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6. edited by David S. Hage University of Nebraska Lincoln,
Nebraska, U.S.A. Handbook of Affinity Chromatography Second Edition
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8. ix Preface Welcome to the Handbook of Afnity Chromatography,
Second Edition. The purpose of this book is to guide scientists,
students, and laboratory workers in the theory, use, and
applications of afnity chromatography. Since its original use
almost 100 years ago, afnity chromatography has become an important
tool in a large variety of areas. This is illustrated in this book
through examples and topics that range from the elds of biology,
biotech- nology, biochemistry, and molecular biology to analytical
chemistry, pharmaceutical sci- ence, environmental science, and
clinical chemistry. overview of afnity chromatography and discusses
important factors to consider in the development of afnity methods,
including the choice of support material, immobilization reviews of
common afnity methods, such as bioafnity chromatography,
immunoafnity chromatography, DNA afnity chromatography, boronate
afnity chromatography, dye- ligand and biomimetic afnity
chromatography, and immobilized metal-ion afnity chromatography.
respectively) explore the preparative, analytical, and biophysical
applications of afnity methods in areas such as biochemistry,
molecular biology, biotechnology, pharmaceutical capillary
electrophoresis, mass spectrometry, microanalytical systems,
chromatographic immunoassays, and molecularly imprinted polymers.
This book is the result of a collaborative effort involving 48
scientists and students from 23 laboratories and organizations
located throughout the world. I would like to thank each of these
individuals for their contributions to this project. I would also
like to thank my wife Jill and my sons Ben and Brian for their
assistance in the preparation of this text. I hope that all who use
this handbook will nd it a valuable addition to their work in
afnity chromatography or related areas. David S. Hage 2006 by
Taylor & Francis Group, LLC This handbook is divided into six
sections. Section I (Chapters 1 to 4) provides an method, and
application or elution conditions. Section II (Chapters 5 to 10)
provides detailed Sections III, IV, and V of this book (Chapters 11
to 16, 17 to 21, and 22 to 25, analysis, proteomics, clinical
testing, and environmental analysis. Section VI (Chapters 26 to 30)
considers recent developments in this eld, including the use of
afnity ligands in
9. xi Contents SECTION I Introduction and Basic Concepts
Chapter 1 An Introduction to Afnity Chromatography
.............................................. 3 David S. Hage and
Peggy F. Ruhn Chapter 2 Support Materials for Afnity
Chromatography........................................ 15 Chapter 3
Immobilization Methods for Afnity
Chromatography............................................................................
35 Chapter 4 Application and Elution in Afnity Chromatography
............................... 79 SECTION II General Afnity
Ligands and Methods Chapter 5 Bioafnity
Chromatography.....................................................................
101 Chapter 6 Immunoafnity Chromatography
............................................................. 127
Chapter 7 DNA Afnity Chromatography
................................................................
173 Chapter 8 Boronate Afnity
Chromatography..........................................................
215 2006 by Taylor & Francis Group, LLC Per-Erik Gustavsson and
Per-Olof Larsson Hee Seung Kim and David S. Hage David S. Hage, Hai
Xuan, and Mary Anne Nelson David S. Hage, Min Bian, Raychelle
Burks, Elizabeth Karle, Corey Ohnmacht, and Chunling Wa David S.
Hage and Terry M. Phillips Robert A. Moxley, Shilpa Oak, Himanshu
Gadgil, and Harry W. Jarrett Xiao-Chuan Liu and William H.
Scouten
10. xii Contents Chapter 9 Dye-Ligand and Biomimetic Afnity
Chromatography .......................... 231 Chapter 10
Immobilized Metal-Ion Afnity
Chromatography................................. 257 SECTION III
Preparative Applications Chapter 11 General Considerations in
Preparative Afnity Chromatography......... 287 Chapter 12 Afnity
Chromatography of Enzymes
................................................... 313 Chapter 13
Isolation of Recombinant Proteins by Afnity
Chromatography...................................................................
347 Chapter 14 Afnity Chromatography in Antibody and Antigen
Purication.........................................................................
367 Chapter 15 Afnity Chromatography of Regulatory and
Signal-Transducing
Proteins............................................................
399 Chapter 16 Receptor-Afnity
Chromatography........................................................
435 SECTION IV Analytical and Semipreparative Applications Chapter
17 Afnity Methods in Clinical and Pharmaceutical
Analysis................... 461 Chapter 18 Afnity Chromatography in
Biotechnology........................................... 487
Chapter 19 Environmental Analysis by Afnity
Chromatography........................... 517 Chapter 20 Afnity
Chromatography in Molecular Biology
................................... 547 2006 by Taylor &
Francis Group, LLC N.E. Labrou, K. Mazitsos, and Y.D. Clonis
Daniela Todorova and Mookambeswaran A. Vijayalakshmi Anuradha
Subramanian Felix Friedberg and Allen R. Rhoads Anuradha
Subramanian Terry M. Phillips Allen R. Rhoads and Felix Friedberg
Pascal Bailon, Michele Nachman-Clewner, Cheryl L. Spence, and
George K. Ehrlich Carrie A.C. Wolfe, William Clarke, and David S.
