**1.2. Ligand**

Ligands are the molecules that bind reversibly to a specific molecule or group of molecules, enabling purification by affinity chromatography [4]. These molecules which play a major role in the specificity and stability of the system are essential for affinity chromatography [13]. The selected ligand must be capable of selectively and reversibly binding to the substance to be isolated and have some groups which are available for modifications in order to be attached to the support material. It is very important to ensure that the modifications do not reduce the specific binding affinity of the ligands. There are general ligands such as dyes, amino acids, Protein A and G, lectin, coenzyme, methal chelates as well as specific ligands such as enzymes and substrates, antibodies and antigens [19].

Affinity ligands are classified as synthetic and biological. Biological ligands are obtained from natural sources such as RNA and DNA fragments, nucleotides, coenzymes, vitamins, lectins, antibodies, binding or receptor proteins, or in vitro from biological and genetic packages, employing display techniques including oligonucleotides, peptides, protein domains and proteins. Synthetic affinity ligands are generated either by de novo synthesis or modification

of existing molecular structures (triaznyl nucleotide-mimetics, purine and pirimidine deriva‐ tives, non-natural peptides, triazinyl dyes, other triazine-based ligands, oligosaccharide and boronic acid analogues). These can be generated by rational design or selected from ligand libraries. Synthetic ligands are generated using three methods;


Many parameters have to be taken into account in order to select appropriate ligand. Table 1 exhibits the advantages and disadvantages of synthetic and biological ligands. Selectivity and affinity are the main advantages of biological ligands. Such ligands can be generated by *in vitro* evolution approaches and selecting from large combinatorial ligand libraries based on biological/genetic packages. Protein ligands display special advantages for example; higher affinities, higher proteolytic stability, preservation of their biological activity when produced by fusion to a different protein or domain. However these ligands can be expensive and unstable to the sterilization and cleanin conditions used in manufacturing process of biologics because of their biological origin, chemical nature and production methods. There is high contamination risk of the end-product with potentially dangerous leaches, in addition to possible contaminants originated from the biological source [20].


**Table 1.** Comparison of biological and synthetic ligands [20]

Another format developed in 1990s was the *continuous bed* or *monolithic support*. Continuous bed supports consist of a single piece of material intersected by pores large enough to support chromatographic flow through the bed. Continuous beds have been developed using many well-known chromatographic materials, such as polyacrylamide, silica, polystyrene/polyme‐ thacrylates, cellulose, and agarose. Most of these continuous beds have two types of pores:

The preparation of a continuous bed is usually straightforward. These beds can often be prepared directly in a chromatographic column, thereby avoiding the time-consuming steps of size classification and column packing that are normally needed with particle-based supports. Reports using continuous beds in affinity chromatography have shown that the

In order to avoid column clogging, various pretreatment methods like filtration and centrifu‐ gation are often necessary to remove particulate matter from samples. To cut down on the need for such methods, a new class of adsorbents has recently been developed to handle viscous and particle-containing feed streams. These materials are known as *expanded-bed adsorbents*. In expanded-bed chromatography the direction of mobile phase flow is upward through the column and is fast enough to fluidize the support particles in the column. This causes the column bed to expand. This expansion makes the interstitial spaces in the column bed larger so that solid contaminants like cells and cell debris can pass through, thereby avoiding column

Another new type of expanded-bed adsorbent uses a thin layer of active material (i.e., derivatized agarose) that surrounds a heavy core. These adsorbents have small diffusion distances for biomolecules along with a higher density than other expanded-bed particles. The advantage of this combination is that it allows better chromatographic efficiencies to be

Ligands are the molecules that bind reversibly to a specific molecule or group of molecules, enabling purification by affinity chromatography [4]. These molecules which play a major role in the specificity and stability of the system are essential for affinity chromatography [13]. The selected ligand must be capable of selectively and reversibly binding to the substance to be isolated and have some groups which are available for modifications in order to be attached to the support material. It is very important to ensure that the modifications do not reduce the specific binding affinity of the ligands. There are general ligands such as dyes, amino acids, Protein A and G, lectin, coenzyme, methal chelates as well as specific ligands such as enzymes

Affinity ligands are classified as synthetic and biological. Biological ligands are obtained from natural sources such as RNA and DNA fragments, nucleotides, coenzymes, vitamins, lectins, antibodies, binding or receptor proteins, or in vitro from biological and genetic packages, employing display techniques including oligonucleotides, peptides, protein domains and proteins. Synthetic affinity ligands are generated either by de novo synthesis or modification

efficiency of these materials is as good as that for particle-based supports.

large flow-carrying pores and smaller diffusion pores.

clogging.

