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Sepsci*1*TSK*Venkatachala=BG I/AFFINITYSEPARATION 3 AFFINITY SEPARATION K.Jones,AffinityChromatographyLtd,Freeport, SeparationandpuriRcationmethodsforbiological Ballsalla,IsleofMan,UK macromolecules vary from the very simple to the esoteric. The type of technique adopted is basically Copyright^ 2000AcademicPress afunctionofsource,thefragilityofthemoleculeand thepurityrequired.Traditionally,highpurityprotein Introduction pharmaceuticalshaveusedmultistageprocessing,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 costsareincurredattheseparation/puriRcationstage. mostwidelyusedvariant.AfRnitychromatographyis Incontrast,thehighlyselectiveindigenousproperties 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- inherentsimplicityanduniversalityofthemethodhas sultsfromanaturallyoccurringphenomenonexisting already generated a wide range of separation tech- withinallbiologicalmacromolecules.Eachbiological nologies, mostly based upon immobilized naturally macromoleculecontainsauniquesetofintermolecu- 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 speciRc to Biological Recognition thepairofmoleculesinvolved.Theinteractivemech- anismcanbeconvertedintoauniversalmutualbind- Asnatureevolved,lifeformshadtodevelopaprotec- ingsystem,whereoneofthebindingpairisattached 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. betweenthecell’sdefencemechanismandtheattack- 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.Itis,however,restrictedalmostexclusively recognize the protein coat of attacking microorgan- to the separationand puriRcationof biological mac- isms and signal killer cells to destroy the invaders romolecules, and is unsuitable for small molecules. beforetheycauseharmtothehost.Equally,ifmicro- AfRnity chromatography or bioselective adsorp- organisms were to survive, they had continually to tionchromatographywasRrstusedin1910,butitwas mutateandchangetheirproteincoatstoavoiddetec- 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.Bythelate1970stheemergenceofrecombinant structure in a speciRc part of the protein, with only DNAtechnologyforthemanufactureofproteinphar- themostminuteofchangesoccurringatthesurfaceof maceuticals provided a new impetus for this highly the protein. Evolution has thus designed a system speciRcchromatographicmethod,implementedbythe where every protein hasa very precise structure,but demand for ever-increasing product purity implicit in onewhichwillalwaysberecognizedbyanother.One regulatory frameworks devised by (amongst others) element of the interacting pair can be covalently the USA’s Food and Drug Administration (FDA). Fi- bondedontoaninertmatrix.Theresultingchromato- 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 andusedtoseparateexclusivelyitsmatchingpartner those with controlled health schemes funded by rev- fromanimpuremixturewhenaddedasasolutionto enue raisedbytaxation.Thesemutuallyincompatible the top of the column. This fact can be stated as pressures indicate the need for more efRcient sep- follows} forevery protein separationproblemthere 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/AFFINITYSEPARATION/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- shownin Figure 1. The ligandof interest, covalently tein.Thewholealsohastobeachievedatanaccept- bonded onto the inert matrix, is contained in the able cost. column, and a solution containing the target (the Thistechniquehasresultedinmanysuccessfulap- ligate) is passed through the bed. The ligand recog- plications, often using antibodies as the afRnity nizestheligatetotheexclusionofallothermolecules, medium (immunoafRnity chromatography), but withtheunwantedmaterialspassingthroughthecol- large scale separations using these ‘natural’ ligands umn packing while the ligate is retained. Once the are largely restricted by cost and regulatoryreasons. bed is saturated with the target molecule (as mea- Although immunoafRnity chromatography is still suredbythebreakthroughpoint),contaminatingspe- widely practised, in recent years the evolution of ciesarewashedthrough,followedbycollectionofthe design technologies has provided powerful new ap- target molecule as