Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications. (2024)

The interactions between biochemicals and chemicals in the body areimportant in many clinical processes. Examples include the binding ofantibodies with antigens, the interactions of hormones with theirreceptors, and the binding of drugs with their biological targets orcarrier agents (1,2). These interactions are usually reversible andrange from having a weak-to-high binding strength, or"affinity." These systems may also be highly selective intheir binding (e.g., an antibody-antigen interaction) or more general innature (e.g., the binding of drugs with a serum transport protein)(1--4). A variety of methods have been employed to study these and othertypes of biological interactions. These techniques have ranged fromequilibrium dialysis and ultrafiltration to x-ray crystallography,absorption or fluorescence spectroscopy, surface plasmon resonance (SPR)spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy (3--5).This review discusses an alternative group of techniques that are basedon affinity chromatography.

Affinity chromatography is a type of liquid chromatography in whichthe stationary phase is an immobilized form of a biologically-relatedbinding agent. This binding agent, or "affinity ligand," isused to retain specific compounds from applied samples (6). The presenceof such an agent results in a separation method that uses the samereversible and selective interactions that are present in manybiological systems. This property has often been employed in affinitychromatography to purify, extract, or remove a given chemical orbiochemical from a sample for either preparative work oranalytical-scale applications (6). The use of a biologically relatedagent as the stationary phase also gives this method the ability tostudy and model the interactions that occur between chemicals andbiochemicals in living systems. This is true for both traditionalaffinity chromatography and high-performance affinity chromatography(HPAC), with the latter making use of HPLC supports and instrumentationto carry out an affinity-based separation or analysis (5--10).

This review will look at various formats that have been used inthese methods to characterize the strength or rate of a biologicalinteraction and the number and types of sites that are involved in thesebinding processes. It will also show how these methods can be used tostudy the interactions between several solutes for the same bindingagent and to screen the interactions of many compounds with a givenbiological target (5--10). An emphasis will be placed on recentapplications of these methods, and particularly those involving HPAC.Finally, recent trends in these methods and possible future directionsfor these techniques will be discussed.

General Approaches in Affinity Chromatography for Binding Studies

There are several methods by which biological interactions can beexamined by affinity chromatography and HPAC (Table 1). One commonapproach is to use zonal elution (8, 10). Zonal elution involves theinjection of a small sample plug onto a chromatographic system, followedby separation of the peaks that result from this injection, as isillustrated in Fig. 1A (11). This is the format that is most commonlyused in other types of liquid chromatography for chemical measurementand identification. However, this format can also be used in affinitychromatography and HPAC to obtain information on a biologicalinteraction by using the peak profile or retention time that isgenerated for a given compound with the immobilized binding agent (5, 8,10).

As will be shown later, zonal elution can be employed with affinitychromatography and HPAC to determine the binding strength of a compoundwith an immobilized agent, the type of competition this compound mayhave with other chemicals for the binding agent, and the affinity andlocation of these binding sites. It is further possible in this type ofexperiment to look at the effects of pH, temperature, ionic strength,and solution composition on a biological interaction (8, 10). Someadvantages of using zonal elution for such work are that it needs only asmall amount of the injected compound for each experiment, the bindingagent can often be reused for hundreds of studies, and it is possible tosimultaneously look at more than 1 compound in a sample by usingappropriate separation or detection conditions to examine anddifferentiate between these compounds as they elute from the column.This method also tends to give good precision and fast analysis times,especially when used in HPAC (8) (Fig. 1A).

Frontal analysis is a second method that is often used in affinitychromatography and HPAC to characterize biological interactions (7, 8,10). This technique is sometimes called frontal affinity chromatography(FAC) (10, 12). In frontal analysis, a solution with a knownconcentration of a chemical or biochemical is passed in a continuousmanner through a column that contains the desired binding agent, as isshown in Fig. 1B. As the binding sites for the applied compound in thecolumn become occupied, more of this compound will elute and produce abreakthrough curve (7, 8, 10). An example of such a curve is alsoprovided in Fig. 1B (13).

The mean position of the curve that is obtained in frontal analysiscan be examined as a function of the concentration of the appliedcompound. This information can then be compared to various bindingmodels to determine the types of interactions that are occurring in thecolumn, the amount of binding sites that are present, and theequilibrium constants for these sites (7, 8, 10, 12). The ability offrontal analysis to simultaneously provide information on both theamount of binding sites and the equilibrium constants for these sites isan important advantage of this method over zonal elution. Frontalanalysis is also valuable for providing information on the overall modelfor an interaction between the applied compound and its binding agent. Adisadvantage of this approach when compared to zonal elution is thatfrontal analysis usually requires more of the applied chemical andadditional time because many solutions of a compound are often needed tocarry out a binding study. However, the time needed for a frontalanalysis study in HPAC can still be relatively fast (7), as illustratedin Fig. 1B.

MEASUREMENT AND COMPARISON OF OVERALL BINDING

One way that affinity chromatography and HPAC can be used is tocharacterize the overall binding of a chemical or biochemical with animmobilized binding agent. Frontal analysis is often used for thispurpose (7, 8, 10). An example is shown in Fig. 2, in which HPAC wasemployed to look at interactions of the drug enantiomers R- andS-propranolol with LDL (14). The data obtained by frontal analysisindicated that a simple nonsaturable binding model (e.g., partitioninginto the nonpolar core of LDL) was present in the case of S-propranolol,while a mixture of saturable binding and nonsaturable interactions waspresent for R-propranolol (e.g., binding with apolipoproteins pluspartitioning into LDL) (14). A similar approach has been used to studythe interactions of R/S-propranolol and verapamil with HDL (15) and thebinding of R/S-propanolol with VLDL (16). HPAC and affinitychromatography have also been employed in examining the binding ofvarious solutes and drugs with the transport proteins[[alpha].sub.1]-acid glycoprotein (AGP) or human serum albumin 1HSA)(10, 17, 18), the binding of urediofibrate-like dual agonists withperoxisome proliferator-activated receptors (19), and the interactionsof many compounds with immobilized enzymes or lectins (12, 20--22).

