1

Supervisor:

dr. Wim T. Kok

Student:

Panagiotis Spyropoulos (10457658)

Recent developments in affinity chromatography Literature Thesis

University of Amsterdam, The Netherlands

2

Table of Contents

1. Introduction ............................................................................................................. 4

1.1 General formats of affinity chromatographic applications ...................................... 5

2. Types and recent developments of affinity chromatography ................................... 7

2.1 Lectin Affinity Chromatography ............................................................................. 7

2.2 Boronate Affinity Chromatography......................................................................... 9

2.3 Immunoaffinity Chromatography .......................................................................... 11

2.4 Immobilized Metal-Ion Affinity Chromatography ................................................ 13

2.5 Protein A, G and L Affinity Chromatography ....................................................... 14

2.6 Analytical Affinity Chromatography..................................................................... 16

2.7 Dye-Ligand Affinity Chromatography .................................................................. 19

2.8 Aptamers ................................................................................................................ 23

2.9 Molecularly Imprinted Polymers ........................................................................... 25

2.10 Bioaffinity Chromatography ................................................................................. 27

2.11 Affinity Capillary Electrophoresis ........................................................................ 29

3. Conclusion ............................................................................................................. 32

References ..................................................................................................................... 33

3

MSc Chemistry

Analytical Sciences

Literature Thesis

Recent Developments in Affinity Chromatography

by

Panagiotis Spyropoulos

April, 2014

Supervisor:

dr. Wim T. Kok

4

1. Introduction

Affinity chromatography is a liquid chromatographic technique in which a specific

binding agent is used for the purification and analysis of sample components [1]. This

occurs by making use of a biological interaction between a protein or a group of

proteins and a specific binding agent coupled to a chromatography matrix [1].

This agent, which is known as the “affinity ligand”, selectively interacts with the

desired analyte in the sample and then it is placed into a solid support within a column

[2]. When this immobilized ligand has been prepared, it can be used for separation or

quantification of the analyte [2].

The most important factor for a successful affinity chromatographic method is the

immobilized ligand. The vast majority of affinity ligands, which are normally used,

come from a biological source like for example antibodies, enzymes, transport

proteins and carbohydrate binding proteins. In addition, synthetic ligands can be used

and more specifically metal ion chelates, boronates and biomimetic dyes [3].

According to the type of the ligand we can characterize different types of affinity

chromatography, like for example lectin affinity chromatography, boronate affinity

chromatography, immunoaffinity chromatography, immobilized metal ion affinity

chromatography and then discuss the developments on each one of them [3].

The type of support is also important for the design and use of the column. When

affinity chromatography is used only for sample pre-treatment or target isolation, a

carbohydrate support is preferred, like for example agarose or cellulose. The main

reason for this choice is the relatively easy modification for ligand attachment and

also the low non-specific binding for a variety of biological compounds. On the other

hand, the low efficiency and not so good mechanical stability of many carbohydrate-

based materials enhance the tendency of them to have better results during off-line

methods and with columns where the pressure and the flow rate is relatively low [4].

According to the type of support, new types of affinity chromatography can be used.

More specifically, high performance affinity chromatography (HPAC), or high

performance liquid affinity chromatography (HPLAC) is another type of affinity

chromatography, where silica particles can be used as HPLC media apart from

traditional carbohydrate based supports [5]. Another approach is affinity monolith

chromatography, in which recently developed monolithic materials are used in order

to attach the affinity ligand [6]. The most important advantage of HPAC depends on

the higher rigidness the efficiency of the particles or the synthetic polymers. As a

result, they have the ability to withstand under higher pressure and flow rate and they

possess better properties related to mass transfer than carbohydrate base supports.

Also, they can be combined with other HPLC columns and analytical methods on-line

and they are more precise, faster and easily automated in comparison with traditional

carbohydrate supports [5].

5

According to Magdeldin and Moser [7] affinity chromatography was firstly

introduced fifty years ago and in recent years the vast majority of purification

techniques (over 60%) contain affinity chromatographic applications.

1.1 General formats of affinity chromatographic applications

The format that is used for both traditional and high pressure affinity chromatographic

applications, in order to separate biomolecules, is the one shown in the following

figure.

Figure 1: General format for affinity chromatography [3].

The first step includes the injection of the sample in the affinity column, under

conditions that permit the target or the analyte of interest to bind to the immobilized

ligand in a robust way. An aqueous buffer with pH and ionic strength, that simulates

the inherent conditions of the ligand and its target, is regularly used in this kind of

applications. During the use of the buffer, the elution of compounds in the sample,

which do not interact much or at all with the ligand, is taking place and as a result a

non retained peak is created [3].

Later, the dissociation of the target from the affinity ligand occurs by using an elution

buffer. A basic requirement of this step is the alteration of the sample composition in

the mobile phase in order to enable the elution of the target. This can be achieved

6

either by changing the pH conditions or by adding a competing agent which is

responsible for the displacement of the target from the column. The collection of the

released target during this elution procedure is also possible and this target can be

analyzed later in order to provide more information. The direct monitoring of the

elution target, by using an HPLC support in the affinity column, is also feasible by an

on-line method. Combinations of both on-line and off-line methods with detection

methods like for example absorbance, fluorescence or mass spectrometry are also

possible. The regeneration of the column before the next application can be made by

passing through the original application buffer, after the elution of the target [8].

The scheme in the figure above is known as the “on/off” or step elution mode of

affinity chromatography and it is responsible for the capture and the elution of the

target [9]. This has been used in a wide range of applications not only in order to

isolate compounds but also for the preparation procedure of the sample, especially in

applications concerning biomedical and pharmaceutical analyses. The main reasons

for this choice are the fact that this issue can be characterized as simple, flexible,

selective, and relatively easy to use [1]. Additionally the automation of the format is

easy when affinity columns, which are suitable for HPAC applications or as a part of

HPLC systems, are used. Ultimately, this issue can be also used in order to detect

analytes directly. In the step of elution mode, on-line absorbance or fluorescence

detectors are used for this reason. Apart from these, mass spectrometric applications

and post column reactors can also be used [10].

Weak affinity chromatography (WAC) or dynamic affinity chromatography is an

approach of affinity chromatography used in various applications in pharmaceutical

and biomedical fields of study, where isocratic elution is operated, assuming that

retained targets have weak or moderate affinity for the immobilized ligand. The

development of these systems enables a single mobile phase to be used not only as the

sample application but also as the elution buffer [9].

Chiral separations which support affinity columns that consist of immobilized

enzymes like for example trypsin, a-chymotrypsin and cellobiohydrolase I or serum

proteins such as human serum albumin (HSA), bovine serum albumin (BSA) and 1-

acid glycoprotein (AGP), are characteristic examples where isocratic elution is used

[11].

Weak affinity chromatographic applications can be also utilized for the investigation

of drug screening assays. More specifically, the fragment-based drug discovery/

design screening of amidines, or arginine mimetic ligands can be achieved by using a

combination of WAC with trypsin and thrombin columns and also techniques, such as

HPLC and mass spectrometry [12]. This type of screening can be used in order to

evaluate ligands or inhibitors of cholera toxin. Moreover, high-throughput screening

of drug–protein interactions have also been investigated by using combinations of

affinity microcolumns with HSA [3].

7

An alternative format used mainly in the biomedical analysis field, is affinity

extraction. This approach contains the removal or extraction of a specific analyte or

group of analytes from the sample with a column which consists of an immobilized

affinity ligand. Then, the elution of the retained targets occurs and they pass to a

second system for analysis. Immunoextraction is a type of this format that is utilized

usually for antibodies which are placed in columns [13]. Ligands, such as boronates

or lectins, can be also used in this type of extraction. In HPAC applications, the

combination of affinity extraction with some ligands, enables the removal of the

targets from the samples really fast, namely in less than a few seconds. This

advantage is extremely helpful for the measurement of the free fractions of drugs and

hormones in serum or drug/protein mixtures, when columns which consist of

immobilized antibodies or HSA as the affinity ligand, are used [14].

An alternative method with relative connection to affinity extraction is affinity

depletion. In particular, an affinity column is used for the removal of abundant

compounds from a highly complicated sample, before the sample components, which

have not been retained, have been analyzed with a second technique. Antibodies are

commonly used within this technique as affinity ligands [13]. As far as the proteomics

field is concerned, the removal of highly abundant proteins is a characteristic

application, like for example HSA and immunoglobulin G (IgG) from serum, before

the analysis of the lower abundance proteins that exist in a sample, by using antibody

columns [3].

2. Types and recent developments of affinity chromatography

Based on the fact that affinity chromatography is widely applied, in this section all the

different types of affinity chromatography, as well as their recent applications and

developments will be presented.