Hage Neil Jordan and Ira S. Krull Mary Anne Nelson and David S.
Hage Luis A. Jurado, Shilpa Oak, Himanshu Gadgil, Robert A. Moxley,
and Harry W. Jarrett
11. Contents xiii Chapter 21 Afnity-Based Chiral Stationary
Phases................................................ 571 SECTION
V Biophysical Applications Chapter 22 Quantitative Afnity
Chromatography: Practical Aspects..................... 595 Chapter
23 Quantitative Afnity Chromatography: Recent Theoretical
Developments..........................................................
629 Chapter 24 Chromatographic Studies of Molecular Recognition and
Solute Binding to Enzymes and Plasma
Proteins................................................................................
663 Chapter 25 Afnity-Based Optical
Biosensors.........................................................
685 SECTION VI Recent Developments Chapter 26 Afnity Ligands in
Capillary Electrophoresis .......................................
699 Chapter 27 Afnity Mass
Spectrometry....................................................................
737 Chapter 28 Microanalytical Methods Based on Afnity
Chromatography.............. 763 Chapter 29 Chromatographic
Immunoassays............................................................
789 Chapter 30 Molecularly Imprinted Polymers: Articial Receptors
for Afnity Separations
......................................................... 837 2006
by Taylor & Francis Group, LLC Sharvil Patel, Irving W. Wainer,
and W. John Lough David S. Hage and Jianzhong Chen Donald J. Winzor
Sharvil Patel, Irving W. Wainer, and W. John Lough Sheree D. Long
and David G. Myszka Niels H. H. Heegaard and Christian Schou Chad
J. Briscoe, William Clarke, and David S. Hage Terry M. Phillips
Annette C. Moser and David S. Hage Karsten Haupt
12. xv Editor David S. Hage is a professor of analytical and
bioanalytical chemistry at the University of Nebraska. He received
his B.S. in chemistry and biology from the University of
WisconsinLa Crosse in 1983 and a Ph.D. in analytical chemistry from
Iowa State Uni- versity in 1987. After nishing a postdoctoral
position at the Mayo Clinic in 1989, he joined the faculty at the
University of Nebraska. His interests include the study of
biological interactions by afnity methods and analytical
applications of afnity chromatography for biological and
environmental agents. He is the author of over 120 research
articles and reviews on the topic of afnity chromatography and
received the 1995 Young Investigator Award from the American
Association for Clinical Chemistry for his work. He has pre- sented
numerous seminars and workshops on afnity methods and is on the
editorial boards of several journals in the eld of chemical
separation and analysis. 2006 by Taylor & Francis Group,
LLC
13. xvii Contributors Pascal Bailon Hoffmann-LaRoche Inc.
Nutley, New Jersey Min Bian Department of Chemistry University of
Nebraska Lincoln, Nebraska Chad J. Briscoe MDS Pharma Services
Lincoln, Nebraska Raychelle Burks Department of Chemistry
University of Nebraska Lincoln, Nebraska Jianzhong Chen Department
of Chemistry University of Nebraska Lincoln, Nebraska William
Clarke Department of Pathology Johns Hopkins School of Medicine
Baltimore, Maryland Y. D. Clonis Laboratory of Enzyme Technology
Department of Agricultural Biotechnology Agricultural University of
Athens Athens, Greece George K. Ehrlich Hoffmann-LaRoche Inc.
Nutley, New Jersey Felix Friedberg Department of Biochemistry and
Molecular Biology College of Medicine Howard University Washington,
DC Himanshu Gadgil Department of Biochemistry University of
Tennessee Health Science Center Memphis, Tennessee Per-Erik
Gustavsson Department of Pure and Applied Biochemistry Center for
Chemistry and Chemical Engineering University of Lund Lund, Sweden
David S. Hage Department of Chemistry University of Nebraska
Lincoln, Nebraska 2006 by Taylor & Francis Group, LLC
14. xviii Contributors Karsten Haupt Compigne University of
Technology Centre de Recherches de Royallieu Compigne, France Niels
Heegaard Department of Autoimmunology Statens Serum Institute
Copenhagen, Denmark Harry W. Jarrett Department of Biochemistry
University of Tennessee Health Science Center Memphis, Tennessee
Neil Jordan Department of Chemistry and Chemical Biology
Northeastern University Boston, Massachusetts Luis A. Jurado
Department of Biochemistry University of Tennessee Health Science