a very pure fraction using an proachestomimicproteinstructures,resultinginthe eluting buffer solution. Finally, the column is development of synthetic ligands able to work in cleansedfromanystronglyadsorbedtracematerials, harsh operational environmentsand at low cost. usually by regeneration with a strong alkali or acid, making it available for many more repeat runs. An The Af\nity Process outstanding advantage of the afRnity 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- Bypermanentlybondingontoaninertmatrixamol- factured by genetically engineered microorganisms ecule(theligand)thatspeciRcallyrecognizesthemol- arelabile,allowingonlyminutequantitiestobepres- 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- phaseresultsinmaximizationofyield,makingmass- stant (K ) for the interaction reSects the comp- ive savings in total productioncosts. d lementarity between ligand and ligate. The optimal Although the technical processing advantages are range of K for afRnity chromatography lies be- clear there is a major difRculty in the appli- d tween 10(cid:1)4 and 10(cid:1)8molL(cid:1)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- continuetointeractsuccessfullywiththeirrespective tages: a lack of stability during use; and high ligates. The ligand can be naturally occurring, an cost.Fortunatelytheseproblemshavenowbeenover- 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. Table1 Affinityligandsandpurifiedproteins Immobilizedligand Purifiedprotein Divalentandtrivalentmetalion Proteinswithanabudanceofhis,trypandcysresidues Lectins Glycoproteins,cells Carbohydrates Lectins Reactivedyes Mostproteins,particularlynucleotide-bindingproteins Nucleicacids Exoandendonucleases,polymerases,othernucleicacid-bindingproteins Aminoacids(e.g.lys,arg) Proteases Nucleotides,cofactorssubstratesandinhibitors Enzymes ProteinsAandG Immunoglobulins Hormones,drugs Receptors Antibodies Antigens Antigens Antibodies Sepsci*1*TSK*Venkatachala=BG I/AFFINITYSEPARATION 5 The beaded agaroses have captured over 85% of thetotalmarketforbiologicalmacromoleculesepar- ations, and are regarded as the industry standard to which all other supports are compared. They have achievedthispositionbyprovidingmanyofthedesir- able characteristics needed, and are also relatively inexpensive.Beadedagarosesdohaveoneseverelim- itation } poor mechanical stability. For analytical applications speed and sensitivity are essential, demanding mechanically strong, very small particles. Beaded agaroses are thus of limited use analytically, Figure1 Schematicdiagramofaffinitychromatography. agapRlledbyhighperformanceliquidchromatogra- phy(HPLC)usingsilicamatrices.Forpreparativeand large scale operations other factors are more impor- Matrices tant than speed and sensitivity. For example, mass BydeRnitionmatricesmustbeinertandplaynopart transferbetweenstationaryphaseandmobilephaseis in the separation. In practice most play a (usually) much less important when compared to the contri- negative role in the separation process.To minimize bution from the chemical kinetics of the binding thesedisadvantagesmatriceshavetobeselectedwith reaction between stationary phase and protein. great care. There is a theoretically perfect matrix, Band spreading is also not a serious problem. When deRned as consisting of monodispersed perfectly combined with the highly selective nature of the shaped spheres ranging from 5 to 500(cid:1)m in dia- afRnity mechanism, these factors favour the com- meter, of high mechanical strength, zero nonspeciRc monuseoflargesized,lowmechanicalstrengthpar- adsorptionand with a range of selectable pore sizes ticles. from10}500nm,averynarrowporesizedistribution In recent years synthetic polymeric matrices have and low cost. This idealized matrix would then pro- been marketed as alternatives. Although nonbiodeg- vide the most efRcient separation under all ex- radable, physically and chemically stable, with good perimentalconditions.As always, a compromisehas permeabilities up to molecular weights greater than tobereached,theusualapproachbeingtoaccentuate 107Da, the advantages provided are generally off- the most attractive characteristics while minimizing set by