Information on the overall interactions of a compound with animmobilized binding agent can also be provided by zonal elution. Forinstance, this can be accomplished by using the retention time([t.sub.R]) or corresponding retention factor (k), where k =([t.sub.R]--[t.sub.M])/[t.sub.M] and [t.sub.M] is the column void time,for a given compound and binding agent. If the rates of association anddissociation between the compound and binding agent are relatively fastcompared to the time the compound spends in the column, the values of[t.sub.R] and k will depend on both the number of binding sites for thiscompound in the column and equilibrium constants for these sites (8).This type of experiment has been used in HPAC to compare the binding ofseveral sulfonylurea drugs and site-selective probes for HSA on columnsthat contained normal or glycated forms of this protein (i.e., as occurduring diabetes) (23). A similar approach has been used to screenvarious drugs for their binding to HSA or AGP (13).

Site-Specific Binding and Competition Studies

Another use of zonal elution in HPAC and affinity chromatographyhas been in characterizing the competition that one compound may havewith another for a given binding agent. This approach has been employedto examine the interactions of many drugs, hormones, and other soluteswith proteins or to measure the binding constants for a compound at aparticular site on these binding agents (8, 10). This experiment isusually carried out by placing a drug or competing agent at a knownconcentration in the mobile phase. A small amount of a second drug,solute, or site-selective probe is then injected in the presence of thismobile phase and onto a column that contains the binding agent. As theinjected compound goes though the column, it will compete for bindingsites with the drug or solute that has been added to the mobile phase.During this process, the solute in the mobile phase will affect theretention of the injected compound if these 2 chemicals share bindingsites or have allosteric effects between their binding regions. Theresulting data can be used to determine the types of interactions thatare present between these chemicals and the equilibrium constants forthese processes (8).

An example of this type of study is provided in Fig. 3 (11), as wasconducted by using HPAC. In this case, the binding of a drug (i.e.,glimepiride) at a specific site on an immobilized protein (i.e., normalHSA or a glycated form of HSA) was examined by placing the drug into themobile phase while injections were made of a second compound that wasknown to bind at only a specific region on the protein (i.e.,L-tryptophan, which binds to Sudlow site II of HSA). In this type ofstudy, the retention factor for the injected compound would be expectedto change as the concentration of the drug is varied in the mobile phaseif these 2 solutes compete directly at the selected site (8). In thissituation, a plot of 1/k vs the concentration of the drug should give alinear relationship with a positive slope. The linear relationships thatwere obtained (Fig. 3) made it possible to determine the equilibriumconstants for glimepiride at Sudlow site II on each type of HSA that wasexamined (11, 24). The same method has been used to examine thecompetition of other site-specific probes and drugs during theirinteractions with normal or modified HSA and with AGP (17, 18, 25--27).Similar approaches have been used to investigate additionalinteractions, including multisite binding and allosteric effects (8,28-32).

An alternative approach that can be used to detect allostericeffects or direct competition is to employ a plot of[k.sub.0]/(k--[k.sub.0]) vs the reciprocal of the competing agent'sconcentration in the mobile phase (33). In this type of plot, [k.sub.0]is the retention factor for the injected solute in the absence of thecompeting agent, and k is the retention factor in the presence of agiven concentration of the same competing agent. This plot results in alinear relationship for systems with either direct competition at asingle site or that have simple allosteric effects between the mobilephase additive and injected compound. The response of this plot can beused to obtain the association equilibrium constant for the competingagent at its binding site and the coupling constant for the effect ofthis competing agent on binding by the injected compound with theimmobilized agent. An important advantage of this method is it can beused to examine both directions of an allosteric effect between 2compounds that bind to the same agent (33, 34). This approach has beenused to study the allosteric interactions between warfarin and tamoxifenon HSA (34), the effect of tolbutamide on the binding of S-warfarin withHSA (27), and the allosteric effects that occur as S-propranolol andwarfarin bind to AGP (17).

FAC coupled with mass spectrometry (FAC-MS) is another approachthat has been employed to examine the competition of compounds forbinding agents in affinity columns (32, 35). In this method, a soluteand competing agent are both placed into the mobile phase and appliedsimultaneously to the column. The breakthrough curve observed for thesolute is then viewed as a function of the concentration of thecompeting agent to look for the presence of direct competition orallosteric effects (32, 35). This method can also be used to compare aseries of competing agents and to obtain the equilibrium constantsbetween these agents and the immobilized agent (19, 35-38). FAC-MS hasbeen used in this format with open-tubular capillaries to comparevarious ureidofibrate-like dual agonists for their binding to domains ofperoxisome proliferator-activated receptors (19). A similar method hasbeen used to determine the binding constants for several flavonoids withthe histone deacetylase SIRT6 and to characterize the ability of theseagents to displace quercetin from SIRT6 (35).

Screening Drug Candidates

A related application of HPAC and affinity chromatography has beenas a tool for screening drug candidates and potential binding partnersfor biological targets. This work has often involved the use of FAC-MS(12). This technique has been employed to screen mixtures of drugcandidates for their binding to enzymes, lectins, antibodies, andreceptors (12, 37--41). The chemicals that have been screened by thisapproach have included peptides, oligosaccharides, and enzyme inhibitors(12, 37--41).