2.1 Lectin affinity chromatography

One type of affinity chromatography, showing a rapid development over the past

decades is lectin affinity chromatography. In this powerful technique the immobilized

lectin is used as stationary phase [3]. Lectins are proteins that bind carbohydrate

residues under specific classification [15]. To be more specific, lectins are divided

into five groups according to their specificity to the monosaccharide. Hence,

according to Sharon [16] they present the highest affinity for mannose,

galactose/Nacetylgalactosamine, N-acetylglucosamine, fucose, and N-

acetylneuraminic acid.

In lectin affinity chromatography the protein is binding to an immobilized lectin

through its sugar moeities (N-linked or O-linked) [7]. Immobilized lectins are

8

extremely important for the separation and isolation of glycoconjugates,

polysaccharides, soluble cell components, and cells which consist of specific

carbohydrate structures. Another use of the immobilized lectins is dedicated to the

removal of any glycoproteins which are contaminated from purified proteins [17].

To be more specific, first of all the glycosylation of the protein takes place and then it

is ready for binding to the affinity support. The next step involves washing away any

remaining contaminants that have not been bound until then. Finally, the purification

of the protein occurs before the elution step, in order to complete the procedure [7].

There are many types of lectin affinity chromatography that can be used in different

scientific fields for research purposes. A significant number of them commercially

exist in an immobilized form. Two characteristic examples of this mode are

Concanavaline A (Con A) Sepharose and Wheat Germ Agglutinin (WGA) which are

widely used to purify glycoproteins [7].

Moreover, a great number of lectins can be used for more than one sugar hence they

can react with a number of different glycoproteins. One such example is Con A,

which does not own specificity and it is also known for binding enough glycoproteins,

polysaccharides and lysosomal hydrolases. Detergents like 0.1% sodium dodecyl

sulfate (SDS), 0.1% sodium deoxycholate, and 0.1% cetyltrimethyl-ammonium

bromide (CTAB) reduce the binding efficiency of Con A [17].

Another lectin with similar characteristics to Con A is Lens culinaris. The

disadvantage of this lectin however, is that its binding is not really strong. One

exception is when sodium deoxycholate is present, since in that case the binding of

the lectin can be efficient and as a result, it can be used in order to purify solubilized

membrane glycoconjugates. In addition, Wheat Germ Agglutinin lectin interacts with

mucins which contain N-acetylglucosamine residues and when 1% sodium

deoxycholate is present the lectin binds efficiently [17].

Finally, a last type of lectin after the isolation from the seeds of Artocarpus

integrifolia, is Jacalin which has a characteristic affinity for D-galactose residues.

According to recent articles and reports it can be used not only for the separation of

IgA1 from IgA2 subclass antibodies, but also for the removal of any IgA that has been

contaminated from preparation procedures of IgG that has been purified [17].

Another important field of study, where lectin columns with additional supports have

been used, is biomedical analysis. Initially coated polystyrene-divinylbenzene support

has been used for the preparation of M-LAC columns. The next step is the

combination of an M-LAC column with an antibody column as part of an automated

HPLC platform [18]. Another alternative is the use of isoelectric focusing with a

digital proteome chip especially for the field of proteomics studies. Generally, the

technique of LC-MS has extremely useful results as it helps the detection of any

changes happened in a wide variety of protein fractions which obtained either from

individuals without health problems or from others with a specific disease [19].

9

One last approach of lectin affinity chromatography is serial lectin affinity

chromatography (S-LAC), where sets of lectin columns are used in order to isolate

and purify glycoproteins and glycoconjugates. In this type, small agarose columns

which contain Con A, WGA or SNA are used and for the extraction step a hydrophilic

interaction column is used that helps for the enrichment of glycoproteins and

glycopeptides before they have been analysed by matrix-assisted laser

desorption/ionization mass spectrometry (MALDI MS) and liquid

chromatography/tandem mass spectrometry (LC–MS/MS) [20].

2.2 Boronate affinity chromatography

Another type of affinity chromatography used successfully with clinical samples,

includes boronates as ligands, hence it is defined as boronate affinity chromatography

[2]. This technique is one of the oldest identified in clinical laboratories and it was

used for diabetes control through the quantitative determination of glycohemoglobin

[2].

Boronate affinity chromatography is mainly based on the interaction between the

hydroxyl group of boronate and the molecules which contain cis-diol groups, such as

carbohydrates, nucleotides, nucleosides, glycoproteins, etc. [21]. The general

interaction between boronic acids and cis-diol-containing compounds is shown in

Figure 2 [22].

Figure 2.The general interaction between boronic acids and cis-diol-containing compounds [22].

Boronates are useful in separation of glycoproteins (e.g., glycohemoglobin) from non-

glycoproteins (e.g., normal hemoglobin), based on the fact that sugars such as glucose

owns cis-diol groups [2].

10

Methods developing this type of affinity chromatography have used both agarose and

supports that can be employed in HPAC techniques, while in other methods other

types of glycoproteins, such as glycated albumin and some apolipoproteins, have been

employed [3]. Moreover, boronate chromatography has also been used for the

analysis and measurement of hemoglobin A1c (HbA1c) which is the basic component

of glycated hemoglobin found in human blood [3].

In addition to the above, other types of glycated proteins in a sample can be examined

using boronate columns and changing the monitoring of the absorbance from 410-415

nm to 280 nm [23]. Last but not least, another method being used is the combination

of boronic acid columns and an immunoassay when the proteins of interest are for

instance the glycated albumin in serum and urine [24] or the glycated apolipoprotein

B in serum [25].

As far as recent developments in boronate affinity chromatography are concerned,

these include the separation of nucleosides, nucleotides and the ribose nucleic acid

mainly because of the 2′, 3′-diol interacting group [21]. Another important application

is the separation of RNA from the mixture of DNA and RNA based on the interaction

with boronate. In this occasion, there is no interaction for DNA molecules due to the

absence of 2′-hydroxyl group and subsequently the esterification of boronate ligand is

not possible. In addition, particular purification of 3′-phosphorylated ribonucleotides

and aminoacylated tRNA from a wide variety of nucleic acids and other biomolecules

can also be achieved [21].

What is more, during the last years developments concerning monolithic matrices

have been providing extremely significant results in the field of chromatographic

separation of biomolecules [26]. Monolithic columns are continuously integrated

porous separation media, without any inter-particular voids. Under pressure

conditions, the open channel network gives the opportunity to the mobile phase to

flow through the pores without restrictions. As a result, convective mass transfer in

less time compared to packed porous bead columns can be achieved. This type of

monolithic columns can be used in order to separate cis-diol containing biomolecules

and more specifically nucleosides and flavin adenine dinucleotide [21].

Another recent development of boronate ligands is the immobilization on 2-D

surfaces for the demonstration of the way that yeast [27] and mammalian cells [28]

are captured. As far as yeast cells are concerned, sugar moieties that are present on the

surface enable the adsorption on boronate grafted siliceous support under pH

conditions of 7.5. Relatively stable monolayer-like structures have been formed on the

surface of the boronate -grafted supports. However, the quantitative detachment is

possible due to competition of the fructose molecules that are present [29].

As far as the mammalian cancer cells are concerned, one characteristic of them is the

adherence on copolymer brushes that contain boronate on glass surface and

capillaries. Quantitative detachment of the attached cells from the grafted plates could

11

take place with 0.1M fructose solution and as a result, new developments concerning

cell separation methods could be examined. Also, either microbial or animal cells

with a sugar moiety localized on the cell wall can be separated [28].

But because monolithic columns’ pore size is a barrier on the movement of

mammalian cells, recent studies have examined the use of monolithic “cryogel”

matrices in order to overcome such limitations. The synthesis procedure of these

matrices occurs in lower temperature than the freezing one of the monomer or the

polymer solution [30]. Cryogels can be characterized for both their open

interconnected porous structure with large pore size and their relatively high

mechanical elasticity [31]. These characteristic properties enable cryogel based

matrices to be used for a wide variety of cell types and biomolecules during affinity

chromatographic separations. Additionally, for matrices of bioreactors and for the

design of tissue engineering scaffolds, the preparation and study of cryogels either

from natural polymers or other biocompatible polymers is extremely significant. Inert

hydrophilic polymers, which can be used for the synthesis procedure of different

matrices, are suggested in order to avoid non-specific interaction [21].