otherquite seriousdisadvantages,exempliRed the limitations, usually by manipulating the experi- by high nonspeciRc adsorption. Inorganic matrices mentalconditionsmostlikelytoprovidetheoptimum have also been used for large scale protein separ- result. ations, notably reversed-phase silica for large scale The relative molecular masses of proteins vary recombinant human insulin manufacture (molecular from the low thousands to tens of millions, making weight approximately 6000Da), but are generally pore size the most important single characteristic of not preferred for larger molecular weight pro- the selected matrix. Very large molecules need very ducts. A very slow adoption of synthetic matrices is openandhighlyporousnetworkstoallowrapidand easy penetration into the core of the particle. Struc- turesofthistypemustthereforehaveverylargepores, Table2 Supportmatrices but this in turn indicates low surface areas per unit volume,suggestingrelativelylownumbersofsurface Supportmatrix Operational pHrange groups to which ligands can be covalently attached. Thematrix must also be biologically and chemically Agarose 2}14 inert. A special characteristic demanded from biolo- Cellulose 1}14 gical macromolecularseparationsmedia is anability Dextran 2}14 to be sanitized on a routine basis without damage. Silica (8 Glass (8 This requires resistance to attack by cleansing re- Polyacrylamides 3}10 agentssuchasmolarconcentrationsofstrongalkali, Polyhydroxymethacrylates 2}12 acidsandchaotropes.Incontrasttoanalyticalsepar- Oxirane}acryliccopolymers 0}12 ations, where silica-based supports are inevitably Styrene}divinylbenzenecopolymers 1}13 used, silica cannot meet these requirements and is Polyvinylalcohols 1}14 N-Acryloyl-2-amino-2-hydroxy-1,2-propane 1}11 generally not favoured for protein separations. PTFE Unaffected Table 2 contains examples of support matrices used in afRnity separations. PTFE,polytetrafluoroethylene. 6 I/AFFINITYSEPARATION/Derivatization indicated as improvements are made to current ma- coupleddirectlytothematrix,butmostrequirecoup- terialsandthepricesofsyntheticsbegintoapproach ling via a previously activated matrix. The afRn- thoseofagarosebeads.Otherfactorsresistanysignif- itymatrixselectedmusthaveanadequatenumberof icant movement towards synthetic matrices. Most appropriatesurfacegroupsontowhichtheligandcan installedprocessingunitsaredesignedforlowperfor- be bonded. The most common surface group is hy- mance applications. Higher performance matrices droxyl.Themajorityofcouplingmethodsinvolvethe would need reinstallation of new, much higher cost activationofthisgroupbyreactingwithentitiescon- highperformanceplant; plant operatorswould need taining halogens, epoxy or carbonyl functional retraining; operating manuals would need rewriting; groups. These surface residues are then coupled to andplantandfactorywouldneedreregistrationwith ligands through primary amines, hydroxyls or thiol the FDA. In combination, the implication is that groups,listed in Table 3. penetration of high performance systems for large Polysaccharides, represented by agarose, have scaleapplicationswillbeslow,andagarosebeadswill a high densityof surfacehydroxylgroups.Tradition continue to dominate the market for protein separ- stilldictatesthatthissurfaceisactivatedbycyanogen ations. bromide, but it is well established that this reagent forms pH-unstable iso-urea linkages, resulting in a poorly performing product. Furthermore CNBr- Covalent Bonding activated agarose needs harsh coupling conditions Abasicrequirementofallchromatographicmediais if high yields of Rnal media are to be obtained, the need for absolute stability under all operational suggesting high wastage of often expensive ligands. conditionsthroughmanycyclesofuse.Consequently Thisfactorisparticularlyevidentwithfragileentities all ligands must be covalently bonded onto the such as the very-expensive-to-produce antibodies, matrix, and various chemistries are available to andyetmanyworkerssimplyreadpreviousliterature achieve this. and make no attempt to examine alternative far A number of factors are involved: superior coupling methods. The advantages of mild couplingregimesaredemonstratedinFigure 2,where 1. The performance of both ligand and matrix are theuseofatriazine-activatedagaroseiscomparedto not impaired as a result of the coupling process. CNBr-activated agarose. Yield is signiRcantly in- 2. Most of the coupled ligand is easily accessible to creased,largelyby couplingunderacidic ratherthan the ligate. alkaline conditions. 