MS has also been combined with zonal elution and affinity columnsfor the high-throughput screening of binding by chemicals to animmobilized agent. The use of this combination with systems that haveweak-to-moderate interactions [i.e., association equilibrium constantsof [10.sup.5]--[10.sup.6] mol/L [(M).sup.-1] or less] is known as weakaffinity chromatography-MS (WAC-MS) (5, 42-45). This is illustrated inFig. 4, where this technique was used to test various drug fragments fortheir binding to the molecular chaperone heat shock protein 90 (HSP90; apotential target for the treatment of cancer and other diseases) (42).WAC-MS has also been used to examine the binding of drugs to albumin(18, 46). In addition, this method has been employed to look at theinteractions of mixtures containing various compound fragments withenzymes such as kinases or proteases (43-45).

Characterization of Binding Sites

The use of affinity chromatography and HPAC in competition studiesis one way in which the interaction sites of a particular drug or solutecan be identified and studied on a protein or other type of bindingagent (8, 32). However, it is possible to also use these methods toobtain even more information about the nature of this interaction andbinding regions. An example is the use of various temperatures duringzonal elution or frontal analysis studies to determine the changes inenthalpy and entropy that occur during the retention of a compound by animmobilized agent. Changes in the pH, ionic strength, or polarity of themobile phase can be employed in a similar manner to see whetheracid/base interactions, hydrogen bonding, coulombic forces, and/ornonpolar interactions contribute to this binding (8).

Another way to characterize a particular site on a binding agent isto compare the retention or affinities of this agent for a series ofrelated compounds. This approach can be used to provide a generalpicture of the structural features that are most important to thebinding process and on the types of forces that create such binding (8,47, 48). This type of data can also be used to create a quantitativestructure-retention relationship, in which the retention factorsmeasured for a series of related molecules are compared to parametersthat describe various structural and physical features of thesecompounds. Once the structural/physical parameters that are mostimportant to this retention are identified, this information can be usedto develop a model of the binding site and in how it interacts with suchcompounds (49-51). Quantitative structure--retention relationships havebeen used with HPAC to study the binding of benzodiazepines to HSA (50),the interactions of [beta]-adrenolytic drugs, antihistamines, and otherdrugs with AGP (8, 10, 51-54), and the ability of various organicmolecules to take part in skin permeation through the use of a columnthat contained keratin (49).

A complementary approach that can be used is to see how theretention of a solute or group of solutes is altered as a change is madein the structure of a binding agent in an affinity column. For instance,this method has been employed in HPAC to look at how modifying specificamino acids (e.g., Cys-34 or Trp-214) on HSA affects the ability of thisprotein to bind to various drugs (55-58). This approach has also beenused to see how the glycation of HSA changes the binding by this proteinto sulfonylurea drugs and other pharmaceutical agents, as is illustratedin Fig. 3 (11, 25, 26). This latter work has recently been performedwith samples of in vivo glycated HSA from individual patients, makingthis approach a possible future tool for personalized medicine (25).

Kinetic Methods

Yet another way affinity chromatography and HPAC can be used is toprovide data on the kinetics of a biological interaction. Manytechniques have been developed for acquiring such information (7, 8,59). One method that can be used for a system with weak-to-moderatebinding is to make band-broadening measurements (59). This can be doneby measuring and comparing the plate heights for a solute at 1 orseveral flow rates on an affinity column and on an inert control column.The difference in these plate height values is then employed todetermine the portion of the plate height that is a result of stationaryphase mass transfer [H.sub.k]). This term can then be directly relatedto the dissociation rate constant ([k.sub.d]) for the solute in itsinteraction with the immobilized binding agent (7, 59).

In a version of this technique that is known as the plate heightmethod, a plot is made of [H.sub.k] as a function of the flow rate (orlinear velocity) and the retention factor of the solute. This plot isthen used to provide the value of [k.sub.d] for the immobilized agentwith the injected solute (7). This approach has been used tocharacterize the association and dissociation rates of HSA with solutesand drugs such as D/L-tryptophan and R/S-warfarin (60, 61). Another formof this technique is peak profiling, which is based on the difference inthe total plate heights that are acquired for a solute on an affinitycolumn and a control column. This difference may be examined at either asingle flow rate or at several flow rates and is again used to find thevalue of the dissociation rate constant (59). This second approach hasbeen used to determine the dissociation rate constants of HSA forcarbamazepine, imipramine, and L-tryptophan (62,63). In addition, thismethod has been used with a second column containing a chiral stationaryphase to determine the dissociation rates for HSA with some chiralmetabolites of phenytoin (64).

The peak decay method is an alternative approach for examining therate of dissociation for biological systems (7, 59). This technique usessmall affinity columns and conditions that promote dissociation overrebinding of a retained solute. One form of this method involves theapplication of a solute to an affinity column, to which the solute hasmoderate-to-strong binding, followed by the application of a displacingagent to quickly elute the retained solute. The result is an elutionprofile that forms a first-order decay curve and in which the slopeprovides the dissociation rate constant for the solute (7, 59). Analternative approach that can be used with systems that haveweak-to-moderate strength interactions is to use small affinity columnsand fast flow rates, which can allow this type of study to be conductedwithout the need for a displacing agent (59, 65). In addition, a stepchange in a factor such as the mobile phase pH can sometimes be used topromote rapid release of the solute, as might be used to compare elutionconditions for an affinity column (17, 66).

The peak decay method has been used in HPAC with small affinitycolumns to determine the dissociation rates of AGP for amitriptyline,lidocaine, and nortriptyline (67). This method has also been used withaffinity monolith columns to determine the dissociation rate constantsfor HSA with a number of drugs (67, 68). A pH step change has beenemployed with the peak decay method to characterize the dissociationrate of thyroxine from antibodies and aptamers that can bind to thishormone (69).