All these reasons enhanced the development of macroporous, inert and open

channeled matrices based on cryogels and combined with a boronate affinity ligand,

which was incorporated via copolymerization and graft polymerization steps, of a

boronate containing polymer. The created grafted cryogels can be valuable, not only

in order to characterize and to separate yeast cells, but also to fractionate both

adherent and non-adherent cells. Finally, an alternative approach for the synthesis of

the cryogel affinity column that contains boronate ligand is the copolymerization

method. This approach is often used in order to separate selectively RNA from the

nucleic acid mixture [21].

2.3 Immunoaffinity chromatography

Immunoaffinity chromatography is a technique where affinity columns which contain

immobilized antibodies or antibody-related agents are used. Moreover, when this

technique is used as a part of an HPLC system, it can be named as “high-performance

immunoaffinity chromatography” (HPIAC) [2].

During immunoaffinity chromatography and HPIAC applications there is a number of

different elution formats that can be used. The step elution mode, shown in Figure 1,

is the most widely used. During this mode, the retained target is being eluted by

changing the pH conditions on the mobile phase. An alternative way is to add an

organic modifier or a chaotropic agent [32]. Most of the times this mode is used when

target compounds are being isolated by immunoaffinity columns before these targets

have been analyzed by another method. Additionally, these targets can be detected

directly if they exist in sufficient levels in the sample. This mode has been used for

the measurement and transferring of different analytes like for example

12

acetylcholinesterase, benzidine, HSA, IgG and insulin. In order to detect these

different analytes, methods like absorbance, fluorescence, and mass spectrometry can

be used. Some additional detection schemes include pulsed amperometry and

radiometric detection [33].

Flow-based immunoassay or “chromatographic immunoassay” is a potential format

that can be also used with immunoaffinity chromatography and HPIAC [33]. There

are two different categories on these methods, namely competitive and non-

competitive immunoassays. In the first one, there is competition between a target

analyte and a labeled analyte analog for particular positions, where they can bind to

an antibody column. The step which follows is the detection of either the retained or

non-retained fraction of the labeled analog, using methods like fluorescence,

absorbance, chemiluminescence, or electrochemical formats. The measurement of the

total amount of target which was present in the original sample is not direct, as it can

be seen from the provided signal [33].

More specifically, the simultaneous injection mode, the sequential injection mode,

and the displacement immunoassay are three different options of immunoaffinity

chromatography for the performance of a competitive binding immunoassay [3].

Analytically, for the simultaneous injection mode the labeled analog and the target are

used at the same time onto a column, which contains only few immobilized antibodies

for these agents. For the sequential injection, initially target application takes place

and later the injection of the labeled analog occurs. For the last one, the displacement

immunoassay, first of all an excess of the labeled analog is adsorbed onto a column

where immobilized antibody exists. Then the application of a sample of the target

takes place to the column and a quantity of the labeled analog is displaced. Each one

of the three modes is employed for different applications. Simultaneous injection

immunoassays have been employed for the analysis of adrenocorticotropic hormone,

caffeine, cortisol, digoxin, gentamicin, HSA, IgG, methotrexate, testosterone, and

theophylline. Sequential injection immunoassays have been implemented for the

measurement of α-amylase, digoxin, HSA, and IgG and finally displacement

immunoassays for the analysis of benzoylecgonine, cocaine, cortisol, phenytoin, and

thyroxine [3].

As far as non-competitive immunoassays are concerned, two similar modes exist, the

one-site immunometric assay and the two-site immunometric assay [34]. The

procedure for the first one involves incubation of the sample with a known excess of

labeled antibodies that bind to the target. Following this, there is the injection of the

mixture onto a column which contains an immobilized analyte analog. The specific

role of this analog is the binding to any antibody that has not been bound to the target

until then and the elution of the binding antibodies as a non-retained fraction. There

are two ways for the final determination of the total amount of target that was present

in the original sample; either by measuring the amount of retained antibodies or non-

retained ones. One-site immunometric assay can be used on its own or as a step of

13

post column detection schemes for analytes such as digoxin, α-fetoprotein,

granulocyte colony-stimulating factor, interleukin-10, and thyroxine [35].

On the other hand, the main characteristic of a two-site immunometric assay or

sandwich immunoassay is the use of two different antibodies, which have the ability

to bind simultaneously to the same target. The first antibody is immobilized to a

support and it is utilized for the extraction of the target. The second one owns a label

which is extremely useful for the detection step. The elution of the retained target and

the labeled antibodies occurs after washing the column from non-retained components

and excess labeled antibodies. These labeled antibodies generate signal which is used

for the direct measurement of the amount of target in the sample. Characteristic

measurements of analytes in this mode are human chorionic gonadotropin, HSA, IgG,

interleukin-5, parathyroid hormone, and thyroid-stimulating hormone [33].

Concerning current developments in immunoaffinity chromatography, the

combination of it with other techniques for the analysis of complex biological

samples, has offered powerful insights in recent years. Two representative examples

of the above method are immunoextraction [13] and immunodepletion [36]. To be

more specific, off-line immunoextraction has been combined with HPLC, gas

chromatography (GC) and other methods for the determination of analytes as α1-anti-

trypsin, BSA, chloramphenicol, cortisol, clenbuterol and phenytoin, etc. On the other

hand, on-line immunoextraction methods, which combine immunoaffinity columns

with GC, mass spectrometry, ion-exchange chromatography, and capillary

electrophoresis, have also been useful leading to determination of drugs, proteins and

other interesting biomedical compounds. However, most of these on-line methods

include immunoextraction and reversed-phase HPLC, which is highly popular in

biological separations. This is due to the fact that an immunoaffinity column can

relatively easily be coupled with a reversed-phase column [13].

2.4 Immobilized Metal Ion affinity chromatography

Immobilized metal ion affinity chromatography (IMAC) firstly introduced by Porath,

Carlsson, Olsson and Belfrage [37], is another type of affinity chromatography which

has been showing huge growth when used in biomedical analysis [3]. IMAC was first

designated as “metal chelate chromatography” [38] and later metal ion interaction

chromatography and ligand-exchange chromatography before gaining its current term

as immobilized metal ion affinity chromatography [39].

IMAC is based on the interactions between immobilized metal ions and specific target

groups from the protein surface, such as amino acids, peptides, proteins, and nucleic

acids [3]. Furthermore, it has been widely approved that concerning amino acids,

histidine, tryptophan and cysteine residues play the most crucial role in the binding of

proteins in IMAC, due to their strong interaction with metal-ions [40]. In IMAC a

matrix is used and the metal ions are immobilized within a column through the use of

14

chelating groups like iminodiacetic acid, nitrilotriacetic acid, carboxymethylated-

aspartic acid, and l-glutamic acid. Metal ions that are often chelated to these groups

include Ni2+, Zn2+, Cu2+ and Fe3+ [40].

IMAC was originally named as “metal chelate chromatography” because it was first

developed for the isolation and separation of metal and histidine containing proteins

[37]. In this case, the sample is passed through an IMAC column, and firstly the

targets that can bind to the immobilized metal ions are retained and later a competing

agent is added or the pH is changed in order for the targets to be eluted [3].

Despite the use of IMAC for the purification of proteins, recently there is a wide

variety of other areas where it is applied [41]. To begin with, Takeda, Matsuoka and

Gotoh [42] report the employment of IMAC for the detection of drugs such as

tetracyclines, quinolones, macrolides, β-lactams, and aminoglycosides. Moreover,

Felix et al. [43] investigate the use of IMAC for detection of biomarkers in the serum,

urine, and tissues in order for diseases to be diagnosed. In this last case, the use of

IMAC has been combined with mass spectrometry in surface-enhanced laser

desorption/ionization (SELDI) [3]. Finally, in their work Sun, Chiu and He [44]

examine IMAC prior to mass spectrometry analysis for the enrichment of

phosphoproteins.

Another recent interest of the researchers towards the immobilized metal ion affinity

chromatography includes the refinement of IMAC for protein purification. IMAC has

not been used a lot for large scale protein purification, therefore researchers are highly

interested in the investigation of the above and more specifically of unexpected

conditions, like for example what happens if there is a leakage of metal ions from

IMAC columns [3]. In that case, the metal ions may interfere with the purity of the

protein raising concern for the researchers [3]. Another issue of concern is the

removal of the histidine tag from the recombinant protein when IMAC is used to

isolate recombinant histidine-tagged proteins, and its possible co-elution with other

proteins from the specific sample [45]. As a result of the above, there is a deep ground

for improvements as far as IMAC applications are concerned. The development of

new chelating ligands for IMAC, as well as methods to control better the selectivity of

IMAC while proteins are isolated from samples, are two examples of new issues that

researchers should investigate towards IMAC applications [3].