3. Chargedorhydrophobicgroupsarenotgenerated onthematrix,soreducingnonspeciRcadsorption. Intermolecular Binding Forces 4. The immobilized ligand concentration is optimal for ligate bonding. Almost all chromatographic separations rely upon 5. There is no leakage of immobilized ligand from the interaction of the target molecule with either the matrix. aliquidphaseoracovalentlybondedmoleculeonthe solid phase, the exceptions being those relying upon Some ligands are intrinsically reactive (or can be molecular size, e.g. molecular sieves and gel Rltra- designed to be so) and contain groups that can be tion. In afRnity separations ligates are inevitably Table3 Activationmaterials Activatingreagent Bondinggrouponligand Cyanogenbromide Primaryamines Tresylchloride Primaryamines,thiols Tosylchloride Primaryamines,thiols Epichlorohydrin Primaryamines,hydroxyls,thiols 1,4-Butanedioldiglycidylether Primaryamines,hydroxyls,thiols 1,1(cid:1)-Carbonyldiimidazole Primaryamines,hydroxyls Cyanuricchloride Primaryamines,hydroxyls Divinylsulfone Primaryamines,hydroxyls 2-Fluro-1-methylpyriinium-toluene-4-sulfonate Primaryamines,thiols Sodiumperiodate Primaryamines Glutaraldehyde Primaryamines Sepsci*1*TSK*Venkatachala=BG I/AFFINITYSEPARATION 7 Figure2 Triazinecoupling.(A) Couplingofhumanserumalbumin(HSA)toready-acitivatedsupportsasafuncitonofpH.(B) Time courseofcouplingofhumanIgGtoready-activatedsupportsat43C. ,CNBr-activatedagarose4XL; ,triazine-activatedagarose 4XL. complex biological macromolecules or assemblies, Fortunately there are a vast number of biological mostly or exclusively consisting of amino acids enti- ligandsthat can interact with morethan onemacro- ties linked together in a speciRc manner. This com- moleculeandconsequentlygroup-speciRcligandsare plexity of structure provides many opportunities to commonplace.Sincegroup-speciRcadsorbentsretain exploit the physicochemical differences between a range of ligates with similar binding requirements, the target molecule and the ligand to be used. Each asingleadsorbentmaybeusedtopurifyanumberof structurecontainsthefourbasicintermolecularbind- ligates. Group-speciRc ligates can be used when the ing forces } electrostatic, hydrogen bonding, hydro- desiredligateispresentinhighconcentration,butthis phobic and van der Waals interactions } spread implies that some preprocessinghas taken place and throughoutthestructureinanexactlydeRnedspatial a concentration step interposed. The use of non/ manner. The degree of accessibility and spatial pre- group-speciRc adsorbents can only offer partial sentation within the pore of the medium, and the separations. This results in having to apply several strength of each force relative to each other, dictate stagesinseries,eachonlycapableofremovingapro- whether these forces are utilized to effect the portion of the impurities. In contrast a highly selec- separation. The biological recognition between spe- tive ligand can exclusively remove the target in one cies is a reSection of the sum of the various mo- step,butoftentheresultingcomplexesareverytight- lecular interactions existing between them, and ly bound, have low binding capacities, are easily this summation is Rxed for the ligate. However, denatured and are expensive to produce. Until re- various ligands may be found that emulate some centlytheseadsorbentswererestrictedto technically or all of the available binding forces to various difRcult isolations. Today the use of computer- degrees. assistedmolecularmodellingsystemsprovidesoppor- AfRnity adsorbents are therefore assigned to tunitiestoinvestigaterelationshipsbetweendesigned one of three broad ligand categories: nonspeciRc, ligands and relevant protein structures. For the Rrst group speciRc or highly speciRc. NonspeciRc ligands time logical design approaches can be applied and have only a