Center Memphis, Tennessee Elizabeth Karle Department of Chemistry
University of Nebraska Lincoln, Nebraska Hee Seung Kim Department
of Chemistry University of Nebraska Lincoln, Nebraska Ira S. Krull
Department of Chemistry and Chemical Biology Northeastern
University Boston, Massachusetts N. E. Labrou Laboratory of Enzyme
Technology Department of Agricultural Biotechnology Agricultural
University of Athens Athens, Greece Per-Olof Larsson Department of
Pure and Applied Biochemistry Center for Chemistry and Chemical
Engineering University of Lund Lund, Sweden Xiao-Chuan Liu
Department of Chemistry California State Polytechnic University
Pomona, California Sheree D. Long Biacore Inc. Piscataway, New
Jersey W. John Lough Institute of Pharmacy and Chemistry University
of Sunderland Sunderland, United Kingdom K. Mazitsos Laboratory of
Enzyme Technology Department of Agricultural Biotechnology
Agricultural University of Athens Athens, Greece Annette C. Moser
Department of Chemistry University of Nebraska Lincoln, Nebraska
Robert A. Moxley Department of Biochemistry University of Tennessee
Health Science Center Memphis, Tennessee David G. Myszka Center for
Biomolecular Interaction Analysis University of Utah Salt Lake
City, Utah Michele Nachman-Clewner Hoffmann-LaRoche Inc. Nutley,
New Jersey 2006 by Taylor & Francis Group, LLC
15. Contributors xix Mary Anne Nelson Department of Chemistry
University of Nebraska Lincoln, Nebraska Shilpa Oak Department of
Biochemistry University of Tennessee Health Science Center Memphis,
Tennessee Corey Ohnmacht Department of Chemistry University of
Nebraska Lincoln, Nebraska Sharvil Patel Bioanalytical and Drug
Discovery Unit National Institute on Aging National Institutes of
Health Baltimore, Maryland Terry M. Phillips Ultramicro Analytical
Immunochemistry Resource Division of Bioengineering and Physical
Sciences Ofce of Research Services National Institutes of Health
Bethesda, Maryland Allen R. Rhoads Department of Biochemistry and
Molecular Biology College of Medicine Howard University Washington,
DC Peggy F. Ruhn MDS Pharma Services Lincoln, Nebraska William H.
Scouten College of Sciences University of Texas at San Antonio San
Antonio, Texas Christian Schou Department of Autoimmunology Statens
Serum Institute Copenhagen, Denmark Cheryl L. Spence
Hoffmann-LaRoche Inc. Nutley, New Jersey Anuradha Subramanian
Department of Chemical Engineering University of Nebraska Lincoln,
Nebraska Daniela Todorova Molecular Interaction & Separation
Technology Labs Universit de Technologie de Compigne Compigne,
France Mookambeswaran A. Vijayalakshmi Molecular Interaction &
Separation Technology Labs Universit de Technologie de Compigne
Compigne, France Chunling Wa Department of Chemistry University of
Nebraska Lincoln, Nebraska Irving W. Wainer Bioanalytical and Drug
Discovery Unit National Institute on Aging National Institutes of
Health Baltimore, Maryland Donald J. Winzor Department of
Biochemistry School of Molecular and Microbial Sciences University
of Queensland Brisbane, Queensland, Australia Carrie A. C. Wolfe
Division of Science and Mathematics Union College Lincoln, Nebraska
Hai Xuan Department of Chemistry University of Nebraska Lincoln,
Nebraska 2006 by Taylor & Francis Group, LLC
16. Section I Introduction and Basic Concepts 2006 by Taylor
& Francis Group, LLC
17. 3 1 An Introduction to Afnity Chromatography David S. Hage
Department of Chemistry, University of Nebraska, Lincoln, NE Peggy
F. Ruhn MDS Pharma Services, Lincoln, NE CONTENTS 1.1 Introduction
...............................................................................................................
3 1.2 History of Afnity
Chromatography.........................................................................
5 1.2.1 Origins of Afnity
Chromatography.............................................................
5 1.2.2 Early Immobilization Methods
.....................................................................
6 1.2.3 Modern Era of Afnity Chromatography
..................................................... 7 1.3
Overview of
Handbook...........................................................................................
11
References.........................................................................................................................
11 1.1 INTRODUCTION Chemical separation is an essential component
of modern research and is widely used to process complex samples.