70 Column Chromatography

**1.2. Ligand**

obtained at higher flow rates [8].

and substrates, antibodies and antigens [19].

Despite the advantages of the affinity chromatography technique, its use is limited due to high cost of affinity ligands and their biological and chemical instability. The develop‐ ment of methods for production of stable synthetic ligands has enabled "utilization of these materials in large scale. For the design of synthetic ligands, information about structure of the target protein and a potential binding site are required, thus a structure-based design can be achieved, in case correct prediction of the ligand's comformation and the binding affinity of the designed ligand. Function-based design can be applied when the structure of the target is not known [9]. Substantially, selection and design of ligands may be performed by using a template which is a part of a natural protein-ligand couple, model‐ ling a molecule which complements the binding sites of the target or directly resembling the natural interactions [2].

suggested as ligands for the affinity chromatography application. It is also of choice to use phage libraries and a screening method known as biopanning. Phage display method allows determining suitable ligands not only for peptides and proteins but also for nonpeptide

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**Figure 8.** Selection of the phage from the phage-displayed combinatorial peptide library [9]

A collection of phage is incubated with the target and retained ligands are considered to be candidates for ligand [9]. At the end of the cycle, the process is repeated in order to increase the amount of the protein which has required binding features. The sequence of the protein is provided from the viral DNA. Phage display libraries are indisputably useful especially for epitope mapping, vaccine development, bioactive peptides and some non-peptide structures and the ligands determined using this method are appropriate for chromatographic analyses, nevertheless there are some cases that limit the use of this technique, such as some optimization problems and some issues as working in large scale as well as the limitation of the application

since peptides does not work unless it is a part of the phage, not in free solution [2].

Although its use in ligand selection for large scale of affinity chromatography is not wide, ribosome display and systematic evolution of ligands by exponential enrichment (SELEX) may

structures (Figure 8).

High selectivity of the biological ligands is a benefit; however these ligands have some handicaps, such as their low binding capacity, cost-efficacy issues, some problems in scale-up and purification process. Hence, synthetic ligands may offer a solution for these issues and enable to provide selectivity, efficacy and inexpensiveness in a body. Biomimetic textile dyes which are developed in 1970s are the most known synthetic ligands. The use of these dyes in biopharmaceutical field is limited due to some issues such as selectivity, purity and toxicity. These complications have led to new researches and developments about biomimetic dyes and new ligand design techniques [2].

The selection of the ligand may be done according to the specific binding site of a target, but this manner of selection may fail owing to the fact that immobilization process may change binding affinity. It is known that the affinity of the target to the ligand is dependent on the features of the target as well as support material, activation and coupling chemistry. Some other techniques other than using free ligand solution in order to predict the conditions of three-dimensional matrix. On the purpose of ligand selection, a great number of alternatives may be tested for binding the target or work with more accurate options by employing ligand design techniques. Therefore the idea to combine chemistry with computational tools has accelerated the developments on this field [2]. Along the development of affinity chromatog‐ raphy techniques, different laboratories are established with the purpose of collection of several ligands for affinity chromatography [9].

Protein-structure-based design of the ligands depends on the correct prediction of the structure of the target protein and the binding site. Apart from this, protein-function-based design is applicable in case the conformation of the target protein is not known. This method is based on the integration of some known properties of the ligand such as an essential molecular structure, a functional group or a derivative of some parts of the structure [9]. The design of a ligand requires several steps to fulfil [2]:


Beyond these design methods, some combinatorial approaches have been developed on the purpose of ligand selection. Synthetic peptide libraries which include all sequences for a length of a protein structure are one of these approaches. By means of these libraries, *in vitro* prediction of the action of the library mixture as it passes through the surface where the protein of concern is immobilized is possible. The ligands which possess affinity to the immobilized protein are suggested as ligands for the affinity chromatography application. It is also of choice to use phage libraries and a screening method known as biopanning. Phage display method allows determining suitable ligands not only for peptides and proteins but also for nonpeptide structures (Figure 8).