The split-peak method is a technique that can be used with affinitycolumns to look at systems with strong binding and relatively slow ratesof dissociation (7, 8, 59). In this technique conditions are used inwhich only part of an injected solute has sufficient time to bind to theaffinity column. These conditions are typically created by using a smallcolumn and/or a high flow rate. The result is a situation in which theinjection of even a pure sample of the solute results in 2 peaks, one ofwhich is highly retained and the second of which elutes as a nonretainedfraction. The change in the relative size of these peaks is thenexamined as the injection flow rate is varied and used to determine theassociation rate constant or mass transfer rate for the solute in thecolumn (7, 8, 59).

The split-peak method has been used in several studies to examinethe binding of immunoglobulins to small affinity columns that containprotein A or protein G (59, 70). This method has been used to optimize aclinical assay based on HPAC for the rapid measurement of IgG-classantibodies in serum by using protein A columns (59). Forms of thismethod have also been employed to examine the binding of HSA toimmobilized anti-HSA antibodies and to characterize the rate of bindingby thyroxine with antibodies or aptamers (59, 66).

Ultrafast affinity extraction is a newer method that can examineboth the rate and degree of binding for a biological interaction (59,71-75). This method looks at an interaction in solution through the useof a small affinity column that can rapidly capture the nonbound form ofa solute in an injected mixture of this solute and a soluble bindingagent. The relative size of the captured fraction is used to determinethe rate or extent of the biological interaction. The immobilized agentused to capture the free form of the solute may be an antibody (72, 74,75) or a more general binding agent such as HSA (i.e., which can be usedto retain many drugs) (71, 73). Conditions are used so that the time thesample spends in the column is small or comparable to the time neededfor dissociation of the solute from its soluble binding agent. Thisoften involves the use of affinity microcolumns and flow rates thatproduce sample residence times in the column that span from the lowmillisecond range up to a few seconds (59).

Ultrafast affinity extraction has been used for systems that rangefrom weak-to-strong binding (59, 72, 75). For instance, this method hasbeen used with columns that contained immobilized antibodies fordetermining the free fraction of warfarin in mixtures of this drug withHSA (74) and, in combination with flow-based displacement immunoassays,the free fractions of thyroxine and phenytoin in clinical samples (72,75). This method has been used with columns containing HSA to measurethe equilibrium constants for a soluble form of this protein with R- orS-warfarin, S-ibuprofen, and imipramine (71). Ultrafast affinityextraction has also been used in a 2-column system and with a chiralstationary phase to measure the free fractions and binding constants ofR- and S- warfarin in drug-protein mixtures and serum (Fig. 5) (73).

Conclusion and Future Directions

This review discussed a variety of ways in which affinitychromatography has been used to examine biological interactions ofinterest in clinical or pharmaceutical analysis. Particular attentionwas given to more recent work and techniques that have involved the useof HPAC. The general principles behind techniques such as zonal elutionand frontal analysis were described, showing how these approaches can beused to examine the strength of a biological interaction, the number andtypes of sites that are involved in these processes, and the effects ofone solute on another during these interactions. Several means forexamining the kinetics of a biological interaction by HPAC and affinitychromatography were also considered. Systems that have been examined bythese methods have ranged from the binding of drugs and hormones toproteins or receptors to the analysis of antibody-antigen,enzyme-inhibitor, and sugar-lectin interactions (6-10, 12, 18).

There are many features of these methods that have made themattractive for such work. For instance, as shown in this review, thesetechniques can be used with many binding agents or formats and can becoupled with numerous detection methods, spanning from absorbancemeasurements to MS (8-10, 12). This is often possible using approacheswith label-free detection, although these methods can also be combinedwith suitable tags for the study of trace analytes (8, 72, 75). Thespeed and precision of these methods, especially when used in HPAC, areother valuable features. The ability to often reuse the same immobilizedbinding agent (i.e., in many cases hundreds of injections) is anotheruseful feature of this approach (8-10).

Many of the recent developments in this field will probablycontinue and further expand the capabilities of these methods. Forinstance, it is expected that columns based on monoliths and supportsfor ultraperformance liquid chromatography will continue to be adaptedfor use in HPAC and affinity chromatography (6, 10, 2022). This shouldallow even more rapid assays to be created with these methods and willprovide greater ease in coupling affinity columns with MS or otheranalytical techniques (5,12,19,38-41). Further work in theminiaturization of affinity columns and systems is also anticipated (5,10, 23, 37). This work has already led to the possibility of carryingout binding studies on relatively exotic binding agents (e.g.,lipoproteins, receptors or modified forms of proteins) and even bindingagents that have been obtained from individual patients (e.g., glycatedHSA) (14-16, 25-27, 36). These efforts, in turn, have resulted in theproposed use of HPAC as a tool in personalized medicine (25).

Another trend that is expected to continue is the creation of newformats for affinity-based binding assays. A specific example that wasdescribed is ultrafast affinity extraction, which can provide a directmeasure of the strength or rate of biological interactions in solutionand which can even examine these processes directly in clinical samples(59, 72-75). The combination of these recent tools and formats withthose that are already available should result in even more futureapplications for affinity chromatography and HPAC in thecharacterization of biological interactions for clinical studies orpharmaceutical analysis.

Author Contributions: All authors confirmed they have contributedto the intellectual content of this paper and have met the following 3requirements: (a) significant contributions to the conception anddesign, acquisition of data, or analysis and interpretation of data; (b)drafting or revising the article for intellectual content; and (c) finalapproval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Uponmanuscript submission, all authors completed the author disclosure form.Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: D.S. Hage, Eastern Analytical Symposium and Abbvie.