Examples range from the trace analysis of a drug or hormone in
blood to the large-scale isolation of a recombinant protein. The
method of liquid chromatography has become particularly popular for
these separations because of its ability to work with a wide range
of substances. When combined with appropriate support materials,
this technique can be used in either high-performance separations
for chemical detection and measurement or in systems designed to
purify a desired product. The wide range of stationary phases and
mobile phases that can be employed in liquid chromatog- raphy also
makes this method quite exible in terms of the types of chemical or
physical properties that can be used as the basis for these
separations. One of the most versatile forms of liquid
chromatography is the technique known as afnity chromatography,
which can generally be dened as a liquid chromatographic 2006 by
Taylor & Francis Group, LLC
18. 4 Hage and Ruhn technique that uses a specic binding agent
for the purication or analysis of sample components [16]. This
technique makes use of the selective and reversible interactions
that occur in many biological systems, such as the binding of an
enzyme with a substrate or an antibody with an antigen. These
interactions are used in afnity chromatography by immobilizing one
of a pair of interacting molecules onto a solid support and placing
it into a column. The immobilized molecule is referred to as the
afnity ligand. This makes up the stationary phase of the afnity
column. Figure 1.1 shows a typical scheme used to perform afnity
chromatography. In this approach, a sample containing the compound
of interest is injected onto the afnity Figure 1.1 Typical
separation scheme for afnity chromatography. (Reproduced with
permission from Hage, D.S., in Handbook of HPLC, Katz, E., Eksteen,
R., Shoenmakers, P., and Miller, N., Eds., Marcel Dekker, New York,
1998, pp. 483498.) Response Apply Elute RegenerateApply Elute (a)
(b) Time (or volume) Regenerate Wash 2006 by Taylor & Francis
Group, LLC
19. An Introduction to Affinity Chromatography 5 column in the
presence of a mobile phase that has the right pH, ionic strength,
and solvent composition for solute-ligand binding. This solvent,
which represents the weak mobile phase of an afnity column, is
referred to as the application buffer. As the sample passes through
the column under these conditions, compounds that are complementary
to the afnity ligand will bind. However, due to the high
selectivity of this interaction, other solutes in the sample will
tend to wash or elute from the column as a nonretained peak. After
all nonretained components have been washed from the column, the
retained solutes are then eluted by applying a solvent that
displaces them from the column or that promotes dissociation of the
solute-ligand complex. This solvent, which represents the strong
mobile phase for the column, is known as the elution buffer. As the
solutes of interest elute from the column, they are either
quantitated directly or collected for later use. The application
buffer is then reapplied to the system and the column is allowed to
regenerate prior to the next sample injection. Due to the strong
and selective binding that characterizes many afnity ligands,
solutes that are quantitated or puried by these ligands can often
be separated with little or no interference from other sample
components. In many cases the solute of interest can be isolated in
only one or two steps, with purication yields of 100-fold to
several thousand- fold being common [26]. In work with hormone
receptors, purication yields approaching one million-fold have even
been reported with afnity-based separations [5]. The wide range of
ligands available for afnity chromatography makes this method a
valuable tool for the purication and analysis of compounds present
in complex samples. Areas in which afnity chromatography has been
used include biochemistry, pharmaceu- tical science, clinical
chemistry, and environmental testing. This book will examine the
variety and types of afnity ligands used in these areas and the
main factors to consider in the development of such a method.
Several specic applications will also be considered, including the
use of afnity chromatography in small- or large-scale purication,
analyte detection, and the characterization of biological
interactions. Finally, a number of new developments in this eld and
in related areas of work will be discussed. 1.2 HISTORY OF AFFINITY
CHROMATOGRAPHY 1.2.1 Origins of Afnity Chromatography Although some
consider afnity chromatography to be a relatively new method, it is
actually one of the oldest forms of liquid chromatography. For
instance, the earliest use of this method was just seven years
after Michael Tswett reported the rst known use of column liquid
chromatography [7]. This occurred in 1910 when Emil Starkenstein
exam- ined the binding of insoluble starch to the enzyme -amylase
[8]. This is also the rst known case in which liquid chromatography
was used for a separation involving a protein. The original studies
with afnity chromatography all made use of insoluble materials that
acted as both the stationary phase and support material. This is
not surprising, since this is the simplest form of afnity
separation. For instance, the insoluble starch used by Starkenstein
acted as both a support material and as a substrate for amylase,
thus leading to this enzymes binding and retention. Similar work
with starch and amylase was con- ducted in the 1920s through 1940s
by other investigators [912], with a 300-fold puri- cation being
obtained in one of these studies [12]. Other examples include the
use of polygalacturonase as a support and ligand for the adsorption
of alginic acid [13], the purication of pepsin through the use of
edestin, a crystalline protein [14], and the isolation of porcine
elastase with powdered elastin [15]. 2006 by Taylor & Francis
Group, LLC
20. 6 Hage and Ruhn As this suggests, much of the earliest work
with afnity supports involved its use in the purication of enzymes.
But research was also being conducted at this time in the selective
purication of antibodies with biological ligands. This arose from
the work by Landsteiner, who showed in 1920 that antibodies can
recognize and bind substances with a specic structure, referred to
as antigens [16]. This type of binding plus the ability of
polyclonal antibodies to form insoluble complexes with antigens led
to the growth in the 1930s of immunoprecipitation as an important
technique for antibody purication [1719]. For instance, this
approach was used by Kirk and Sumner to isolate antibodies against
urease and to demonstrate that these antibodies were proteins [17].
This approach was similar to the work being performed in the
purication of enzymes in that a ligand (i.e., the antigen) was used
to create a specic biological interaction with the target of
interest (the antibodies). However, rather than having the ligand
be the same as the solid used for this isolation, this solid was
formed as a result of the binding process. 1.2.2 Early
Immobilization Methods Although the use of insoluble ligands and
immunoprecipitation proved the potential for biological
interactions as a means for chemical isolation, this approach was
still limited to solutes for which an appropriate ligand and/or
support was available. However, this began to change in the 1940s
and 1950s as synthetic techniques became available for placing a
broader range of ligands onto insoluble materials. These efforts
began by employing solids that contained a noncovalently adsorbed
layer of ligand. For instance, in 1935 DAlessandro and Soa used
antigens coated on kaolin and charcoal for the isolation of
antibodies associated with syphilis and tuberculosis [20]. A
similar type of support was used by Meyer and Pic in 1936 [21].