ling a molecule which complements the binding sites of the target or directly resembling

High selectivity of the biological ligands is a benefit; however these ligands have some handicaps, such as their low binding capacity, cost-efficacy issues, some problems in scale-up and purification process. Hence, synthetic ligands may offer a solution for these issues and enable to provide selectivity, efficacy and inexpensiveness in a body. Biomimetic textile dyes which are developed in 1970s are the most known synthetic ligands. The use of these dyes in biopharmaceutical field is limited due to some issues such as selectivity, purity and toxicity. These complications have led to new researches and developments about biomimetic dyes and

The selection of the ligand may be done according to the specific binding site of a target, but this manner of selection may fail owing to the fact that immobilization process may change binding affinity. It is known that the affinity of the target to the ligand is dependent on the features of the target as well as support material, activation and coupling chemistry. Some other techniques other than using free ligand solution in order to predict the conditions of three-dimensional matrix. On the purpose of ligand selection, a great number of alternatives may be tested for binding the target or work with more accurate options by employing ligand design techniques. Therefore the idea to combine chemistry with computational tools has accelerated the developments on this field [2]. Along the development of affinity chromatog‐ raphy techniques, different laboratories are established with the purpose of collection of

Protein-structure-based design of the ligands depends on the correct prediction of the structure of the target protein and the binding site. Apart from this, protein-function-based design is applicable in case the conformation of the target protein is not known. This method is based on the integration of some known properties of the ligand such as an essential molecular structure, a functional group or a derivative of some parts of the structure [9]. The design of a

**1.** Determination of the binding site or possible biological interactions to use as a template

**5.** Optimisation and chromatographic evaluotion of the adsorbent following the immobili‐

Beyond these design methods, some combinatorial approaches have been developed on the purpose of ligand selection. Synthetic peptide libraries which include all sequences for a length of a protein structure are one of these approaches. By means of these libraries, *in vitro* prediction of the action of the library mixture as it passes through the surface where the protein of concern is immobilized is possible. The ligands which possess affinity to the immobilized protein are

the natural interactions [2].

72 Column Chromatography

new ligand design techniques [2].

several ligands for affinity chromatography [9].

ligand requires several steps to fulfil [2]:

**4.** Selection of the ligand of interest,

**2.** Initial design of the ligand using this template,

**3.** Preparation of a ligand library and chromatographic evaluation,

for the modelling,

zation.

**Figure 8.** Selection of the phage from the phage-displayed combinatorial peptide library [9]

A collection of phage is incubated with the target and retained ligands are considered to be candidates for ligand [9]. At the end of the cycle, the process is repeated in order to increase the amount of the protein which has required binding features. The sequence of the protein is provided from the viral DNA. Phage display libraries are indisputably useful especially for epitope mapping, vaccine development, bioactive peptides and some non-peptide structures and the ligands determined using this method are appropriate for chromatographic analyses, nevertheless there are some cases that limit the use of this technique, such as some optimization problems and some issues as working in large scale as well as the limitation of the application since peptides does not work unless it is a part of the phage, not in free solution [2].

Although its use in ligand selection for large scale of affinity chromatography is not wide, ribosome display and systematic evolution of ligands by exponential enrichment (SELEX) may be mentioned as another approach and a potential ligand design and selection method due to its versatility and rapidity [9]. Ribosome display method enables to select and develop a protein library *in vitro* [2]*.* The principle of ribosome display is depicted in Figure 9. A collection of DNA encoding the selected peptide is exposed to an *in vitro* transcription and translation process, then in favourable condition, the complex of mRNA, peptide and ribosome since the stop codon does not exist. Thereafter the complex is passed thtough the immobilised target and the peptides which possess high affinity to the target are retained. At the end of this process, it is possible to seperate mRNA usually by EDTA, then by means of reverse tran‐ scription, DNA are attained and amplified [9].

ligand activity which result in multisite attachment, inappropriate orientation or steric hindrance can be observed if the correct procedure is not pursued [21]. Several methods are available to couple a ligand to a pre-activated matrix. The correct selection of coupling method

