 - 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

Separation and Purification of Biochemicals 661

high-value target molecule present in very low concentra- lective removal of the target molecule from most contam-
tions, i.e., in the microgram to milligram per liter range, inants. The construction of a recombinant protein having
in a complex environment such as a cell culture super- a fused tag to facilitate its purification may provide a more
natant. This is, for example, the case for most recombinant economical and safer production process even though the
therapeutic proteins expressed in mammalian cells. Such cloning may be more elaborate and the tag need to be
cell culture supernatants tend to contain many other pro- removed before the final purification of a pharmaceutical.
teins in much higher abundance than the target molecule, The affinity ligand may be covalently linked to the sta-
thus necessitating the use of biospecific interactions to tionary phase surface via a hydroxyl, amino, or carboxyl
capture the target product. In such cases and on the function. Frequently, a spacer arm is used to anchor the
production scale, AC presents a rather attractive single- ligand to the matrix in order to improve accessibility. A
step alternative to multistep processes incorporating IEC, variety of preactivated stationary phases are commercially
HIC/RPC, and SEC separations. Alternatively, AC may available for the convenient attachment of affinity ligands.
be used with the aim of specific removal of a contami- Immobilization of the affinity ligand tends to lower its
nant from the product. The application potential of AC in affinity for the target molecule compared to the value mea-
general is wide. At present, however, the costs of the affin- sured in free in solution often by more than one order of
ity ligand tend to prevent its use save for the high-value magnitude. Ligand leakage and the concomitant loss in
products of molecular biotechnology. capacity and product contamination are serious problems
AC is usually performed in the frontal mode under con- in affinity chromatography, and so is the fact that many
ditions where only the target molecule binds to the station- affinity columns will also bind denatured or otherwise
ary phase, while all other feed components move through malformed product molecules or product fragments.
the bed unretained. The surface of the affinity sorbent
should be highly hydrophilic and without functions that 1. Immobilized Metal Affinity Chromatography
promote nonspecific interactions. The most commonly
used supports are based on agarose, porous glass, silica, Immobilized metal affinity chromatography (IMAC) has
polyacrylamide, methacrylate, and cellulose. Fibrous sup- been used to purify albumin, monoclonal antibodies, im-
ports were developed specifically for preparative applica- munoglobulins, blood factors, interferons, enzymes, and
tions. Nonspecific adsorption can be further reduced by many other proteins and polypeptides. Lately the tech-
carefully choosing the operating conditions regarding the nique has gained general significance in preparative pro-
pH and salt concentration of the mobile phase and the tein purification since fusion proteins carrying a polyhisti-
additives used. After the impurities have left the column, dine tag can be created, which have a very high affinity to
the product is eluted in a suitable buffer. Desorption is IMAC columns. Protein IMAC is based on the interaction
normally achieved by altering the pH, increasing the salt between a metal ion-based electron acceptor (Lewis acid)
concentration, or introducing a chaotropic agent. Temper- anchored to the stationary phase and an electron donor
ature changes, or reversible denaturation, are also used (Lewis base) on the surface of the protein. The proteins in
occasionally to cause desorption. most cases interact through their surface histidine and—to
Typical affinity ligands in AC include antibodies, anti- a lesser extent—their tryptophane residues. As mentioned
gens, lectins, receptors, enzyme inhibitors, hormones, and above, recombinant proteins can be expressed by adding
triazine dyes. Protein A and Protein G are widely used as an oligohistidine tag and thus impart to them high affin-
group-specific ligands for the isolation of antibodies, even ity toward the immobilized metal ions. This approach may
though they exhibit some subclass specificity. Antibodies have advantages over the use of larger tags such as Protein
themselves are also powerful ligands in AC, due to the A, which may be difficult to remove to obtain the correctly
fact that they can be readily raised against most prod- folded and biologically active protein product.
2+
ucts of biotechnological importance. A lot of research has Metal ions of intermediate polarizability such as Cu ,
2+
2+
been invested in the last years to design new and improved Ni ,Zn , and Co 2+ are particularly suited for interac-
affinity ligands, either derived from a natural molecule or tion with proteins as they may interact not only with the
constructed de novo, e.g., via combinatorial chemistry, nitrogen in amino and imino groups, but also with oxygen
with the aim of improving the stability, the specificity, and sulfur. Metal ions are immobilized on the stationary
but also the price of the AC stationary phases. Recent ad- phase by chelating agents, such as the two-dentate ligand
vances in genetic engineering have also helped to expand IDA (iminodiacetic acid) bound to the support. The na-
the scope of AC. It is possible to fuse an affinity tag such ture of the chelating agent is of consequence. If the immo-
as a Protein A or a polyhistidine sequence to a recombi- bilization of the metal ion involves several coordination
nant protein, which increases the product affinity for the sites, the metal is bound strongly to the chromatographic
corresponding affinity column considerably and allows se- surface and bleeding is less likely to occur. At the same