However, it was soon realized that a more stable system would be
obtained by chemi- cally bonding the ligand to the support. This
was rst used by Landsteiner and van der Scheer in 1936, when they
adapted a diazo-coupling technique used to prepare hapten
conjugates [22]. In this case, they attached a number of haptens to
a solid material based on chicken erythrocyte stroma, with this
material then being used for the isolation of antibodies for these
haptens (see Figure 1.2). Work with other, more durable support
materials also began to appear.A key develop- ment in this area
occurred in 1951, when Campbell and coworkers used an activated
form Figure 1.2 An early example of the use of immobilized antigens
for antibody purication. These were used by Landsteiner and van der
Scheer in 1936 for performing cross-reactivity studies of immune
sera. (Reproduced with permission from Landsteiner, K. and van der
Scheer, J., J. Exp. Med., 63, 325339, 1936.) 1 cc of suberanilic
acid immune serum absorbed with azostromata made from
p-Aminoadipanilic acid p-Aminosebacanilic acid Unabsorbed immune
serum p-Aminoadipanilic acid p-Aminosuberanilic acid
p-Aminosebacanilic acid Azoproteins made from casein and 0 0 + +
+++ +++ ++ +++ +++ ++++ ++++ ++++ + ++ 0 0 +++ +++ 2006 by Taylor
& Francis Group, LLC
21. An Introduction to Affinity Chromatography 7 of cellulose
(p-aminobenzylcellulose) for immobilizing the protein serum
albumin. This material was then used to isolate antialbumin
antibodies from rabbit serum [23]. Similar work appeared by Lerman
in the preparation of immobilized hapten supports [24] and in 1953
through the use of a covalently immobilized ligand for purifying
the enzyme mush- room tyrosinase (see Figure 1.3) [25]. Other
studies in immunopurication later began to appear with ligands
attached to such supports as polyaminostyrene [26] and glass beads
[27]. By 1966, several reviews on such work had appeared [2834].
After the report by Lerman [25], the use of immo- bilized ligands
for enzyme isolation received only limited attention for some time,
probably due to the rise of ion exchange with cellulose in the
1950s and 1960s as a separation tool in enzymology. However, this
began to change in the mid-1960s, when Arsenis and McCormick began
to use cellulose-based afnity supports for the isolation of
avokinase and avin mononucleotide-dependent enzymes [35, 36]. 1.2.3
Modern Era of Afnity Chromatography The next major development came
about in the late 1960s. There were three things that led to this
event. The rst was the creation of beaded agarose supports by
Hjerten in the mid-1960s [37]. This provided a more efcient and
exible support than cellulose for use with biopolymers in liquid
chromatography. The second development was the discovery of the
cyanogen bromide immobilization method. This was reported in 1967
by Axen, Porath, and Ernback, providing a more convenient and
general approach that could be used to attach proteins and peptides
to polysaccharides [38]. The third development was a 1968 report by
Cuatrecasas, Wilchek, and Annsen in which agarose and the cyanogen
bromide method were used together to create immobilized nuclease
inhibitor columns [39]. These columns were then used for purifying
the enzymes staphylococcal nuclease, -chymotrypsin, Figure 1.3
Early examples of immobilized inhibitors that were used by Lerman
in 1953 for the isolation of tyrosinase. (Reproduced with
permission from Lerman, L.S., Proc. Natl. Acad. Sci. U.S.A., 39,
232236, 1953.) Cellulose O NN NN OH OH Cellulose O COOHNN NN OH
COOH NN OH AsO3H2 Cellulose O NN OH HO OH Cellulose O NN OH
Cellulose O NN OH NN CH2 CH3 NNCellulose O CH2 NNCellulose O CH2
2006 by Taylor & Francis Group, LLC
22. 8 Hage and Ruhn and carboxypeptidase A. An example of such
a separation is shown in Figure 1.4. This was the same report in
which the term afnity chromatography was rst used to describe this
separation technique [39]. The techniques and applications that
appeared in this last report led to a rapid increase in interest in
the use of afnity ligands in liquid chromatography. This is
illustrated and 2001 that have contained the phrase afnity
chromatography. The number of such reports has grown from four in
1968 to approximately 1000 per year over most of the last decade.