Research Funding: D.S. Hage, Portions of this work wereinstitutionally supported by the NIH under grants R01 GM044931 and R01DK069629.

Expert Testimony: None declared.

Patents: None declared.

Other Remuneration: D.S. Hage, Elsevier.

References

(1.) Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbook ofclinical chemistry and molecular diagnostics. 5th Ed. St Louis (MO):Saunders; 2012.

(2.) Clarke W, editor. Contemporary practice in clinical chemistry.3rd Ed. Washington: AACC Press; 2016.

(3.) Williams MA, Daviter T, editors. Protein-ligand interactions,methods and applications. New York: Springer; 2013.

(4.) Cantor CR, Schimmel PR. Biophysical chemistry, part 2:techniques for the study of biological structure and function, part 2.San Francisco: Freeman; 1980.

(5.) Ohlson S, Duong-Thi MD. Emerging technologies for fragmentscreening. In: Erlanson DA, Jahnke W. Fragment-based drug discovery:lessons and outlook. 1st Ed. Weinheim: Wiley-VCH Verlag; 2016. p 173-95.

(6.) Hage DS, editor. Handbook of affinity chromatography, 2nd Ed.Boca Raton: CRC Press; 2006.

(7.) Chaiken IM. Analytical affinity chromatography. Boca Raton:CRC Press; 1987.

(8.) HageDS, Chen J. Quantitative affinity chromatography:practical aspects. In: Hage DS, editor. Handbook of affinitychromatography, 2nd Ed. Boca Raton: CRC Press; 2006. p 595-628.

(9.) Winzor DJ. Quantitative affinity chromatography: recenttheoretical developments. In: Hage DS, editor. Handbook of affinitychromatography, 2nd Ed. Boca Raton: CRC Press; 2006. p 629-62.

(10.) Zheng X, LiX, Beeram S, Podariu M, Matsuda R, Pfaunmiller EL,et al. Analysis of biomolecular interactions using affinitymicrocolumns: a review. J Chromatogr B AnalytTechnol Biomed Life Sci2014;968:49-63.

(11.) Matsuda R, Li Z, Zheng X, Hage DS. Analysis of multisitedrug-protein interactions by high-performance affinity chromatography:binding of glimepiride to normal or glycated human serum albumin. JChromatogr A2015;1408:133-44.

(12.) Schriemer DS. Biosensor alternative: frontal affinitychromatography. Anal Chem 2004;76:440A-8A.

(13.) Bi C, Jackson A, Vargas-Badilla J, Li R, Rada G, Anguizola J,et al. Entrapment of [alphal.sub.1]-acid glycoprotein inhigh-performance affinity columns for drug-protein binding studies. JChromatogr B 2016;1021:188-96.

(14.) Sobansky MR, Hage DS. Identification and analysis ofstereoselective drug interactions with low density lipoprotein byhigh-performance affinity chromatography. Anal Biochem Chem2012;403:563-71.

(15.) Chen S, Sobansky MR, Hage DS. Analysis of drug interactionswith high-density lipoprotein by high-performance affinitychromatography. Anal Biochem 2010;397:107-14.

(16.) Sobansky MR, Hage DS. Analysis of drug interactions with verylow density lipoprotein by high-performance affinity chromatography.Anal Biochem Chem 2014; 406:6203-11.

(17.) Anguizola J, Bi C, Koke M, Jackson A, Hage DS. Oncolumnentrapment of [alphal.sub.1]-acid glycoprotein for studies ofdrug-protein binding by high-performance affinity chromatography. AnalBioanal Chem 2016; 408:5745-56.

(18.) Hage DS, Anguizola JA, Jackson AJ, Matsuda R, Papastavros E,Pfaunmiller E, etal. Chromatographicanalysis of drug interactions in theserum proteome. Anal Methods 2011;3:1449-60.

(19.) Temporini C, Pochetti G, Fracchiolla G, Piemontese L,Montanari R, Moaddel R, et al. Open tubular columns containing theimmobilized ligand binding domain of peroxisome proliferator-activatedreceptors [alpha] and [gamma] for dual agonists characterization byfrontal affinity chromatography with mass spectrometry detection. JChromatogr A 2013;1284:36-43.

(20.) Tetala KK, Chen B, Visser GM, van Beek TA. Single stepsynthesis of carbohydrate monolithic capillary columns for affinitychromatography of lectins. J Sep Sci 2007; 30:2828-35.

(21.) Kovarik P, Hodgson RJ, Covey T, Brook MA, Brennan JD.Capillary-scale frontal affinity chromatography/MALDI tandem massspectrometry using protein-doped monolithicsilica columns. Anal Chem2005;77:3340 -50.

(22.) Hodgson RJ, Chen Y, Zhang Z, Tleugabulova E, Long H, Zhao X,et al. Protein-doped monolithic silica columns for capillary liquidchromatography prepared by the sol-gel method: applications to frontalaffinity chromatography. Anal Chem 2004;76:2780-90.

(23.) Jackson AJ, Anguizola J, Pfaunmiller EL, Hage DS. Use ofentrapment and high-performance affinity chromatography to compare thebinding of drugs and site-specific probes with normal and glycated humanserum albumin. Anal Bioanal Chem 2013;405:5833-41.

(24.) Anguizola J, Matsuda R, Barnaby OS, Hoy KS, Wa C, Debolt E,et al. Review: glycation of humanserum albumin. Clin Chim Acta2013;425:64-76.

(25.) Anguizola J, Joseph KS, Barnaby OS, Matsuda R, Alvarado G,Clark W, et al. Development of affinity microcolumns for drug-proteinbinding studies in personalized medicine: interactions of sulfonylureadrugs with in vivo glycated human serum albumin. Anal Chem2013;85:4453-60.