2.5 Protein A, G and L affinity chromatography

Proteins A, G and L are either native or recombinant proteins that originate from

microbes and their main characteristic is the specific binding to immunoglobulins,

including immunoglobulin G (IgG) that represents the 80% of serum

immunoglobulins [7]. Hence, protein A, G and L affinity column chromatography are

extremely helpful techniques, in order to isolate and purify IgGs from serum and other

biological fluids [46].

15

In particular, native and recombinant protein A is cloned in Staphylococcus aureus,

while recombinant proteins G and L are cloned in Streptococcus and

Peptostreptococcus magnus, respectively. The area of binding depends on the protein

that is used. Analytically, a specific binding in the Fc region of IgG is observed for

both proteins A and G, while the specific binding of protein L is taking place to the

kappa light chains of IgG [7].

According to many reports of affinity chromatographic applications where proteins A,

G and L are used, the category of matrix that is widely utilized is beaded agarose, like

for example Sepharose CL-4B or agarose cross linked with 2,3-dibromopropanol and

desulphated by alkaline hydrolysis under reductive conditions, polyacrylamide, and

magnetic beads [47,48].

Proteins A, G and L are characterized for their extensive binding with the IgG

subclass. This binding affinity varies in different immunoglobulin species. More

specifically, protein A declares stronger affinity binding for cat, dog, pig and rabbit

IgC and protein G for human or mouse IgG. In order to purify various IgG samples of

different mammals, the application of a recombinant protein of A and G is also

possible. As far as protein L is concerned, the specific binding of it to the kappa light

chain of immunoglobulins, which can be also found in other immunoglobulins like

IgG, IgM, IgA, and IgE, makes them appropriate in order to purify different classes of

antibodies. The binding of IgGs from the majority of species to proteins A and G

occurs under neutral pH and ionic strength conditions. A pH less than 2.7 is suggested

for the elution of immunoglobulins after their purification step [7].

Therefore, the main difference of these ligands is the binding to antibodies from

different species and classes. More specifically, the binding between protein G and

human IgG3 is stronger compared to the one with protein A. Moreover, there is a

weak interaction between protein A and human IgM, while there is no interaction

observed concerning protein G. Another protein that can be also used in this kind of

columns is A/G, a recombinant protein which is a mixture of both ligands’ activities

[2].

Protein A, G and L can be an extremely significant tool for scientists in the field of

analysis of immunoglobulins and mainly IgG-class antibodies, in humans. For this

reason, over the years numerous studies and reviews have been conducted around

protein A, G and L chromatographic techniques. For instance, Kitsiouli et al. [46]

examined protein G affinity chromatographical techniques and in particular the levels

of separation and purification of IgG from other proteins, while Hage [8] investigated

other applications of protein A and G, such as their use in order for antibodies to

adsorb onto supports that are going to be used in immunoaffinity chromatography.

This application is used mainly either for high antibody activities or when often

replacement of the antibodies in the column is wanted [2]. Concerning some of the

latest studies, Salvalaglio et al. [49] examined the design of mimetic ligands with

properties similar to those of protein A, while Holland et al. [50] investigated the

16

accuracy of the Protein G analysis of IgG for product specification and quality control

of colostrum products.

2.6 Analytical affinity chromatography

Analytical affinity chromatography, or else quantitative affinity chromatography or

biointeraction affinity chromatography is a method of affinity chromatography which

is used for studying interactions in biological systems [3]. A large number of articles

and reports, which have been written recently, examine this specific binding between

drugs and a wide range of agents including serum proteins, enzymes, and receptors

[1]. Moreover, specific interactions between biomolecules and lectins, aptamers and

antibodies have also been investigated. This type of affinity chromatography can

provide extremely significant and detailed information about the overall extent of

binding, the equilibrium constants for an interaction, the number and types of sites

involved in the binding process, and the rate constants for the interaction [51].

In many reports and studies, scientists in order to achieve these measurements, utilize

the zonal elution method. This method involves the injection of a small quantity of

sample of the drug or of the solute of interest into the mobile phase, where it can be

present either only the buffer or a competing agent with a standard concentration [2].

The determination of the changes on the elution time and the retention factor (k9 or

else capacity factor) of the solute that has been injected according to the concentration

values of the competing agent, is the best way for the analysis of the results. Closely

related studies have been conducted, for the investigation of the effect of interactions

between drugs and proteins to a wide range of solvent conditions, and also for the

development of quantitative structure-retention relationships that can be used in order

to further explore these binding processes [2].

The vast majority of reports and studies concerning HPLC affinity chromatographic

applications, and more specifically the zonal elution method, for binding between

drugs and hormones with proteins, are based on the investigation of the way that other

solutes displace drugs and hormones from proteins [2].

During these studies, injections of a target or probe are performed, while a competing

agent with a standard concentration in the mobile phase is present [1]. Exact

identification of the type of competition that takes place between these two solutes,

can occur by investigating the relationship between the change of the retention factor

of the target and the concentration of the competing agent. There are occasions where

data from previous works can be used for the determination of the association

equilibrium constants and also for the number of sites which are involved in the

interaction [52].

Characteristic examples include work that has examined the displacement of d,l-

thyronine and d,l-tryptophan from HSA by bilirubin or caprylate [53], the competition

17

of R/S-warfarin with racemic oxazepam, lorazepam, and their hemisuccinate

derivatives on an HSA column [54], the straight or allosteric competition of octanoic

acid on immobilized HSA for the binding sites of R/S-warfarin, phenylbutazone,

tolbutamide, R/S-oxazepam hemisuccinate, ketoprofen A/B, and suprofen A/B [55],

the competition either of R-warfarin and l-tryptophanV with d-tryptophan [56] or l-

thyroxine and related thyronine compounds on HSA [57], the displacement of R- and

S-ibuprofen by one another at their binding regions on HSA [58], and finally, the

effects of fatty acids on the binding of HSA with various drugs like for example

warfarin, phenylbutazone, tolbutamide, acetohexamide, and gliclazide [59].

Another possibility of the zonal elution method is the comparison of a wide variety of

coumarin and indole derivatives as potential probes for Sudlow sites I or II of HSA

[60]. During studies that have been conducted the last years, modifications on zonal

elution method have been made in order to investigate the allosteric interactions

which happen on HSA between ibuprofen and benzodiazepines, l-tryptophan and

phenytoin, or warfarin and tamoxifen [61]. Moreover, modifications have been made

on specific binding regions of HSA using either synthetic methods [62] or natural

processes, like for instance glycation [63]. Site-specific probes have also been used

for the investigation of changes that take place in solute interactions of a number of

competition studies [3].

Frontal analysis or frontal affinity chromatography is another method that can be used

especially for binding studies [64]. The main characteristic of this technique is the

continuous application of a target of known concentration to an affinity column,

where an immobilized binding agent is present. After taking place the saturation step,

the formation of a breakthrough curve occurs. The key point to the whole procedure is

either the mean position or the shape of the curve [63]. The determination of the exact

type of interaction that happens in the column depends on the mean position of the

curve, which can fit in a wide range of binding models. The parameters of the ideal

binding are also important and can provide valuable information on the equilibrium

constants and the number of binding sites that exist [1]. The shape of the

breakthrough curve, finally, is extremely helpful for studying interaction kinetics [65].

Some important applications of frontal analysis include the investigation of binding of

HSA to R/S-warfarin or d/l-tryptophan [3], the comparison of the binding between

monomeric and dimeric HSA for various drugs [66], the examination of the effects

based on chemical modification on the binding of site-specific probes with HSA [62]

and also the examination of multi-site binding of thyroxine with HSA [67]. In recent

studies, frontal analysis has been used for the examination of the total binding

between HSA and drugs like acetohexamide, carbamazepine, imipramine, lidocaine,

phenytoin, tolbutamide and verapamil [68]. Moreover, the interactions of a number of

coumarin and indole derivatives with HSA have been investigated [60]. Experiments

with the same method have been conducted for the investigation of interactions

between propranolol, carbamazepine, and lidocaine with AGP [69] and also

propranolol and verapamil with high-density lipoprotein [70].

18

Rapid developments and significant results have been also derived with the frontal

analysis method concerning the detection of multi-site binding [3]. The effect of

changing, either the mobile phase condition or the temperature on the affinity, and the

number of binding sites which are involved in a solute-ligand interaction have also

been examined. One example is the changes in binding by d/l-tryptophan to HSA

depending on pH [71]. The topic of many reports in the field of biological analysis is

the examination of the effect of temperature on biological interactions. More

specifically studies and experiments have been conducted about the interactions of

R/S-warfarin, d/l-tryptophan with HSA [72], propranolol and carbamazepine with

AGP [69] and also propranolol with high density lipoprotein [70]. This mode has been

extremely used for the determination of overall changes in drug or solute interactions

after modifying the binding regions of HSA [73].