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Before ligands are coupled matrix is activated. Among several methods used for activation, the cyanogen bromide activation is the most frequently preferred. Activation using this method produces a highly reactive cyanate ester [7]. The ligands are attached to the support via primary aromatic or aliphatic amino groups. High toxicity of cyanogen bromide is the disadvantage of this method [13,19]. Subsequent coupling of ligands to the activated matrix results in an isourea linkage. Despite the popularity of this method, the isourea linkage of the ligands causes several problems during the purification procedure, including nonspecific binding due to charge and leakage of the ligand because of instability of the isourea bond. *N*hydroxysuccinimide (NHS) esters have also been used for immobilizing ligands. The prepa‐ ration of active esters requires a matrix that contains carboxylic groups. Such matrices can be easily obtained from agarose by activation of the hydroxyl groups with different reagents, including cyanogen bromide, activated carbonates, etc. and successive reaction with ω-amino acids of different sizes depending on the length of the spacer arm required. The NHS ester is then prepared by mixing the carboxylic matrix with dicyclohexylcarbodiimide and NHS. Due to the stability problem a different method based on *N,N,N′,N′-*Tetramethyl (succinimido) uronium tetrafluoroborate can be also used. The covalent attachment of ligands to such activated carriers provides the production of stable amide bonds. Another method for activating polysaccharides is the use of *N′N*-disuccinimidyl carbonate (DSC), which forms highly reactive carbonate derivatives with polymers containing hydroxyl groups. These derivatives react with nucleophiles under mild, physiological conditions (pH 7.4), and the procedure results in a stable carbamate linkage of the ligand coupled to the carrier. The immobilization of different ligands (e.g., enzymes, enzyme inhibitors, antigens and antibodies) on activated carbonate carriers has been achieved, together with excellent maintenance of biological activity of the proteins [7]. Pre-activated commercial matrices are also available (Table 2) to avoid many steps and problems of chemical activation process. A wide range of coupling chemistries, involving primary amines, sulfhydryls, aldehydes, hydroxyls and carboxylic acids are available for covalently attaching ligands to the matrices [12]. The use of commercially available, pre-activated media is recommended to save time and avoid the use

of the potentially hazardous reagents that are required in some cases [4].

After the activation of the support material, it is ready for the immobilization process of the ligand. In case the ligand is a small molecule, steric hindrance will occur between the immo‐ bilized support and the compound of interest (Figure 10). This may reduce or totally block specific binding of the substance. Use of the supports having a spacer arm attached or attachment of a spacer molecule to the support before immobilization of the ligand generally solves this problem. Spacer arm keeps ligand at a suitable distance from the surface of the support (Figure 9), thus the substance of interest will not be prevented to attach to the immobilized ligand. It is possible to bind spacer arms directly to the support prior to the imobilization of the ligand. Then a secondary reaction provides the attachment of ligand to

depends on the ligand characteristics [4].

SELEX is a widely used technique for screening of aptamers which are nucleic acid ligands. According to this method, a pool of DNA with a random sequence region attached to a constant chain is constituted by amplification then transcribed to RNA. RNA pool is separated accord‐ ing to the affinity of RNA molecules to a target protein. DNA molecules obtained by reverse transcription from retarded RNA molecules are amplified and the cycle is repeated.

The selection of the ligand may be designed according to the structure of the target protein as well. Under the favor of combination of the structure-based design and combinatorial chemistry, the efforts to synthesis a convenient structure are minimized [9].

**Figure 9.** Ribosome display method [9]

### **1.3. Immobilization**

The immobilized ligand is an essential factor that determines the success of an affinity chromatographic method [12]. The method which is used for affinity ligand immobilization is important because actual or apparent activity of the final column can be affected. Decrease in ligand activity which result in multisite attachment, inappropriate orientation or steric hindrance can be observed if the correct procedure is not pursued [21]. Several methods are available to couple a ligand to a pre-activated matrix. The correct selection of coupling method depends on the ligand characteristics [4].