(26.) Matsuda R, Anguizola J, Joseph KS, Hage DS. Analysis of druginteractions with modified proteins by high-performance affinitychromatography: binding of glibenclamide to normal and glycated humanserum albumin. J Chromatogr A 2012;1265:114-22.

(27.) Joseph KS, Hage DS. Characterization of the binding ofsulfonylurea drugs to HSA by high-performance affinity chromatography. JChromatogr B 2010;878:1590-8.

(28.) Bergstrom M, Astrom E, Pahlsson P, Ohlson S. Elucidating theselectivity of recombinant forms of Aleuria aurantia lectin using weakaffinity chromatography. J Chromatogr B Analyt Technol Biomed Life Sci2012; 885-886:66-72.

(29.) Engstrom HA, Johansson R, Koch-Schmidt P, Gregorius K, OhlsonS, Bergstrom M. Evaluation of a glucose sensing antibody using weakaffinity chromatography. Biomed Chromatogr 2008;22:272-7.

(30.) Winzor DJ. Determination of binding constants by affinitychromatography. J Chromatogr A 2004;1037: 351-67.

(31.) Kakita H, Nakamura K, Kato Y. High-performance affinitychromatography of a chick lectin on an adsorbent based on hydrophilicpolymer gel. J Chromatogr 1991; 543:315-26.

(32.) Hage DS. High-performance affinity chromatography: a powerfultool for studying serum protein binding. J Chromatogr B2002;768:3-30.

(33.) Chen J, Hage DS. Quantitative analysis of allostericdrug-protein binding by biointeraction chromatography. Nature Biotechnol2004;22:1445-8.

(34.) Chen J, Hage DS. Quantitative studies of allosteric effectsby biointeraction chromatography: analysis of protein binding to lowsolubility drugs. Anal Chem 2006;78:2672-83.

(35.) Singh N, Ravichandran S, Norton DD, Fugmann SD, Moaddel R.Synthesis and characterization of a SIRT6 open tubular column:predicting deacetylation activity using frontal chromatography. AnalBiochem 2013; 436:78-83.

(36.) Moaddel R, Wainer IW. The preparation and development ofcellular membrane affinity chromatography columns. Nature Protocols2009;4:197-205.

(37.) Schriemer DC, Bundle DR, Li L, Hindsgaul O. Microscalefrontal affinity chromatography with mass spectrometric detection: a newmethod for the screening of compound libraries. Angew Chem Int Ed1998;37: 3383-97.

(38.) Zhang B, Palcic MM, Mo H, Goldstein IJ, Hindsgaul O. Rapiddetermination of the binding affinity and specificity Of the mushroomPolyporussquamosus lectin using frontal affinity chromatography coupledto electrospray mass spectrometry. Glycobiology 2001;11:141-7.

(39.) Calleri E, Fracchiolla G, Montanari R, Pochetti G, LavecchiaA, Loiodice F, et al. Frontal affinity chromatography with MS detectionof the ligand binding domain of PPAR[gamma] receptor: ligand affinityscreening and stereoselective ligand-macromolecule interaction. JChromatogr A 2012;1232:84-92.

(40.) Zhang B, Palcic MM, Schriemer DC, Alarez-Manilla G, Pierce M,Hindsgaul O. Frontal affinity chromatography coupled to massspectrometry for screening mixtures of enzyme inhibitors. Anal Biochem2001;299:173-82.

(41.) Slon-Usakiewicz JJ, Ng W, Dai JR, Pasternak A, Redden PR.Frontal affinity chromatography with MS detection (FAC-MS) in drugdiscovery. Drug Discov Today 2005; 10:409-16.

(42.) Meiby E, Simmonite H, le Strat L, Davis B, Matassova N, MooreJD, et al. Fragment screening by weak affinity chromatography:comparison with established techniques for screening against HSP90. AnalChem 2013; 85:6756-66.

(43.) Duong-Thi MD, Meiby E, Bergstrom M, Fex T, Isaksson R, OhlsonS. Weak affinity chromatography as a new approach for fragment screeningin drug discovery. Anal Biochem 2011;414:138-46.

(44.) Meiby E, Knapp S, Elkins JM, Ohlson S. Fragment screening ofcyclin G-associated kinase by weak affinity chromatography. Anal BioanalChem 2012;404:241-725.

(45.) Duong-Thi MD, Bergstrom M, Fex T, Isaksson R, Ohlson S.High-throughput fragment screening by affinity LCMS. J Biomol Screening2013;18:160-71.

(46.) Ohlson S, Shoravi S, Fex T, Isaksson R. Screening fortransient biological interactions as applied to albumin ligands: a newconcept for drug discovery. Anal Biochem 2006;359:120-3.

(47.) Loun B, Hage DS. Characterization of thyroxinealbumin bindingusing high-performance affinity chromatography. II. Comparison of thebinding of thyroxine, triiodothyronines and related compounds at thewarfarin and indole sites of human serum albumin. J Chromatogr B BiomedAppl 1995;665:303-14.

(48.) Ohlson S, Bergstrom M, Pahlsson P, Lundblad A. Use ofmonoclonal antibodies for weak affinity chromatography. J Chromatogr A1997;758:199 -208.

(49.) Turowski M, Kaliszan R. Keratin immobilized on silica as anew stationary phase for chromatographic modelling of skin permeation. JPharm Biomed Anal 1997;15: 1325-33.

(50.) Kaliszan R, Kaliszan A, Noctor TAG, Purcell WP, Wainer IW.Mechanism of retention of benzodiazepines in affinity, reversed-phaseand adsorption high-performance liquid chromatography in view ofquantitative structure retention relationships. J Chromatogr A1992;609:69 -81.