Another approach of the frontal analysis method is its combination with mass

spectrometry, namely frontal affinity chromatography-mass spectrometry (FAC-MS)

[74]. This format can be characterized as an extremely significant tool, especially for

competitive binding experiments, due to its ability to distinguish mixtures of

compounds and their capability of binding to a given immobilized ligand [75].

Examples of binding agents that have been used with this technique are lectins,

enzymes and galactosaminoglycans [3].

As it has been also stated above, analytical affinity chromatography can provide

valuable information on the study of the kinetics of a biological interaction. Several

modes have been found for this type of research. First of all, the plate height method

is one example, where two different columns, one containing the affinity ligand of the

study and another one without ligand, are used [3]. The main function of this method

involves the measurement of the band broadening. The plot of the measured plate

heights of the analyte, versus the flow rate or the linear velocity, can help in order to

examine the contribution of the plate height based on stationary phase mass transfer,

which is connected to the dissociation rate between the target and the affinity ligand

[51]. Two characteristic experiments of this mode are the studies of the kinetics of

R/S-warfarin and d/l-tryptophan with HSA [76].

A variation on the plate height method is peak profiling, where measurements for

retention times and peak widths are conducted for both a target and a non-retained

solute on an affinity column [51]. As a first step, there is a comparison of these results

and then they are used for the determination of the rate constant, which is used for

target dissociation from the affinity ligand. The results for the non-retained solute are

used for the correction of band–broadening processes apart from the target with ligand

interaction [3]. One characteristic application of the method is the estimation of the

dissociation rate constants for l-tryptophan, carbamazepine, imipramine and

phenytoin metabolites from HSA [77]. Moreover, single or multiple flow rates have

been used in peak profiling methods and there have been tremendous developments in

the combination of this technique with solutes, which can interact with a number of

ways with the column, like for example binding to both the affinity ligand and the

19

support [3]. Finally, the combination of the method with chiral separations can assist

in the simultaneous examination of the interaction kinetics of HSA with the

enantiomers of drug metabolites [77].

A second approach of analytical affinity chromatography, which has been used for the

examination of the kinetics of biological interactions, is the peak decay method [3].

In this approach, firstly the injection of a pulse of a target onto a small affinity column

takes place. Then, in order to prevent the re-association of the target, which is

released from the affinity ligand, the mobile phase passes through really fast. The

decay curve which results under the appropriate conditions, helps the measurement of

the dissociation rate constant for the target from the ligand [51]. Recent applications

and reports refer to this method as it is used for the estimation of the dissociation rate

constants for a wide range of rugs from HSA [78]. It is also mentioned as an

important high- performance method, where affinity microcolumns and affinity

monoliths are used, in order to take the desired results or measurements [79].

Both zonal elution and frontal analysis methods have used curve fitting as a tool, in

order to extract valuable information on the kinetics of biological interactions [51].

More specifically, the profiles of frontal analysis methods have been used for the

examination of association kinetics for interactions between target and antibodies in

immunoaffinity columns [65]. These profiles have also been used for the examination

of interactions between targets and aptamers [3]. Apart from frontal analysis methods,

zonal elution experiments, which were conducted under non-linear elution conditions,

have also been used for the determination of the kinetics on studies with affinity

columns. One characteristic example of zonal elution experiment, which is referred to

many reports and articles, is the investigation of interactions between a wide range of

inhibitors with immobilized nicotinic acetylcholine receptor membrane columns [3].

2.7 Dye-ligand affinity chromatography

An alternative approach of affinity chromatography with extremely significant results

is dye-ligand affinity chromatography. In this type of affinity chromatography,

synthetic dyes compose the group of ligands than are employed in the technique [3].

The initial motivation for scientists to investigate more about dye ligand affinity

chromatography was given after the interactions that took place between Blue

Dextran, a Cibaron Blue and dextran conjugate, which is used as a void marker in

size-exclusion chromatography, and particular kinases. Until then, only purification of

various proteins by size-exclusion chromatography with Blue Dextran, like for

example, erythrocyte pyruvate kinase, phosphofructokinase, glutathione reductase,

and several coagulation factors had been initiated. The final conclusion of these

studies was that the main reason for protein binding was Cibacron Blue F3G-A, a

major reactive dye [80].

20

The first study, concerning the direct and covalent immobilization of Cibacron Blue

on Sephadex G-200 and also the purification of yeast pyruvate kinase with this

affinity sorbent, was conducted by Roschlau and Hess [81]. Later, there were many

articles and reviews dealing with this concept, which has been used in a wide variety

of applications in order to purify a wide range of proteins with a number of matrices

having this blue ligand.

In recent years, dye ligands have been widely used in affinity chromatography and

they have led to important results concerning protein purification [82]. The fact that

they are inexpensive, easily available, highly resistant to chemical or biological

degradation and readily immobilized constitutes some of their advantages [83].

The most popular dyes used for the purification of proteins are triazinyl-based

reactive dyes. The synthesis procedure is mainly based on Cyanuric chloride or

(1,3,5-trichloro-sym-triazine). In particular, the fact that electronegative atoms are

present is the reason for the three carbon atoms to be characterized as extremely

positive, and as a result they are very sensitive to nucleophilic attacks. The attachment

of chromophore molecules to this is extremely simple and so dichlorotriazinyl dyes

can be also formed [80].

A characteristic example of this category of dyes is the Procion MX series (from

Imperial Chemical Industries). These series can also react later with aniline,

sulfanilates or other nucleophilic substituents in order to produce monochlorotriazinyl

dyes like for example Cibacron (from Ciba-Geigy) or Procion H (from ICI). The

result of the coupling between two dichlorotriazinyl molecules and a bifunctional

molecule like diaminobenzene is the formation of bifunctional triazinyl dyes.

Characteristic examples are Procion H-E (from ICI), monofluoro-triazinyl (Cibacron,

Ciba-Geigy), trichloropyrimidnyl (Drimarene, Sandoz), and difluorochloropyrimidnyl

(Lavafix, Bayer and Drimarene, Sandoz) [80].

Apart from dyes based on triazinyl groups, one extremely significant series is the

Remazol series from Hoechst. This series is mainly based on the attachment to the

matrix due to the vinyl sulfone active groups that contain and because of this mode

they can be utilized as dye ligands in order to purify proteins [80].

Another type of ligand that can be also used in chromatographic applications is the

“biomimetic dye”. This ligand can be characterized as an extremely specific

unmodified dye. The main principle of this mode is the use of combinatorial

chemistry and computational methods for screening dyes and generally ligands for a

specific target. This mode can be a valuable tool for researchers in studies where it is

necessary to purify protein-based pharmaceuticals [3]. The production of these dyes

involves either personalization or redesign of the dye structure. This is the main

reason that textile dyes become more specific for target proteins. There are enough

studies and reports for this phenomenon from the beginning of 1980s, for example by

Lowe et al. [84], until recently with extremely important and successful results

21

especially for the recovery of a particular enzyme as suggested in the report of Clonis

et al. [85]. For the preparation of the first biomimetic dye, benzamidine was linked to

a chlorotriazine link which is really reactive with the help of a diaminomethylbenzene

group. This biomimetic dye was used in order to separate trypsin from chymotrypsin.

In the fields of bioinformatics and molecular modeling, extremely important

improvements concerning the synthesis of new series of biomimetic dye ligands, have

been noticed. In particular, anthraquinone-moiety-containing aromatic sulfonated

dyes, like Cibacron Blue 3GA, Procion Blue H-B and MX-R and Vilmafix Blue A-R

have a tendency to bind to the nucleotide-binding site and mimic the natural binding

of anionic coenzymes like NADH and FAD, in a wide range of proteins.

Anthraquinone dichlorotriazine dyes (such as VBAR) behave as affinity labels for

MDH (malate dehydrogenase) and LDH (lactate dehydrogenase). Improvement of the

way that l-lactate dehydrogenase is being purified can be made with the appropriate

biomimetic design of the ligand. In order to achieve this result, a three-dimensional

structural model of LDH is used as a guide and also some structural changes of the

dye molecules have occurred [80].

Biomimetic ligands, like for example mercaptopyruvic-, m-aminobenzoic-, and

amino-ethyloxamic-biomimetic dyes have been used for the purification of ketoacid-

group recognizing enzymes, such as formate dehydogenase, oxaloacetate

decarboxylase and oxalate oxidase. One last, recent application of molecular

modeling is the design of triazine non-dye ligands for Protein A, human IgG and

insulin precursor [80].