be mentioned as another approach and a potential ligand design and selection method due to its versatility and rapidity [9]. Ribosome display method enables to select and develop a protein library *in vitro* [2]*.* The principle of ribosome display is depicted in Figure 9. A collection of DNA encoding the selected peptide is exposed to an *in vitro* transcription and translation process, then in favourable condition, the complex of mRNA, peptide and ribosome since the stop codon does not exist. Thereafter the complex is passed thtough the immobilised target and the peptides which possess high affinity to the target are retained. At the end of this process, it is possible to seperate mRNA usually by EDTA, then by means of reverse tran‐

SELEX is a widely used technique for screening of aptamers which are nucleic acid ligands. According to this method, a pool of DNA with a random sequence region attached to a constant chain is constituted by amplification then transcribed to RNA. RNA pool is separated accord‐ ing to the affinity of RNA molecules to a target protein. DNA molecules obtained by reverse

The selection of the ligand may be designed according to the structure of the target protein as well. Under the favor of combination of the structure-based design and combinatorial

The immobilized ligand is an essential factor that determines the success of an affinity chromatographic method [12]. The method which is used for affinity ligand immobilization is important because actual or apparent activity of the final column can be affected. Decrease in

transcription from retarded RNA molecules are amplified and the cycle is repeated.

chemistry, the efforts to synthesis a convenient structure are minimized [9].

scription, DNA are attained and amplified [9].

74 Column Chromatography

**Figure 9.** Ribosome display method [9]

**1.3. Immobilization**

Before ligands are coupled matrix is activated. Among several methods used for activation, the cyanogen bromide activation is the most frequently preferred. Activation using this method produces a highly reactive cyanate ester [7]. The ligands are attached to the support via primary aromatic or aliphatic amino groups. High toxicity of cyanogen bromide is the disadvantage of this method [13,19]. Subsequent coupling of ligands to the activated matrix results in an isourea linkage. Despite the popularity of this method, the isourea linkage of the ligands causes several problems during the purification procedure, including nonspecific binding due to charge and leakage of the ligand because of instability of the isourea bond. *N*hydroxysuccinimide (NHS) esters have also been used for immobilizing ligands. The prepa‐ ration of active esters requires a matrix that contains carboxylic groups. Such matrices can be easily obtained from agarose by activation of the hydroxyl groups with different reagents, including cyanogen bromide, activated carbonates, etc. and successive reaction with ω-amino acids of different sizes depending on the length of the spacer arm required. The NHS ester is then prepared by mixing the carboxylic matrix with dicyclohexylcarbodiimide and NHS. Due to the stability problem a different method based on *N,N,N′,N′-*Tetramethyl (succinimido) uronium tetrafluoroborate can be also used. The covalent attachment of ligands to such activated carriers provides the production of stable amide bonds. Another method for activating polysaccharides is the use of *N′N*-disuccinimidyl carbonate (DSC), which forms highly reactive carbonate derivatives with polymers containing hydroxyl groups. These derivatives react with nucleophiles under mild, physiological conditions (pH 7.4), and the procedure results in a stable carbamate linkage of the ligand coupled to the carrier. The immobilization of different ligands (e.g., enzymes, enzyme inhibitors, antigens and antibodies) on activated carbonate carriers has been achieved, together with excellent maintenance of biological activity of the proteins [7]. Pre-activated commercial matrices are also available (Table 2) to avoid many steps and problems of chemical activation process. A wide range of coupling chemistries, involving primary amines, sulfhydryls, aldehydes, hydroxyls and carboxylic acids are available for covalently attaching ligands to the matrices [12]. The use of commercially available, pre-activated media is recommended to save time and avoid the use of the potentially hazardous reagents that are required in some cases [4].

After the activation of the support material, it is ready for the immobilization process of the ligand. In case the ligand is a small molecule, steric hindrance will occur between the immo‐ bilized support and the compound of interest (Figure 10). This may reduce or totally block specific binding of the substance. Use of the supports having a spacer arm attached or attachment of a spacer molecule to the support before immobilization of the ligand generally solves this problem. Spacer arm keeps ligand at a suitable distance from the surface of the support (Figure 9), thus the substance of interest will not be prevented to attach to the immobilized ligand. It is possible to bind spacer arms directly to the support prior to the imobilization of the ligand. Then a secondary reaction provides the attachment of ligand to


**Table 2.** Activated commercially available resins of affinity chromatography

the spacer. The substance of interest doesn't be able to bind the ligand unless the spacer arm is long enough, but it is also possible to shorten the spacer arm in salt buffer [19].