(51.) Kaliszan R, Nasal A, Turowski M. Quantitativestructure-retention relationships in the examination of the topographyof the binding site of antihistamine drugs on [alphal.sub.1]-acidglycoprotein. J Chromatogr A 1996;722:25-32.

(52.) Karlsson A, Aspegren A. Enantiomeric separation of aminoalcohols on protein phases using statistical experimental design: acomparative study. J Chromatogr A 2000;866:15-23.

(53.) Fitos I, Visy J, Simonyi M, Hermansson J. Chiralhigh-performance liquid chromatographic separations of vinca alkaloidanalogues on [alphal.sub.1]-acid glycoprotein and human serum albumincolumns. J Chromatogr A 1992;609:163-71.

(54.) Gyimesi-Forras K, Szasz G, Gergely A, Szabo M, Kokosi K.Optical resolution of a series of potential cholecystokinin antagonist4(3H)-quinazolone derivatives by chiral liquid chromatography onalpha(1)-acidglycoprotein stationary phase. J Chromatogr Sci2000;38:430-4.

(55.) Noctor TAG, Wainer IW. The in situ acetylation of animmobilized human serum albumin chiral stationary phase forhigh-performance liquid chromatography in the examination ofdrug-protein binding phenomena. Pharm Res 1992;9:480-4.

(56.) Chattopadhyay A, Tian T, Kortum L, Hage DS. Development oftryptophan-modified human serum albumin columns for site-specificstudies of drug-protein interactions by high-performance affinitychromatography. J Chromatogr B 1998;715:183-90.

(57.) Bertucci C, Nanni B, Raffaelli A, Salvadori P. Chemmodification of human albumin at [Cys.sub.34] by ethacrynicacid:structural characterisation and binding properties. J Pharm Biomed Anal1998;18:127-36.

(58.) Zheng X, Podariu M, Bi C, Hage DS. Development of enhancedcapacity affinity microcolumns by using a hybrid of proteincross-linking/modification and immobilization. J Chromatogr A2015;1400:82-90.

(59.) Zheng X, Bi C, Li Z, Podariu M, Hage DS. Analytical Methodsfor kinetic studies of biological interactions: a review. J Pharm BiomedAnal 2015;113:163-80.

(60.) Loun B, Hage DS. Chiral separation mechanisms inprotein-based HPLC columns. 2. Kinetic studies of (R)and (S)-warfarinbinding to immobilized human serum albumin. Anal Chem 1996;68:1218-25.

(61.) Yang J, Hage DS. Effect of mobile phase composition on thebinding kinetics of chiral solutes on a protein-based HPLC column:interactions of D- and L-tryptophan with immobilized human serumalbumin. J Chromatogr A 1997;766:15-25.

(62.) Schiel JE, Ohnmacht CM, Hage DS. Measurement of drug-proteindissociation rates by high-performance affinity chromatography and peakprofiling. Anal Chem 2009;81:4320-33.

(63.) Tong Z, Schiel JE, Papastavros E, Ohnmacht CM, Smith QR, HageDS. Kinetic studies of drug-protein interactions by using peak profilingand high-performance affinity chromatography: examination of multi-siteinteractions of drugs with humanserum albumin columns. J Chromatogr A2011;1218:2065-71.

(64.) Tong Z, Hage DS. Characterization of interaction kineticsbetween chiral solutes and human serum albumin by using high-performanceaffinity chromatography and peak profiling. J Chromatogr A 2011;1218:6892-7.

(65.) Chen J, Schiel JE, Hage DS. Non-competitive peak decayanalysis of drug-protein dissociation by high-performance affinitychromatography and peak profiling. J Sep Sci 2009;32:1632-41.

(66.) Pfaunmiller E, Moser AC, Hage DS. Biointeraction analysis ofimmobilized antibodies and related agents by high-performanceimmunoaffinity chromatography. Methods 2012;56:130-5.

(67.) Yoo MJ, Hage DS. High-throughput analysis of drugdissociation from serum proteins using affinity silica monoliths. J SepSci 2011;34:2255-63.

(68.) Yoo MJ, Hage DS. Use of peak decay analysis and affinitymicrocolumns containing silica monoliths for rapid determination ofdrug-protein dissociation rates. J Chromatogr A 2011;218:2072-8.

(69.) Nelson MA, Moser AC, Hage DS. Biointeraction analysis byhigh-performance affinity chromatography: kinetic studies of immobilizedantibodies. J Chromatogr B 2010;878:165-71.

(70.) Hage DS, Walters RR, Hethcote HW. Split-peak affinitychromatographic studies of the immobilization-dependent adsorptionkinetics of protein A. Anal Chem 1986;58:274-9.

(71.) Mallik R, Yoo MJ, Briscoe CJ, Hage DS, Analysis ofdrug-protein binding by ultrafast affinity chromatography usingimmobilized human serum albumin. J Chromatogr A 2010;1217:2796-803.

(72.) Ohnmacht CM, Schiel JE, Hage DS. Analysis of free drugfractions using near infrared fluorescent labels and an ultrafastimmunoextraction/displacement assay. Anal Chem 2006;78:7547-56.

(73.) Zheng X, Yoo MJ, Hage DS. Analysis of free fractions forchiral drugs using ultrafast extraction and multidimensionalhigh-performance affinity chromatography. Analyst 2013;138:6262-5.

(74.) Clarke C, Choudhuri AR, Hage DS. Analysis of free drugfractions by ultra-fast immunoaffinity chromatography. Anal Chem2001;73:2157-64.