The main principle of the interactions between dye ligands and proteins is the specific

binding of dye molecules, which behave similarly to natural ligands, to the active

points of some protein molecules. A really important characteristic is the adsorption

of these proteins onto dye-ligand affinity sorbents. This fact enables the ligands to

easily interact with other parts of the proteins. The binding of the vast majority of the

proteins is not specific and involves extremely complicated combinations of different

kinds of bonds like for example electrostatic, hydrophobic or hydrogen. Moreover, all

of them have a great impact as far as the structure of the dyes is concerned [83].

As technology develops rapidly, dye-ligand affinity chromatography has been used in

many studies and it can be characterized as a significant tool for scientists and

researchers. Apart from the traditional mode, new types like affinity partitioning,

affinity precipitation and also expanded-bed chromatography have been developed

[1].

Affinity precipitation is mainly based on the particular affinity interactions of a

regular precipitation method. The main characteristics of this technique are the

selective precipitation of the complex created by the binding of a soluble ligand with

the target molecule, and as a result the increased degree of purification during the

elution of the target molecule. In order to achieve this, researchers have used

22

reversibly soluble polymers such as carboxymethyl cellulose and also

heterobifunctional ligands, where the conjugation of the affinity ligand is possible.

Changes in temperature, pH conditions and ionic strength are the main reason for the

precipitation of this complex. One characteristic example is the precipitation of lactate

dehydrogenase by using CB3GA conjugated to carboxyme thyl cellulose. The result

of this exceptional precipitation procedure is a 23-fold purification from an

unprocessed porcine extract [1].

Another mode of dye ligand affinity chromatography, that is also widely used, is

polymer-shielded dye-affinity chromatography (Figure 3). According to this, water-

soluble polymers can be used in order to improve the chromatographic process in a

significant degree. This technique is based on the creation of a complex between a

nonionic water-soluble polymer, such as poly(vinylpyrrolidone) or poly(vinyl

alcohol) and the dye matrix. These polymers are used in order to protect the dye from

non specific interactions with the target protein. The main advantage of this technique

is the really fast purification and this is the main reason that this technique has been

used for the purification of a large number of enzymes. One specific application of

this mode is to isolate lactate dehydrogenase from a crude porcine muscle extract as a

part of an expanded-bed system with the support Streamline-CB3GA. This

combination results in a 4.1-fold purification [1].

Figure 3: Polymer-shielded dye-affinity chromatography [1].

23

Dye ligand affinity chromatography has been used in various applications in order to

purify pharmaceutical proteins. Some characteristic applications are the purification

of human component factor B, fact or C2, factor II, factor IX, trypsin, chymotrypsin,

and proteinase 3. In detail, the purification of human recombinant alpha-interferon on

a mimetic dye-ligand matrix and also the purification of follicle-stimulating hormone,

pituitary gonadotropins, ricin A chain, and human serum albumin have been

investigated from scientists in different studies all these years. Albumin prepared by

CB3GA adsorbents have not been used in applications concerning the field of clinical

chemistry due to safety and toxicity regulations, but they are commonly used either

as a stabilizer for recombinant products or as an element of cell culture media [86].

Conventional affinity chromatographic applications with dye-ligands have been used

in order to remove small hydrophobic molecules and ions from biological fluids.

More specifically the coupling of CB3GA to poly(EGDMA-2-hydroxyethyl

methacrylate) (pHEMA) micro-beads was used for the removal of bilirubin from

human plasma in a packed-bed system. Additionally, this coupling can be also utilized

in order to remove A13+ and Fe3+ from human plasma. Another application of a dye

ligand column is the separation of G-DNA structures of which guanosine-rich

oligodeoxyribonucleotide is the main component. These separations are conducted on

a Reactive Green 19-agarose resin where Li+ ions are present [1].

Last but not least, the combination of an affinity extraction system with a reversed

micelle system has been used in order to extract lysozyme. The reversed micelle has

been generated by using a CB3GA-lecithin conjugate in n-hexane and enabled the

recovery of lysozyme in a high degree. As biospecific and steric-hindrance effects are

both present, this mode can be characterized as extremely selective and it is

appropriate in order to purify these low-molecular-weight proteins [1].

Finally, dye ligands have been used in order to remove toxic macromolecules such as

prion proteins, human immunodeficiency virus-1, or hepatitis B particles from

biological fluids. In particular, the azo dye Congo Red has been used due to the

specific binding of prion proteins and immobilized CB3GA has been utilized because

of the adsorption of envelope glycoproteins and also for the retention of the vast

majority of hepatitis B particles (more than 99.5%) from a human plasma sample [1].

2.8 Aptamers

Aptamers are synthetic nucleic acids which are characterized for their specific binding

to targets and as a consequence they are used in many affinity chromatographic

applications in order to recognize molecules [87].

The process which is used for the identification of aptamers to various chemical

targets is SELEX (systematic evolution of ligands by exponential enrichment). More

specifically, large amounts of nucleic acids with a functional role are being isolated in

24

a repetitive way through in vitro selection and then amplification follows through

polymerase chain reaction [88]. The category of targets contains both nucleic acid and

nonnucleic acid binding proteins, like for example growth factors and also enzymes,

antibodies and peptides. Another characteristic of aptamers is that they have been

related to the recognition of small molecules like for example amino acids,

nucleotides, drugs and dyes. This collection between target and aptamer was the

motive for in vivo and in vitro developments [89].

According to various reports aptamers were discovered in the beginning of 1990s. A

number of studies have provided information about their generation, which has

occurred all these years towards a broad range of targets such as peptides, amino

acids, small molecules and proteins, including cell membrane proteins. The affinity

exhibition of aptamers for their targets is high as it can be understood from the low

nanomolar to picomolar range of their Kd values [90].

Based on the study of Proske et al. [90], the discrimination between isoforms, with a

really close relationship or distinct structure states of the same target molecule, is

possible due to the exceptional specificity of the aptamers. Nonetheless, according to

Marro et al. [91] the selection of aptamers, which are responsible for the recognition

of murine and human protein targets with equal affinities, is also possible. This fact

enables their use in both preclinical and clinical developments.

Aptamers compared to antibodies, have the tendency of binding to functional domains

of the target protein such as substrate binding pockets or allosteric sites, thus the

modulation of the biological function of the molecule can be achieved. This benefit is

the motive for many studies and research projects concerning aptamer affinity

chromatography during the last years [90]. According to Farokhzad et al. [92], the

binding and the inhibitory behavior on carrier material is retained even after the

immobilization step. The same happens during the delivery into animals as Nimjee et

al. [93] reported, and certainly during the expression into cells.

According to a number of different reports many applications of aptamers in the field

of biotechnology provide extremely successful results. More specifically, Romig et al.

[89] examined the purification processes, Burgstaller et al. [94] the validation of the

target, Green et al. [95] the drug discovery area and finally, Nimjee et al. [93] the

diagnostics and therapy fields, respectively.

Consequently, and as technology develops rapidly, it is shown that aptamers can be

used in numerous applications. This significant progress in the field of aptamer

technology has provided extremely useful results to scientists, concerning different

fields of research, such as purification processes, target validation, diagnostics, drug

discovery, and therapeutic approaches [90].

As Zhao et al. [87] report, various analytical techniques such as electrochemistry,

quartz crystal microbalance, fluorescence, mass spectrometry, capillary

25

electrophoresis (CE) and polymerase chain reaction enabled the demonstration of

chromatographic assays which are based on the use of affinity aptamers.

In affinity chromatographic applications, aptamers can be used on stationary phase in

order to separate and quantify targets [96]. The benefits of this use are obvious when

antibodies are utilized as affinity ligands and more specifically the modification and

immobilization steps become easier and also the applications are more stable. In

addition, applications of aptamers to toxic compounds provide improved results and

the selection conditions are more flexible [97]. Moreover, batches of synthetic

aptamers are much more reproducible [96]. The vast majority of chromatographic

applications, which are based on aptamers, are connected to small molecules [87].

Apart from this, aptamers in affinity chromatographic applications can be used in

order to separate and detect proteins. The immobilization step of aptamers can take

place in open tubular capillaries, polymer monolithic columns and also microchannels

or columns that are packed with microbeads [87]. Finally, a number of novel

approaches for elution and immobilization steps, relied on the principles of

oligonucleotide chemistry, are also possible [97].

The main problem of using aptamers with low affinity is that during CE separation the

dissociation of the aptamer-target complex is possible. Solution to this problem has

been given by using aptamers with both weak and strong binding affinity in order to

avoid the restriction of affinity CE. One important advantage of using aptamers in

affinity chromatographic applications, mainly for the analytes which are present in

complex sample matrix, is the capture of the target analytes of interest in a selective

and efficient way [87].

