Affinity Separation 3

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 R between them. This recognition is highly speci cto Biological Recognition the pair of molecules involved. The interactive mechanism 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 maceuticalsprovidedanewimpetusforthishighly 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. 4 I / AFFINITY SEPARATION / Derivatization The matching pair must be identiRed, and one afRnity method is not restricted to protein separ- of them isolated in a pure form. Covalent bond- ations; nucleic acids and whole cells can also be ing onto an inert matrix in a stable manner must separated. always allow the ‘docking’ surface of the protein to The simplicity of the chromatographic process is be positioned to make it available to the target pro- shown in Figure 1. The ligand of interest, covalently tein. The whole also has to be achieved at an accept- bonded onto the inert matrix, is contained in the able cost. column, and a solution containing the target (the This technique has resulted in many successful ap- ligate) is passed through the bed. The ligand recog- plications, often using antibodies as the afRnity nizes the ligate to the exclusion of all other molecules, medium (immunoafRnity chromatography), but with the unwanted materials passing through the col- large scale separations using these ‘natural’ ligands umn packing while the ligate is retained. Once the are largely restricted by cost and regulatory reasons. bed is saturated with the target molecule (as mea- Although immunoafRnity chromatography is still sured by the breakthrough point), contaminating spe- widely practised, in recent years the evolution of cies are washed through, followed by collection of the design technologies has provided powerful new ap- target molecule as a very pure fraction using an proaches to mimic protein structures, resulting in the eluting buffer solution. Finally, the column is development of synthetic ligands able to work in cleansed from any strongly adsorbed trace materials, harsh operational environments and at low cost. usually by regeneration with a strong alkali or acid, making it available for many more repeat runs. An R The Af\nity Process outstanding advantage of the af nity process is an ability to concentrate very dilute solutions while The afRnity method is critically dependent upon stabilizing the captured protein once adsorbed onto the ‘biological recognition’ existing between species. the column. Many of the in-demand proteins manu- By permanently bonding onto an inert matrix a mol- factured by genetically engineered microorganisms ecule (the ligand) that speciRcally recognizes the mol- are labile, allowing only minute quantities to be pres- ecule of interest, the target molecule (the ligate) can ent in the fermentation mix before they begin to be separated. The technique can be applied to any deteriorate. An ability to capture these very small biological entity capable of forming a dissociable quantities while stabilizing them in the adsorbant complex with another species. The dissociation con- phase results in maximization of yield, making mass- S stant (Kd) for the interaction re ects the comp- ive savings in total production costs. lementarity between ligand and ligate. The optimal Although the technical processing advantages are R R range of Kd for af nity chromatography lies be- clear there is a major dif culty in the appli- tween 10\4 and 10\8 mol L\1. Most biological cation of afRnity chromatography as understood ligands can be used for afRnity purposes provid- by most practitioners today. Most ligands described ing they can be immobilized, and once immobilized in Table 1 suffer from two primary disadvan- continue to interact successfully with their respective tages: a lack of stability during use; and high ligates. The ligand can be naturally occurring, an cost. Fortunately these problems have now been over- engineered macromolecule or a synthetic molecule. come, and afRnity chromatography is now accep- Table 1 provides some examples of immobili- ted as the major separations technology for zed ligands used to purify classiRed proteins. The proteins. Table 1 Affinity ligands and purified proteins Immobilized ligand Purified protein Divalent and trivalent metal ion Proteins with an abudance of his, tryp and cys residues Lectins Glycoproteins, cells Carbohydrates Lectins Reactive dyes Most proteins, particularly nucleotide-binding proteins Nucleic acids Exo and endonucleases, polymerases, other nucleic acid-binding proteins Amino acids (e.g. lys, arg) Proteases Nucleotides, cofactors substrates and inhibitors Enzymes Proteins A and G Immunoglobulins Hormones, drugs Receptors Antibodies Antigens Antigens Antibodies Sepsci*1*TSK*Venkatachala=BG I / AFFINITY SEPARATION 5 The beaded agaroses have captured over 85% of the total market for biological macromolecule separations, and are regarded as the industry standard to which all other supports are compared. They have achieved this position by providing many of the desir- able characteristics

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