Page 8 - Subyek Encyclopedia - Encyclopedia of Separation Science

Sepsci*1*TSK*Venkatachala=BG
I / AFFINITY SEPARATION 3

AFFINITY SEPARATION

K. Jones, Affinity Chromatography Ltd, Freeport, Separation and puriRcation methods for biological
Ballsalla, Isle of Man, UK macromolecules vary from the very simple to the
esoteric. The type of technique adopted is basically
Copyright ^ 2000 Academic Press
a function of source, the fragility of the molecule and
the purity required. Traditionally, high purity protein
Introduction pharmaceuticals have used multistage processing, but
this is very inefRcient as measured by the well-
Of the collection of separation technologies known documented fact that 50}80% of total production
as ‘afRnity’, afRnity chromatography is by far the costs are incurred at the separation/puriRcation stage.
most widely used variant. AfRnity chromatography is In contrast, the highly selective indigenous properties
becoming increasingly important as the speed of the of the afRnity method offer the alternative of
revolution taking place in biotechnology processing very elegant single-step puriRcation strategies. The
increases. The concept of an ‘afRnity’ separation re- inherent simplicity and universality of the method has
sults from a naturally occurring phenomenon existing already generated a wide range of separation tech-
within all biological macromolecules. Each biological
nologies, mostly based upon immobilized naturally
macromolecule contains a unique set of intermolecu-
occurring proteinaceous ligands. By comparing the
lar binding forces, existing throughout its internal
‘old’ technologies of ‘natural’ ligands or multistage
and external structure. When alignment occurs be-
processing with the ‘new’, exempliRed by synthetic
tween a speciRc site of these forces in one molecule
designed ligands, the most recent advances in af-
with the site of a set of forces existing in another
Rnity processing can be described.
(different) molecule, an interaction can take place
between them. This recognition is highly speciRcto Biological Recognition
the pair of molecules involved. The interactive mech-
anism can be converted into a universal mutual bind- As nature evolved, life forms had to develop a protec-
ing system, where one of the binding pair is attached tive mechanism against invading microorganisms if
to an inert matrix, packed into a column and used they were to survive. Thus there is a constant battle
exclusively to capture the other matching molecule. between the cell’s defence mechanism and the attack-
When used in this (afRnity) mode, the technique is ing microorganisms, a battle resolved by the cells
probably the simplest of all chromatographic generating antibodies (the immunoglobulins) able to
methods. It is, however, restricted almost exclusively recognize the protein coat of attacking microorgan-
to the separation and puriRcation of biological mac- isms and signal killer cells to destroy the invaders
romolecules, and is unsuitable for small molecules. before they cause harm to the host. Equally, if micro-
AfRnity chromatography or bioselective adsorp- organisms were to survive, they had continually to
tion chromatography was Rrst used in 1910, but it was mutate and change their protein coats to avoid detec-
only in the 1960s that afRnity chromatography as tion by existing antibodies. The ‘attack and destroy’
practised today was developed as a puriRcation tech- process is a function of changes in the molecular
nique. By the late 1970s the emergence of recombinant structure in a speciRc part of the protein, with only
DNA technology for the manufacture of protein phar- the most minute of changes occurring at the surface of
maceuticalsprovided a new impetus forthishighly the protein. Evolution has thus designed a system
speciRc chromatographic method, implemented by the where every protein has a very precise structure, but
demand for ever-increasing product purity implicit in one which will always be recognized by another. One
regulatory frameworks devised by (amongst others) element of the interacting pair can be covalently
the USA’s Food and Drug Administration (FDA). Fi- bonded onto an inert matrix. The resulting chromato-
nally, the need to reduce the cost of drugs is under graphic medium can then be packed into a column,
constant scrutiny by many Governments, particularly and used to separate exclusively its matching partner
those with controlled health schemes funded by rev- from an impure mixture when added as a solution to
enue raised by taxation. These mutually incompatible the top of the column. This fact can be stated as
pressures indicate the need for more efRcient sep- follows } for every protein separation problem there
aration systems; the afRnity technique provides the is always an afTnity solution. The process of
promise of meeting all necessary requirements. producing a satisfactory medium is quite difRcult.