One particular requirement in order to improve the sensitivity of the applications by

taking advantage of the preconcentration of targets is that targets with a strong

binding to the aptamer affinity column have to be eluted in a suitable way. What it is

also wanted is the capture and the elution of targets from the column stationary phase

without any damage of the latter. As a result, the affinity column can be used again

for similar purposes [87].

2.9 Molecularly Imprinted Polymers

Molecularly Imprinted Polymers (MIPs) is another group consisted of ligands and

supports whose use in affinity methods experiences huge growth in recent years.

Molecular imprinting is a technique which enables the production of artificial binding

sites in macroporous polymer particles, on surfaces or in membranes. These sites

demonstrate selective rebinding of the imprint or template molecules that are used

during their manufacture process [98]. All these years molecularly imprinted

polymers have been used in numerous applications like chromatographic media,

artificial antibodies, sensors and catalysts [99].

26

Molecularly imprinted materials own intrinsic properties in comparison with

enzymes, antibodies and biological receptors. They can be characterized as robust,

cheap and there is the possibility to use them in occasions where no recognizing

biomolecule is achievable [98].

During the preparation procedure of an imprinted polymer, the initial role of a

template or an imprint molecule is the management of the configuration of functional

monomers. Then the polymerization step with a high degree of cross linking follows,

and these monomers are frozen into position. Subsequently the template is removed,

and as a result cavities with counterbalancing size, shape and placement of functional

groups are made. Reversibility is the only requirement for the interactions between

the template and these functional monomers, which can be either covalent or non-

covalent [100].

In the vast majority of applications cavities consist of organic polymers based on

acrylate, acrylamide or styrene, yet the constitution of the cavities in silica is under

investigation. The production of them as bulk polymers is possible with many

different uses into microscopic particles, such as micron-scale beads or other bead

materials, combinations between micron-scale beads and thin surface layers, and

finally, as membranes [101].

Imprinted polymers have been widely applied during the last years. More specifically,

novel-cross linkers and monomers have been used in order to improve the

performance of such polymers [102]. In addition, imprinted polymer particles have

been used as stationary phase components in a number of thin-layer chromatographic

separations [103]. Last but not least, they have been used in many affinity capillary

electrophoresis applications [104].

In recent years, molecularly imprinted polymers are used as affinity based separation

media for sample preparation, namely molecularly imprinted SPE (MISPE) media.

There is a variety of modes concerning the molecularly imprinted SPE method, such

as online, offline, microextraction in packed syringe and also on-column extraction,

which all can be used in a significant number of pharmaceytical, environmental,

bioanalytical and food analysis applications. The greatest advantage of MISPE media

compared to the conventional SPE media is that they are more selective [99].

During a MISPE protocol procedure the loading, washing and elution steps of the

solvents play a crucial role. More specifically, in online modes the choice of washing

and eluting solvents is limited due to the RP LC separation that can take place. On the

other hand, in offline modes the selection of the solvents does not affect so much the

eluate, because later it will be separated and detected from an analyte in GC, LC, or

CE [105].

Additionally, molecularly imprinted polymers can be used either in compound

specific or in group specific extraction modes. In the first occasion, particular

extraction of the target compound is taking place by the application of a prepared MIP

27

for a target compound or its structurally related compound. In the second one, the

extraction of a target compound and its compounds with similar structural

characteristics occurs, such as a drug and its metabolites in biological fluids, a

herbicide and its degradation products in environmental samples and finally, an active

compound in crude extracts of traditional folk medicines and herbs [106].

As technology develops rapidly, fully automated and minituarized MISPE systems are

expected to be used in even more applications in the future. When MISPE

applications with high efficiency are needed, compound-specific and group-specific

MIPs are preferred to natural receptors and antibodies. Finally, another field of study,

which is expected to be investigated in future studies by researchers, is the preparation

of MIPs for a large molecule like for example a protein and a biologically important

compound [99].

2.10 Bioaffinity Chromatography

Bioaffinity chromatography is a type of affinity chromatography in which biological

compounds such as immunoglobulin-binding proteins, enzymes, lectins,

carbohydrates, avidin/biotin system and antibodies are used as ligands [1].

Immunoglobulin-binding proteins, namely protein A which is produced by

Staphylococcus aureus and protein G which is produced by streptococci, are the

ligands that are used in the vast majority of bioaffinity chromatographic applications

[107]. However, enzymes and enzyme inhibitors can also be used as affinity ligands

[1]. Immobilized enzymes are widely utilized in many applications, concerning

pharmaceutical and food industries. Furthermore, they are used in order to purify

enzyme inhibitors, as well as for the removal of impurities from unprocessed extracts.

In a similar way, enzyme inhibitors can be utilized for the purification of enzymes

from crude extracts [107]. The immobilization of enzymes on monolithic stationary

phases enables them to be used in a wide range of applications concerning bioaffinity

chromatography [108].

Nonetheless, as technology develops rapidly the demands for improvements and

advances in order to separate biomolecules in a more efficient way, as well as the

overwhelming of the restrictions, which specific stationary phases have, has

motivated researchers and scientists in order to search for materials that can replace

stationary phases used last decades [107].

Svec and Fréchet [109] developed continuous methacrylate rods from glycidyl

methacrylate (GMA) and ethylene dimethacrylate (EDMA) as monomer and

crosslinker. These two discoveries had a great impact, as they provided the platform

for non-particulate stationary phases and subsequently the generation of the name

‘‘monoliths’’.

28

Monoliths are continuous stationary phases which form as a homogeneous column in

a single piece and their preparation is taking place in various dimensions either with

agglomeration-type or with fibrous microstructures [110]. Therefore, affinity

monolith chromatography (AMC) is a liquid chromatographic technique, where a

monolithic support and a biologically related binding agent, as a stationary phase, are

used. This method can provide extremely useful results concerning the selective

separation, the analysis and the investigation of particular sample compounds [111].

There are two categories of monolithic materials, namely ‘‘organic’’ and

‘‘inorganic’’, according to the material that they are made from. The preparation of

organic monoliths is taking place when monomers, crosslinkers, porogens and an

initiator are polymerized in situ. Some characteristic examples of this category are

methacrylate and acrylamide (AAm) based polymers, poly(styrenedivinylbenzene),

agarose and cryogels. For the preparation of inorganic monoliths (silica), the sol-gel

method is applied or bare silica particles. The preparation schemes of monolithic

structure can have various formations like for example rods, CIM disks, capillaries

and microfluidic chips. The procedure involves initially the covalent immobilization

step and later in order to achieve the coupling of the ligand onto the stationary phase,

either biospecific adsorption or entrapment, are used [107].

Another classification, concerning monolithic materials is regarding their pores. There

are two caharacteristic types; macropores (the flow through pores) and

mesopores/micropores. As Tetala and van Beek [107] report, “the macropores have

diameters larger than 50 nm and enable the flow of mobile phase through the column

directing the sample to access the network of mesopores, which have pore diameters

in the range of 2–50 nm. Micropores have pores smaller than 2 nm”.

In the vast majority of affinity monolithic chromatographic applications the GMA-

EDMA copolymer system is used mainly because of its epoxide groups which are

available for ligand immobilization [112]. A typical GMA-EDMA monolith solution

consists of a monomer (GMA), crosslinker (EDMA), initiator and two porogenic

solvents [107]. One significant drawback of this system, however, is the low surface

area of GMA-EDMA monolith. In order to avoid this limitation relatively smaller

pore sizes should be used [113]. In their study, Kornyšova et al. [113] implement a

simple three-step method, where HEMA-PDA-DATD copolymers were used for the

formation of the monolithic bed. In this approach the attaching ligands can be

characterized more porous. The original monolith was composed of a monomer

(HEMA) and crosslinkers (PDA and DATD), an initiator and a catalyst.

A recent category of affinity monolithic stationary phases is the cryogels. The

exceptional characteristics of these phases are their hydrophilicity and their

macropores size in the range of 10–100 mm which is really large in comparison with

macropores of GMA-EDMA (1.5 mm) and silica monoliths (2 mm), respectively

[114]. A typical cryogel monolithic solution consists of AAm, allyl glycidyl ether and

29

N,N’-methylene bisacrylamide along with aqueous ammonium persulfate as initiator

and TEMED as accelerator [107].

An alternative approach of monolithic materials that is used in many applications is

the silica monoliths. These are present at two forms for ligand immobilization and

more specifically commercially available bare silica (e.g. Chromolith-Si) or

aminopropyl silica (e.g. Chromolith-NH2) and also sol–gel entrapment method [115].