superRcial likeness to biological ligands consequently stable inexpensive ligands have now and binding is usually effected by just one of the become available. fourbindingprocessesdescribedabove.Althoughion exchangematerialscanbeusedinasimilarmannerto Analytical Scale-Up afRnity adsorbents, they only exhibit the single forceofelectrostaticbinding.Theyarethuslimitedto Modern biotechnology uses two different types relativelyindiscriminatebinding.Inthiscasetheonly of chromatography. Analytical separations require criterion for binding is that of an overall charge. that run time is minimized, while resolution and 8 I/AFFINITYSEPARATION/Derivatization sensitivityaremaximized.Incontrast,forpreparative used to determine degradation of the target protein andprocessapplications,theobjectiveistomaximize (for example deamidated and oxidized elements); to purity, yield and economy. These techniques have identifypreviouslyunidentiRedcomponents;toestab- developedseparately,simplybecausebiologicalmac- lish the chromatographic identity between recom- romolecules pose unique difRculties, making binant and natural materials; to develop orthogonal them unsuitablefor ‘standardized’analysis. A major methods for the identiRcation of unresolved impu- inSuenceonthis divisionhasbeenthatscale-up usu- rities; and for many other demanding analytical ally occurs very much earlier in the development of approaches. a process, causing biochemists to turn to the traditional low performance methods of ion exchange Af\nity versus Traditional Media (IE), hydrophobic interaction (HI) and gel permeation chromatography (GPC). The highly efR- Whenprojectsaretransferredfromresearchtodevel- cient afRnity chromatography method was gener- opment two sets of chromatographic techniques are ally ignored, primarily because of the difRculty of carried forward: analysis, usually based on RP- havingtodevelopauniqueligandforeachseparation HPLC; and larger scale serialized separation steps, rather than having ‘off-the shelf’ column pack- often incorporating traditional methods of ion ex- ings immediately available from external suppliers. change, hydrophobicinteraction and gel permeation For analytical purposes high performance afRnity chromatography.Majordecisionshavetobetakenat liquid chromatography (HPALC) is a rarity, a func- this juncture } to scale up the separation processes tion of the limited availability of suitable matrices developedduringtheresearchphaseortoinvestigate (Table 2) and the afRnity process itself. alternatives. Regulatory demand and shortened pat- Where quantiRable high speed chromatography is entlifetimescompelmanagementsto‘fasttrack’new required, reversed-phase HPLC (RP-HPLC) has no products. Commercial pressure is at a maximum. equal. Unfortunately there is no general purpose BeingRrsttomarkethasthehighestpriorityinterms method for biomolecules to parallel the inherent of technical andcommercial reward. Very little time power of RP-HPLC. The success of RP-HPLC for is left to explore other separation strategies. It is analysis can be judged from the large number of known that serial application of IE, HI and GPC published applications developed for the ‘Rrst wave’ inevitably leads to very high manufacturing costs, of protein pharmaceuticals manufactured by geneti- but which comes Rrst? Most often the decision is callyengineeredmicroorganisms.Theseextracellular takentobeginmanufactureusingunoptimizedsepar- (relatively) low molecular weight proteins include ations as deRned in research reports. It is only in human insulin, human growth hormone and the in- retrospect that very high production costs become terferons. However, as molecular size and fragility apparent. By then it is too late } regulatory systems increase, so difRculties in using HPLC increase, are Rrmly in place. aprimaryreasonwhymuchanalysisisstillconducted There is an alternative. If researchers were more on low performance systems. Low performance sys- awareofprocesseconomicsandtheconsequencesof tems are easily scaled up; RP-HPLC is not. regulatory demand, selection of superior separation Biological separation systems must be aseptically processes could then result. Although most