**Figure 10.** Spacer arm, keeping ligand at a suitable distance from the surface of the support

Properties of an ideal spacer arm are listed below:


Compounds which have diamine groups such as hexanediamine, propanediamine and ethylenediamine are the most preferred spacer arms used in affinity cromatography. Some other examples of spacer arms are shown in Table 3 [19].

The following step is the immobilization of ligands on the activated matrix by isourea bonds. Immobilization through isourea linkage has some disadvantages including nonspesific binding of the ligand because of the instability of the bonds. Another method for immobiliza‐ tion is to use active esters such as *N-*hydroxy-succimide (NHS) esters. The carboxyl groups required for preparation of active esters can be prepared by activation of hydroxyl groups of

12

agarose. The ligands attach to this type of matrix via amide bonds. It is also possible to activate polysacharides by formation of highly reactive carbonate derivatives. In this case the polymer which contains hydroxyl groups is activated by the use of *N'N-*disuccinimidyl carbonate (DSC). The resultant carbonate derivatives create stable carbamate bonds with nucleophiles under mild, physiological conditions. Immobilization methods can be categorized as follow

O C

NH CH

NH

R

NH R NH2

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C NH

NH (CH2)2 NH (CH2)2 NH2

CH

COOH

R

O

**Name Structure**

Diamine H2N R NH2

O C

O C

Polyether -0(CH2)2O(CH2)2O(CH2)2OH

**Table 3.** Some examples for spacer arms and their structures

Amino acid NH2RCOOH

NH

NH

General types of immobilization methods

Covalent methods Noncovalent methods Coordination methods

Non‐specific adsorption Biospecific adsorption Entrapment

**Figure 11.** Immobilization methods used in affinity chromatography

12

(Figure 11) [7].

Alkylamine

Polypeptides

Polyamine

**Table 3.** Some examples for spacer arms and their structures

the spacer. The substance of interest doesn't be able to bind the ligand unless the spacer arm

ligand

**1.** It should be long enough (at least 3 atoms) to keep the substance at an appropriate distance.

**3.** It should have bifunctional group for the reaction with both support and the sample [9]. Compounds which have diamine groups such as hexanediamine, propanediamine and ethylenediamine are the most preferred spacer arms used in affinity cromatography. Some

The following step is the immobilization of ligands on the activated matrix by isourea bonds. Immobilization through isourea linkage has some disadvantages including nonspesific binding of the ligand because of the instability of the bonds. Another method for immobiliza‐ tion is to use active esters such as *N-*hydroxy-succimide (NHS) esters. The carboxyl groups required for preparation of active esters can be prepared by activation of hydroxyl groups of

Spacer arm

is long enough, but it is also possible to shorten the spacer arm in salt buffer [19].

**Product name Functional group specifity**

UltraLink lodoacetyl resin -SH

Affi-Gel 10 and 15 -NH2 Pierce CDI-activated resin -NH2

CNBr-activated Sepharose 4 Fast Flow -NH2

Thiopropyl SepharoseTM 6B -SH Tresyl chloride-activated agarose -NH2, -SH

**Table 2.** Activated commercially available resins of affinity chromatography

CarboLink Coupling resin -CHO, C=O ProfinityTM Epoxide resin -NH2, -OH, -SH

Epoxy-activated SepharoseTM 6B -NH2, -OH, -SH

EAH SepharoseTM 4B -COOH, -CHO

**Figure 10.** Spacer arm, keeping ligand at a suitable distance from the surface of the support

Properties of an ideal spacer arm are listed below:

**2.** It should be inactive not to cause a non-specific binding.

other examples of spacer arms are shown in Table 3 [19].

76 Column Chromatography

ligand

agarose. The ligands attach to this type of matrix via amide bonds. It is also possible to activate polysacharides by formation of highly reactive carbonate derivatives. In this case the polymer which contains hydroxyl groups is activated by the use of *N'N-*disuccinimidyl carbonate (DSC). The resultant carbonate derivatives create stable carbamate bonds with nucleophiles under mild, physiological conditions. Immobilization methods can be categorized as follow (Figure 11) [7].

**Figure 11.** Immobilization methods used in affinity chromatography