Silica monoliths can be characterized as mechanically strong, with efficient results

and a high surface area. On the other hand, the restriction in the pH range (pH 2–8)

and also the decrease in the size represent the most important drawbacks of this type

of materials [116]. As far as the sol-gel method is concerned, the entrapment of the

ligand is taking place in a single step and the activity of the ligand remains without

any change. Nevertheless, the denaturation of the ligands, due to the alcoholic

byproducts which are released during the polymerization process, as well as the

decreased diameter of the column constitute important disadvantages of the method

[117].

Concluding, bioaffinity monolithic chromatography is expected to be used in a wide

range of applications in the future. This is due to the fact that this method has become

rough, not complicated and it can be performed in a conventional way without being

mandatory the investigation into depth for every application [107]. New forms are

going to be operated in order to recognize bacteria and also aptamers are going to be

used more often. Moreover, the investigation of new types of monoliths will also

include the study of present or alterative types of polymers, in order to come out with

a wider range of pore sizes, surface areas and new morphologies that can be used in

this type of affinity chromatography [111]. Finally, monolithic stationary phases are

expected to have a great impact on future applications, for instance if organic

monolithic supports will be combined with hybrids of silica [111].

2.11 Affinity Capillary Electrophoresis

One significant advantage of capillary electrophoresis (CE) is the separation of a

broad range of analytes at the same moment. Affinity Capillary Electrophoresis

(ACE) is a technique used in order to separate substances which participate either in

specific or in non-specific affinity interactions during the electrophoresis process, by

using a capillary electrophoresis format. The molecules can be free in solution or they

can be immobilized to a solid support [118].

There are three modes of affinity capillary electrophoresis, namely the non-

equilibrium electrophoresis of equilibrated sample mixtures, the dynamic equilibrium

affinity electrophoresis and the affinity-based capillary electrophoresis (CE) or

capillary electrochromatography (CEC) separations on immobilized selectors. In the

first one, the receptor and the ligand are in the sample and the electrophoresis

separation buffer is empty of both receptor and ligand. A portion of the total sample

30

solution is introduced into the capillary in order to begin the process. In the second

one, the receptor protein is in the sample and the ligand is placed in the

electrophoresis separation buffer. Inside the capillary binding and separation of the

affinity components is taking place. Finally, there are the affinity-based CE or CEC

separations on immobilized selectors. During this kind of separations the

immobilization of the receptor protein is taking place on the walls of a portion of the

capillary or microbeads, or membranes, or microchannels. The affinity related capture

of the ligand which exists either in a diluted solution or in a more complicated

mixture on the receptor is taking place. Later, components which are not connected

with the matrix are washed away and in addition the release of particular ligand from

adsorbed state occurs. The final step contains the performance of the separation by

capillary electrophoresis [118].

The use of ACE during recent years has provided significant results and advances in

biology and analytical chemistry. To be more specific, developments of CE in the

immunochemistry field are based on two different axes. The first one is the

miniaturization of immunoassays (lab-ona-chip) which enables rapid, relatively easy

and with high sensitivity measurements in the field of clinical chemistry. The second

one relies on CE applications in order to characterize in depth immunoreagents taking

into account the reactivity and the binding strength. One characteristic example is the

custom-made antibodies for particular affinity applications. One specific requirement

for the miniaturization process of immunoassays is the achievement of appropriate

detection limits which can be compared to the performance of traditional ELISAs. In

ELISA subpicomolar detection limits and parallel sample processing are necessary

[119].

There are also CE applications for analysis of proteins. Binding studies based on CE

are characterized from adsorption problems. A reproducible protein analysis demands

the appropriate working out conditions. One characteristic limitation of CE

applications is that the ideal conditions for protein separations, such as acidic PH, in

most of the cases are not the appropriate ones in order to study physiologically

relevant binding interactions. Low affinity interactions are used for the petides and

proteins analysis under the appropriate buffer conditions. The role of the new buffer

additives used is the connection with the analytes and by that means the shielding

from wall binding. One characteristic example is the use of mono and diquaternized

diamines in order to separate peptides and proteins [120].

Another approach of CE applications is the planning and characterization of

intramolecular interactions which are involved in protein folding. The determination

of various conformations contributed to long lifetimes, and also the wide range of

protein folding is the main reason for acceptable changes in the shape/size, charge

distribution, or exposure of interacting domains to change electrophoretic velocities

[121].

31

Furthermore, microelectrophoretic methods have been used for the investigation of

enzyme-substrate reactions which can be characterized as noncovalent molecular

interactions. Both the screening and the quantitative online characterization of

substrate formation and the inhibitor action can be conducted by electrophoretical

mixing. Moreover, the electrophoretically mediated microanalysis (EMMA) setup has

been used for the separation of zones of reactants and products [122].

In the field of biological reactions, carbohydrates are extremely crucial. Lectins, a

group of proteins with specific carbohydrate-binding capabilities, have been used in

many CE applications in order to identify and characterize parts of carbohydrates

[123]. On the other hand, the characterization of lectins is also possible by taking into

account the specific binding when panels of well-defined carbohydrates in CE are

used [124].

CE applications are evident for lipids, as well. The main problem however, is that

lipids in aqueous buffers can be characterized as insoluble and also they do not have

enough chromophores. Lipoconjugates such as lipoproteins and glycolipids, are

hydrophilic enough and as a result they are able for ordinary analysis techniques. In a

different way, the analysis of pure lipids can be conducted as micellar preparation, as

vesicles/liposomes, by creating mixed micelles when detergents are present and

finally by utilizing nonaqueous CE [125].

As far as the molecular biology field is concerned, CE applications have been used for

the investigation of DNA sequences. In addition there has been an increase in the

number of binding studies where DNA or RNA as analytes are involved. Two

subfields which have been developed in the last years contain the characterization of

small molecular ion–DNA interactions and drug–DNA/RNA interactions respectively

[126].

In addition, there are many studies and reports concerning the analysis of drug–

protein interactions. Drugs, protein–drug binding, enantiomers, and small molecules

CE-FA applications have been used in the vast majority of these studies. Moreover,

there are many studies based on HSA analyses because of the specific role that it has

about the administration of a wide range of drugs in humans. The determination of

protein binding and consequently the bioavailability of the drug candidates, that may

be used, have been examined with CE-FA methods [127].

Last but not least, CE applications have been used for the analysis of particles,

organelles, microorganisms, and eukaryotic cells, therefore also for their interactions,

based mainly on the inhomogeneous nature of the analytes [128].

32

3. Conclusion

As it has been shown throughout this review, in general affinity chromatography is an

alternative approach of traditional applications which take place in order to quantify

selective samples and examine a number of clinical samples. The greatest advantage

of this method is that for every compound in the field of clinical chemistry that has to

be analyzed, an independent affinity system can be created. There are many available

choices of ligands and operating formats used either for direct or indirect solute

determination.

Affinity chromatography provides valuable and important information about

separation methods in the fields of biomedical and pharmaceutical analysis. For this

reason, during the recent years, there has been reported significant growth on studies

focused on affinity chromatography. A number of different modes have been

examined until now, changing different parts of the whole mechanism, like for

example the type of support onto the column (traditional carbohydrate or HPLC

supports), the elution method (step or isocratic elution), and the affinity ligands

(lectins, boronates, antibodies, immobilized metal ions, dye ligands or aptamers).

Moreover, combinations with other analytical techniques such as reversed phase

HPLC, or mass spectrometry especially for the detection procedure can provide

satisfying results [3].

Additionally, affinity chromatography has been used in many new applications or

innovations which have helped many researchers to broaden their horizons of

knowledge and reconsider in a different way of view aspects of the technique. More

specifically, biological interactions and the competitive action between different

solutes for the binding sites are two characteristic examples. It is generally believed

that affinity chromatography will develop rapidly the following years and a large

number of new applications and modes are going to be investigated with this

technique. Researchers are almost sure that this technique will solve many problems

and will answer many questions manly during analysis of different biological and

pharmaceutical agents in really complex samples.

Finally, valuable information can be provided after using affinity chromatography in

clinical laboratories. A number of significant tests and applications can be conducted,

such as analysis of chiral drugs or examination of drug and hormone-protein binding.

The vast majority of these are specialized tests and it is almost sure that researchers

and scientists are going to use affinity chromatography even more in the future in

order to explore or investigate even more clinical samples or clinical applications.

Therefore, the future of affinity chromatography is undoubtedly promising not only

due to the impressive progress on bio-informatic tools, but also to the construction of

libraries which enable scientists and researchers to select robust, high-affinity ligands

[129]. The rapid development of high-throughput screening technologies also

enhances this option for the future.

33

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