resear- cleanthroughouttheprocess.Themixturesareinevi- chersarefullyawareoftheadvantagesofsingle-step tablycomplexandusuallycontainmanycontamina- afRnity methods, paradoxically the high selectiv- ting similarly structured species. Such species can ity advantage of afRnity chromatography is also a adsorb very strongly onto the medium, demanding weakness. Suitable off-the-shelf afRnity adsor- post-usewashingwithverypowerfulreagentstoster- bents are often unavailable, in which case an adsor- ilize and simultaneously clean the column. Silica- benthastobecustomsynthesized.Sincethemajority basedmatricescannotsurvivethistypeoftreatment, of biochemists haveno desire (or time) to undertake hence scale-up of analytical procedures is generally elaborate chemical synthesis, antibody-based adsor- precluded. The Rrst wave of commercial protein bents are commonlyused. However, raising suitable pharmaceuticalshavegenerallyprovedtoberelative- antibodiesandpurifyingthembeforeimmobilization ly stable under high stress conditions. On the other onto a preactivated support matrix is an extremely hand intracellular proteins, often of high molecular laboriousprocedure. In addition, proteins are so of- weight, are unstable. Analysis by high performance ten tightly bound to the antibody that subsequent RP-HPLC methods then becomes problematic. De- elution involves some degree of denaturationand/or mand for fast, high resolution, analytical methods lossofacitivity.Idealmediarequiretheincorporation will continue to increase for on-line monitoring and ofelements of bothnonselective andselective adsor- processvalidation.Suchtechniqueshavealreadybeen bentstoprovideadsorbentswithageneralapplicabil- Sepsci*1*TSK*Venkatachala=BG I/AFFINITYSEPARATION 9 ity. If stable, highly selective and inexpensive af- asimplesynthesizedentityorahighmolecularweight Rnityligandswereavailable,thenopportunitieswould protein. The afRnity technique is theoretically of exist for researchers to develop efRcient high yield universal application and any protein can be separ- separationsevenintheearliestphasesofinvestigation. ated whatever its structure and origin. As always, These systems could then be passed forward to pro- there are major limitations. The most effective duction with the knowledge that optimally efR- afRnity ligands are other proteins. Unfortunately cient separations are immediately achievable. such proteins are difRcult to Rnd, identify, isolate Production costs of any pure material reSect the andpurify.Thisresultsinhighcosts.Anevengreater absolute purity level required and the difRculty of deterrent is that most proteins are chemically, cata- achieving it. Therapeutic proteins have high purity lytically and enzymically unstable,a particularlyun- requirements and the larger the administered dose, attractivefeatureiftheyaretobeusedforthemanu- the purer it has to be. Since many protein pharma- facture of therapeutic substances; and regulatory ceuticalswillbeusedathighdoselevels,puritiesneed authorities generally reject applications using pro- to exceed 99%, occasionally up to 99.999%. That teinaceous ligands. these purities can be met by traditional methods is In anticipating that one day stable inexpensiveaf- possible,butitiswidelydocumentedthattheapplica- Rnitymediawouldbeindemand,ateamledbyC.R. tionof such methodsmassively increasesproduction Lowe began an investigation into which synthetic costs.Between50and80%oftotalproductioncosts ligand structures offered the greatest possibility oftherapeuticproteinsareincurredatthepuriRcation ofdeveloping inexpensivestable ligands. It was con- stage.Themanufacturingcostofaproductisdirectly cludedthatstructuresthatcouldbemanipulatedinto related to its concentration in the mother liquor; speciRcspatialgeometriesandtowhichintermolecu- the more dilute it is, the higher the cost of recov- lar binding forces could easily be added offered ery. Since traditional puriRcation processes on their the highest chance of success. Model compounds owncannotselectivelyconcentrateatargetproteinto were already available; the textile dyes. the exclusion of all others, they have to be used in series. The number of stages required can vary