An even greater number of reports (i.e., roughly three times the
number shown in Figure 1.5) have included the use of related
phrases like immunoafnity chromatog- raphy. These results clearly
demonstrate the widespread use of afnity chromatography and the
impact it has had on modern separations. Following the
reintroduction of afnity chromatography in 1968, a variety of
ligands and applications for this method began to appear. For
instance, the next six years saw the introduction of such methods
as DNA-cellulose afnity chromatography [40], boronate afnity
chromatography [41], dye-ligand afnity chromatography [42], and
immobilized metal-ion afnity chromatography [43]. The ligands that
are used in these techniques and other ligands and approaches that
are now commonly used in afnity chromatography. Figure 1.4 One of
the rst examples of modern afnity chromatography (Reproduced with
permis- sion from Cuatrecasas, P.,Wilchek, M., andAnnsen, C.B.,
Proc. Natl. Acad. Sci. U.S.A., 68, 636643, 1968.) Absorbancy,280m
0.2 0.2 2 4 6 8 10 12 14 16 1.0 0.6 0.2 0.6 0.6 Euent, ML
Carboxypeptidase A (Unsubstituted Sepharose) Carboxypeptidase A
(L-Tyrosyl-D-Tryptophan Sepharose) Carboxypeptidase B
(L-Tyrosyl-D-Tryptophan Sepharose) 2006 by Taylor & Francis
Group, LLC the types of targets they retain are summarized in Table
1.1. This same table also shows by Figure 1.5, which shows the
number of publications that have appeared between 1968
23. AnIntroductiontoAfnityChromatography9 Figure 1.5 Number of
articles per year that appeared from 1968 to 2001 and contained the
phrase afnity chromatography. A total of 26,132 articles were
identied in this search, which was performed using CAPLUS and
MEDLINE. The inclusion of other phrases, such as afnity
chromatographic, immunoafnity chroma- tography, and immunoafnity
chromatographic led to the identication of 75,742 articles from
this same period of time. NumberofArticlesPublished 1200 1000 800
600 400 200 0 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986
1988 1990 1992 1994 1996 1998 2000 Publication Year 2006 by Taylor
& Francis Group, LLC
24. 10HageandRuhn Table 1.1 Common Ligands Used in Afnity
Chromatography Method Ligand Retained Solutes Immunoafnity
chromatography Antibodies Antigens (e.g., drugs, hormones,
peptides, proteins, viruses, cell components) Antigens Antibodies
against the antigens Afnity chromatography of enzymes Inhibitors,
substrates, cofactors, coenzymes, and miscellaneous ligands Enzymes
Lectin afnity chromatography Lectins Sugars, glycoproteins, and
glycolipids Protein A/protein G afnity chromatography Protein A,
protein G, and related ligands Antibodies and antibody fragments
DNA afnity chromatography DNA or RNA DNA/RNA-binding proteins,
complementary nucleotides Boronate afnity chromatography Boronates
Carbohydrates, nucleosides, nucleotides, nucleic acids,
glycoproteins, and catechols Dye-ligand afnity chromatography
Synthetic dyes Proteins and enzymes Biomimetic afnity
chromatography Ligands generated using combinatorial chemistry or
from peptide, phage display, ribosome display, or aptamer libraries
Various targets, including proteins and enzymes Immobilized
metal-ion afnity chromatography Metal-ion chelates Metal-binding
amino acids, peptides, proteins, and nucleotides 2006 by Taylor
& Francis Group, LLC
25. An Introduction to Affinity Chromatography 11 1.3 OVERVIEW
OF HANDBOOK This book is designed to introduce the reader to the
topic of afnity chromatography and to the most common methods that
are employed in this technique. The rst section of this handbook
deals with the basic components of an afnity chromatographic
system. This followed by a review of immobilization methods for
attaching ligands to such supports under the topics of bioafnity,
immunoafnity, and DNA afnity chromatography. Examples dyes and
biomimetic compounds, and immobilized metal-ion chelates,
respectively. isolation of targets. This was the original
application of afnity chromatography and remains one of the largest
uses for this technique. In this section, a review of general items
to consider in the design of preparative chromatographic systems is
provided in Chapter 11. ligands in the isolation of enzymes,
recombinant proteins, antibodies and antigens, regula- tory or
signal-transducing proteins, and targets for receptors. Along with
its traditional use in preparative work, afnity chromatography has
also looks at some specic areas in which this method continues to
play a signicant role in research and analysis, including clinical
testing and pharmaceutical analysis, biotechnology, environmental
analysis, molecular biology, and chiral separations, respectively.
Another growing eld has been the use of afnity columns to
characterize and study biological interactions. This approach is
sometimes referred to as quantitative afnity chro- aspects of the
experiments involved in such studies are considered (Chapter 22).
An in-depth afnity-based biosensors for such work is considered
(Chapter 25). to afnity chromatography or are special applications
of this technique. The discussion in this nal section includes the
use of afnity ligands in such areas as capillary electro- systems
based on afnity chromatography and the use of afnity columns in
chromatographic- ularly imprinted polymers. REFERENCES 1. Ettre,
L.S., Nomenclature for chromatography, Pure Appl. Chem., 65,
819872, 1993. 2. Hage, D.S.,Afnity chromatography, inHandbook of
HPLC, Katz, E., Eksteen, R., Shoenmakers, P., and Miller, N., Eds.,
Marcel Dekker, New York, 1998, pp. 483498. 3. Turkova, J., Afnity
Chromatography, Elsevier, Amsterdam, 1978. 4. Scouten, W.H., Afnity
Chromatography: Bioselective Adsorption on Inert Matrices, Wiley,
New York, 1981. 2006 by Taylor & Francis Group, LLC begins with
a discussion of support materials that are used in this method
(Chapter 2), (Chapter 3). The selection of application and elution
conditions are then examined in Chapter 4. Common ligands employed
in afnity chromatography are examined in Section II More-detailed
presentations are then made in Chapters 12 to 16 regarding the use
of afnity been playing an increasing role in the analysis of
chemicals. Section IV (Chapters 17 to 21) Section III (Chapters 11
to 16) discusses the use of afnity chromatography for the of
synthetic or nonbiological ligands are then given in Chapters 8 to
10, including boronates, (Chapters 5 to 10). A variety of
biological ligands are rst considered in Chapters 5 to 7
matography. This area is examined in Section V (Chapters 22 to 25).