(75.) Clarke W, Schiel JE, Moser A, Hage DS. Analysis of freehormone fractions by an ultrafast immunoextraction/ displacementimmunoassay: studies using free thyroxine as a model system. Anal Chem2005;77:1859-66.

David S. Hage [1] *

(1) Department of Chemistry, University of Nebraska-Lincoln,Lincoln, NE.

* Address correspondence to the author at: Chemistry Department,University of Nebraska, 704 Hamilton Hall, Lincoln, NE, 68588-0304. Fax402-472-9402; e-mail [emailprotected].

Received September 13, 2016; accepted December 2, 2016.

Previously published online at DOI: 10.1373/clinchem.2016.262253

[C] 2016 American Association for Clinical Chemistry

[2] Nonstandard abbreviations: SPR, surface plasmon resonance; NMR,nuclear magnetic resonance; HPAC, high-performance affinitychromatography; FAC, frontal affinity chromatography; AGP, [alpha]1-acidglycoprotein; HSA, human serum albumin; FAC-MS, FAC with massspectrometry; M, mol/L; WAC-MS, weak affinity chromatography-MS; HSP90,heat shock protein 90.

Caption: Fig. 1. Examples of (A) zonal elution and (B) frontalanalysis experiments for binding studies that were carried out by HPAC.The results given to the right in (A) illustrate the shift in retentionthat was observed for small injections of R-warfarin (i.e., a drug thatbinds to Sudlow site I of human serum albumin) onto a column containingimmobilized human serum albumin in the presence of variousconcentrations of the glimepiride in the mobile phase; the verticaldashed line represents the mean position of the peak for R-warfarin inthe absence of glimepiride. The results given to the right in (B) showthe breakthrough curves that were obtained for the application ofvarious concentrations of carbamazepine to a column that containedimmobilized [alpha.sub.1]-acid glycoprotein. Adapted from Matsuda et al.(11) and Bi et al. (13) with permission from Elsevier.

Caption: Fig. 2. Comparison of data obtained by frontal analysisfor examining the binding of R-propranolol and 5-propranolol with anHPAC column containing immobilized LDL (left) and a general modelshowing the types of interactions each of these enantiomers had with LDL(right). The graph on the left is based on data obtained from Sobanskyand Hage (14).

Caption: Fig. 3. Use of zonal elution-based competition studies inHPAC to examine the direct competition of a drug or solute with aninjected probe for specific binding sites on an immobilized protein, asillustrated by the image on the left for the competition of glimepiridewith L-tryptophan at Sudlow site II on columns that contained normalHSA([??]) or 2 glycated preparations of this protein ([??], [??]). Theimage on the right shows common sites of modification in glycated HSAand the location of the 2 major drug binding sites on this protein,Sudlow sites I and II. Reproduced from Matsuda et al. (11) and Anguizolaet al. (24) with permission from Elsevier.

Caption: Fig. 4. Use of WAC to screen the binding of drugfragments, as illustrated by employing a column containing theimmobilized ATPase domain of HSP90. (A), chromatograms obtained forfragments 46-62, out of a total of 111 drug fragments tested. (B),results-obtained for all 111 drug fragments when screened for theirbinding to HSP90 by using WAC, NMR spectroscopy, SPR spectroscopy, an FP(fluorescence polarization) assay, or a thermal shift assay (Tm), withsome results also being included based on x-ray crystallography and ITC(isothermal titration calorimetry). The hits for binding are indicatedby green (or dark gray) and non-hits are represented by red (or lightgray). Adapted from Meiby et al. (42) with permission from the AmericanChemical Society.

Caption: Fig. 5. Scheme for the simultaneous isolation of a freedrug fraction and separation of the various chiral forms of a drug inthis fraction by using ultrafast affinity extraction and an HPLC chiralstationary phase. This particular example uses an affinity microcolumncontaining immobilized HSA for ultrafast affinity extraction. Reproducedfrom Zheng et al. (73) with permission from The Royal Society ofChemistry.

Table 1. Methods for examining biological interactions by affinitychromatography and HPAC.Technique General principleZonal elution Small plug of a chemical/biochemical is injected onto an affinity column; the profiles or retention times of the resulting peaks are used to provide information on how the injected solutes are interacting with the immobilized agent in the column.Frontal analysis Solution of a chemical/biochemical is passed in a continuous manner through an affinity column; as sites in the column become occupied, a breakthrough curve forms that can provide information on the solute's interactions with the immobilized agent in the column.Plate height and Measurement and comparison of plate heights, suchpeak profiling as for a solute and non-retained solute on anmethods affinity column (containing an immobilized agent) and a control column.Peak decay Measurement of solute dissociation from a smallmethod affinity column under conditions that promote dissociation over rebinding of the solute to the immobilized agent in the column.Split-peak Measurement of the non-retained (or retained) peakmethod area for a solute on an affinity column under conditions where only part of the injected solute has sufficient time to bind to the immobilized agent.Ultrafast Capture and measurement by a small affinity columnaffinity of the non-bound form of a solute in the presenceextraction of a binding agent in solution.Technique ApplicationsZonal elution Characterization of overall binding Competition studies Site-specific studies Screening of drug candidates Characterization of binding sitesFrontal analysis Characterization of overall binding Competition studies Screening of drug candidates Characterization of binding sitesPlate height and Measurement of dissociation rate constants forpeak profiling systems with weak-to-moderate bindingmethodsPeak decay Measurement of dissociation rate constants formethod systems with weak-to-strong bindingSplit-peak Measurement of association rate constants formethod systems with strong binding and slow dissociation ratesUltrafast Measurement of binding strength and-oraffinity dissociation rate constants for systems with weak-extraction to-strong binding