be- Synthetic Ligands tween fourand15. Each step representsa yield loss, andincursaprocessingcost.Yieldsoflessthan20% Textiledyeshadalreadyprovedtobesuitableligands arenotuncommon.Figure 3showsanenzymepuriR- forprotein separations.Blood proteins,dehydrogen- ed in multiple stages and by a one-step afRnity ases,kinases,oxidases,proteases,nucleases,transfer- process. ases and ligases can be puriRed by a wide variety of Itwas theselimitationsthatcausedbiochemiststo dyes. However, they did not prove to be the break- examine highly selective ligands. Almost any com- throughsoeagerlyawaited.Anessentialfeatureofall pound can be used as an afRnity ligand provided chromatographic processes is exact repeatability it can be chemically bonded onto a support matrix fromcolumntocolumn,yearafteryear.Textiledyes and,onceimmobilized,itretainsitsabilitytointeract are bulk chemicals, most of which contain many with the protein to be puriRed. The ligand can be by-products, co-produced at every stage of the dye Figure3 Comparisonofmultistepversusaffinityseparation. 10 I/AFFINITYSEPARATION/Derivatization manufacturingprocess.Thisfact alonemakesrepro- ducibility problematic. Furthermore, the bonding process between dye and matrix was poorly re- searched.Thisresultedinextensiveleakage.Allcom- mercially available textile dye products leak exten- sively, especially under depyrogenating conditions (Figure 4). Despite these limitations, it was recog- nizedthatdye-likestructureshadapowerfulunderly- ingabilitytoseparateaverydiverserangeofproteins. Their relatively complex chemical structures allow spatial manipulation of their basic skeletons into aninRnite variety of shapesand conRgurations.Pro- teinsare complexthree-dimensional(3-D)structures and folds are present throughout all protein structures. An effective ligand needs to be shaped in Figure5 Schematicrepresenttionofligand}proteininteraction. such a manner that it allows deep insertion into W, electrostatic interaction; X, hydrogen bonding; Y, van der a suitable surface Rssure existing within the 3-D Waals interaction; Z, hydrophobic interaction. ***, original structure (Figure 5). In contrast, if the ligand only backbone; ---, new structure added; 2, original backbone interacts with groups existing on external surfaces, move; ,fieldsofinteraction. then nonspeciRc binding results and proteins other than the target are also adsorbed. A much more selectiveapproachistoattempttoinsertaligandinto ing;Y,vanderWaals;Z,hydrophobic)alignwiththe an appropriate fold of the protein, and add binding binding areas in the protein fold, idealized afRn- groupsto correspondwith those presentin a fold of ityreagentsresult.Theuseofspacerarmsminimizes the protein. If all four of the basic intermolecular steric hindrance between the carrying matrix and forces(Figure 5:W,electrostatic;X,hydrogenbond- protein. Figure4 Leakageofbluedyefromvariouscommercialproducts. ,0.1molL(cid:1)1NaOH; ,0.25molL(cid:1)1NaOH; ,1molL(cid:1)1 NaOH.Key:A,MimeticBlue1A6XL(affinitychromatography);B,Affi-GelBlue(Bio-Rad);C,BlueTrisacryl-M(IBF);D,FractogelTSK AF-Blue(Merck);E,C.I.ReactiveBlue2polyvinylalcohol-coatedperfluropolymersupport;F,BlueSepharoseCL-6B(Pharmacia);G, immobilizedCibacronBlueF3G-A(Pierce);H,CibacronBlueF3G-A"Si500(Serva);I,ReactiveBlue2-SepharoseCL-6B(Sigma). Sepsci*1*TSK*Venkatachala=BG I/AFFINITYSEPARATION 11 The Rnal step is to design appropriate bonding purities,substantialincreasesincolumnlifetime,and technologiesto minimize potential leakage. Until re- improvements in batch-to-batchreproducibility. centlythistypeofmodellingwasapurelytheoretical exercise. It was only the introduction of computer- Rational Design of Af\nity Ligands assistedmolecularmodellingtechniquesthatallowed the theory to be tested. Before the arrival of logical Modi\cationof ExistingStructures modellling the discovery of selective ligands was en- tirely based upon empirical observation, later fol- The Rrst example of a rational design of new bio- lowed by a combination of observation, experience mimetic dyes used the interaction between horse andlimitedassistancefromearlycomputergenerated liveralcoholdehydrogenase(ADH)andanaloguesof