First, various practical look at the theory behind this method is
then given (Chapter 23), and its use in the study of enzymes and
plasma proteins is reviewed (Chapter 24). Finally, the use and
development of Section VI (Chapters 26 to 30) presents several new
methods that are closely related phoresis (Chapter 26) and mass
spectrometry (Chapter 27). The creation of microanalytical based
immunoassays are also described in this section (Chapters 28 and
29). The last chapter (Chapter 30) explores a new approach for
creating afnity columns using molec-
26. 12 Hage and Ruhn 5. Parikh, I. and Cuatrecasas, P., Afnity
chromatography, Chem. Eng. News, 63, 1732, 1985. 6. Walters, R.R.,
Afnity chromatography, Anal. Chem., 57, 1099A1114A, 1985. 7.
Tswett, M., The chemistry of chlorophyll, phylloxanthin,
phyllocyanin, and chlorophyllane, Biochem. Zeit., 5, 632, 1907. 8.
Starkenstein, E., Ferment action and the inuence upon it of neutral
salts, Biochem. Z., 24, 210218, 1910. 9. Ambard, L., Amylase: Its
estimation and the mechanism of its action, Bull. Soc. Chim. Biol.,
3, 5165, 1921. 10. Holmbergh, O., Adsorption of -amylase from malt
by starch, Biochem. Z., 258, 134140, 1933. 11. Tokuoka, Y., Koji
amylase, IX: Existence of -amylase, J. Agric. Chem. Soc. Japan, 13,
586594, 1937. 12. Hockenhull, D.J.D. and Herbert, D., The amylase
and maltase of Clostridium acetobutylcium, Biochem. J., 39, 102106,
1945. 13. Lineweaver, H., Jang, R., and Jansen, E.F., Specicity and
purication of polygalacturonase, Arch. Biochem., 20, 137152, 1949.
14. Northrup, J.H., Crystalline pepsin, VI: Inactivation by - and
-rays from radium and by ultraviolet light, J. Gen. Physiol., 17,
359363, 1934. 15. Grant, N.H. and Robbins, K.C., Porcine elastase
and proelastase, Arch. Biochem. Biophys., 66, 396403, 1957. 16.
Landsteiner, K., Specic serum reactions induced by the addition of
substances of known constitution (organic acids), XVI: Antigens and
serological specicity, Biochem. Z., 104, 280299, 1920. 17. Kirk,
J.S. and Sumner, J.B., The reaction between crystalline urease and
antiurease, J. Immunol., 26, 495504, 1934. 18. Marrack, J.R. and
Smith, F.C., Quantitative aspects of immunity reactions: The
combination of antibodies with simple haptenes, Brit. J. Exp.
Pathol., 13, 394402, 1932. 19. Heidelberger, M. and Kabat, E.A.,
Quantitative studies on antibody purication, II: The dissociation
of antibody from pneumococcus-specic precipitates and specically
aggluti- nated pneumococci, J. Exp. Med., 67, 181199, 1938. 20.
dAlessandro, G. and Soa, F., The adsorption of antibodies from the
sera of syphilitics and tuberculosis patients, Z. lmmunitats., 84,
237250, 1935. 21. Meyer, K. and Pic, A., Isolation of antibodies by
xation on an adsorbent-antigen system with subsequent regeneration,
Ann. Inst. Pasteur, 56, 401412, 1936. 22. Landsteiner, K. and van
der Scheer, J., Cross reactions of immune sera to azoproteins, J.
Exp. Med., 63, 325339, 1936. 23. Campbell, D.H., Luescher, E., and
Lerman, L.S., Immunologic adsorbents, I: Isolation of antibody by
means of a cellulose-protein antigen, Proc. Natl. Acad. Sci.
U.S.A., 37, 575578, 1951. 24. Lerman, L.S., Antibody chromatography
on an immunologically specic adsorbent, Nature, 172, 635636, 1953.
25. Lerman, L.S., A biochemically specic method for enzyme
isolation, Proc. Natl. Acad. Sci. U.S.A., 39, 232236, 1953. 26.