models. Although several novel structures evolved the textile dye Cibacron Blue F3G-A (Figure 7). It duringthisperiod,ageneralapproachtothedesignof hadbeenestablishedthattheparentdyebindsinthe new structures remained elusive. At this time only NAD#-binding site of the enzyme, with the an- veryfew3-Dproteinstructureswereavailable,again thraquinone, diaminobenzene sulfonate and triazine greatly restricting application of rational design ap- rings (rings A, B and C, respectively, in Figure 7) proaches. As more sophisticated programmes, simu- apparentlyadoptingsimilar positionsto thoseof the lation techniques, protein fragment data and many adenine adenosine ribose and pyrophosphategroups more proteinstructureswere released, logical design of NAD(cid:1). The anthraquinone ring (A) binds in methods were revolutionized. However, many mil- a wide apolar fold that constitutes, at one end, the lionsofproteinsare involvedinlife processes,andit adenine bridging site, while the bridging ring (B) is isclearthatmanyyearswillelapsebeforethemajor- positionedsuchthatitssulfonategroupinteractswith ityofthesewillbefullydescribedbyaccuratemodels. the guanidinium side chain of Arg271 (Figure 8). Consequentlyintuition and experience will continue Ring C binds close to where the pyrophosphate toplayamajorroleinthedesignofsuitableligands. bridge of the coenzyme binds with the reactive Of availablerationally designedsyntheticmolecules, triazinylchlorineadjacenttothenicotinamideribose- theMimetic(cid:2)(cid:3)rangecancurrentlyseparateover50% binding site. The terminal ring (D) appears to be of a randomly selected range of proteins. Stability boundin a fold between the catalytic and coenzyme under depyrogenating conditions has been demon- binding domains, with a possible interaction of the strated for these products (Figure 6). This results in sulfonatewiththesidechainofArg369.Thebinding minimal contamination from ligand and matrix im- ofdyetohorseliverADHresemblesADPbindingbut differs signiRcantly at the nicotinamide end of themoleculewiththemid-pointpositionofringDdis- placedfromthemid-pointpositionofthenicotinamide ringofNAD#byabout1nm.Consequentlyanumber ofterminal-ringanaloguesofthedyeweresynthesized andcharacterizedinanattempttoimprovethespeciR- cityofdyebindingtotheenzyme.Table 4listssomeof the analogues made by substituting }R in the D ring (Figure 7), together with their dissociation constants. These data show that small substituents bind more tightlythanbulkiergroups,especiallyifsubstitutedin theo-orm-positionswithaneutraloranionicgroup. Further inspection of the computer model given as Figure 8showedthatthedyeanaloguesweretooshort and rigid to bind to horse liver ADH in an identical manner to the natural coenzyme, NAD#. Conse- quentlyanaloguesoftheparentdyeweredesignedand synthesized with central spacer functionalities to increase the length and Sexibility of the molecule (Figure 9). This product proved to be some 10 times superior to any previously synthesized compound. This work provided the Rrst proof that rationally Figure6 Comparison of ligand leakage from mimetic ligand designed molecules could be converted into stable, affinityadsorbentA6XL( )andconventionaltextiledyeagarose inexpensive, chromatographic media,whileproviding ( ). the most remarkable separations. 12 I/AFFINITYSEPARATION/Derivatization Figure7 Principalstructuralelementsoftheanthraquinonedye,CibacronBlueF3G-A. De NovoDesign mimicthebindingofnaturallyoccurringanionichet- erocycles such as NAD#, NAPD#, ATP, coenzyme Most early efforts in proving the rational design A, folate, pyridoxal phosphate, oligonucleotides technology was based upon dye structures. To date and polynucleotides. However, some proteins, all dyes considered have been anionic, presumably particularlyproteolyticenzymes,interactwithcationic because the charged chromophores of these ligands substrates. The trypsin-like family of enzymes forms Figure8 Putativebindingpocketfortheterminal-ringanalogue(m-COO(cid:1))ofCibacronBlueF3G-Ainthecoenzymebindingsiteof horseliveralcoholdehydrogenase(ADH).Thesitelieslateraltothemaincoenzymebindingsiteandcomprisesthesidechainsoftwo juxtaposedcationicresiduesArg47andHis51.