Manecke, G. and Gillert, K.E., Serologically specic adsorbents,
Naturwissenschaften, 42, 212213, 1955. 27. Sutherland, G.B. and
Campbell, D.H., The use of antigen-coated glass as a specic
adsorbent for antibody, J. Immunol., 80, 294298, 1958. 28. Isliker,
H.C., Chemical nature of antibodies, Adv. Prot. Chem., 12, 387463,
1957. 29. Kabat, E.A. and Mayer, M.M., Experimental
Immunochemistry, 2nd ed., Charles C Thomas, Springeld, IL, 1961,
pp. 781797. 30. Manecke, G., Reactive polymers and their use for
the preparation of antibody and enzyme resins, Pure Appl. Chem., 4,
507520, 1962. 31. Sehon, A.H., Physicochemical and immunochemical
methods for the isolation and charac- terization of antibodies,
Brit. Med. Bull., 19, 183191, 1963. 2006 by Taylor & Francis
Group, LLC
27. An Introduction to Affinity Chromatography 13 32. Weliky,
N., Weetall, H.H., Gilden, R.V., and Campbell, D.H., Synthesis and
use of some insoluble immunologically specic adsorbents,
Immunochemistry, 1, 219229, 1964. 33. Weliky, N. and Weetall, H.H.,
Chemistry and use of cellulose derivatives for the study of
biological systems, Immunochemistry, 2, 293322, 1965. 34. Silman,
I.H. and Katchalski, E., Water-insoluble derivatives of enzymes,
antigens, and anti- bodies, Annu. Rev. Biochem., 35, 873908, 1966.
35. Arsenis, C. and McCormick, D.B., Purication of liver avokinase
by column chromatog- raphy on avine-cellulose compounds, J. Biol.
Chem., 239, 30933097, 1964. 36. Arsenis, C. and McCormick, D.B.,
Purication of avin mononucleotide-dependent enzymes by column
chromatography on avin phosphate cellulose compounds, J. Biol.
Chem., 241, 330334, 1966. 37. Hjerten, S., The preparation of
agarose spheres for chromatography of molecules and particles,
Biochem. Biophys. Acta, 79, 393398, 1964. 38. Axen, R., Porath, J.,
and Ernback, S., Chemical coupling of peptides and proteins to
polysaccharides by means of cyanogen halides, Nature, 214,
13021304, 1967. 39. Cuatrecasas, P., Wilchek, M., and Annsen, C.B.,
Selective enzyme purication by afnity chromatography, Proc. Natl.
Acad. Sci. U.S.A., 68, 636643, 1968. 40. Alberts, B.M., Amodio,
F.J., Jenkins, M., Gutmann, E.D., and Ferris, F.L., Studies with
DNA-cellulose chromatography, I: DNA-binding proteins from
Escherichia coli, Cold Spring Harbor Symp. Quant. Biol., 33,
289305, 1968. 41. Weith, H.L., Wiebers, J.L., and Gilham, P.T.,
Synthesis of cellulose derivatives containing the dihydroxyboryl
group and a study of their capacity to form specic complexes with
sugars and nucleic acid components, Biochemistry, 9, 43964401,
1970. 42. Staal, G., Koster, J., Kamp, H., Van Milligen-Boersma,
L., and Veeger, C., Human erythro- cyte pyruvate kinase, its
purication and some properties, Biochem. Biophys. Acta, 227, 8692,
1971. 43. Porath, J., Carlsson, J., Olsson, I., and Belfrage, B.,
Metal chelate afnity chromatography, a new approach to protein
fraction, Nature, 258, 598599, 1975. 2006 by Taylor & Francis
Group, LLC
28. 15 2 Support Materials for Afnity Chromatography Per-Erik
Gustavsson and Per-Olof Larsson Department of Pure and Applied
Biochemistry, Lund University, Lund, Sweden CONTENTS 2.1
Introduction
.............................................................................................................
16 2.2 Properties of Support
Materials..............................................................................
16 2.2.1 Chemical
Inertness......................................................................................
16 2.2.2 Chemical
Stability.......................................................................................
20 2.2.3 Mechanical
Stability....................................................................................
21 2.2.4 Pore
Size......................................................................................................
21 2.2.5 Particle
Size.................................................................................................
22 2.2.5.1 Zonal Elution
Chromatography...................................................
23 2.2.5.2 Adsorption-Desorption Afnity Purication
............................... 24 2.2.5.3 Partial Loading in
Adsorption-Desorption Afnity Purication
.....................................................................
24 2.2.5.4 Particulate Contaminants: Large Beads/Expanded Beds
....................................................... 25 2.2.6
Standard Commercially Available Afnity
Supports.................................. 25 2.3 Afnity Supports
with Special Properties
.............................................................. 26
2.3.1 Nonporous
Supports....................................................................................
26 2.3.2 Membranes
..................................................................................................
26 2.3.3 Flow-Through
Beads...................................................................................
28 2.3.4 Continuous Beds
.........................................................................................
29 2.3.5 Expanded-Bed
Adsorbents..........................................................................
30 2.4 Summary and Conclusions
.....................................................................................
30 Symbols and
Abbreviations..............................................................................................
31
References.........................................................................................................................
32 2006 by Taylor & Francis Group, LLC
29. 16 Gustavsson and Larsson 2.1 INTRODUCTION A key item in
the selection and design of an afnity chromatographic method is the
choice of support material. The supports used in afnity
chromatography must meet several requirements. Ideally, such a
support should be inexpensive and allow solutes to have rapid,
unhindered access to the immobilized afnity ligand. In addition,
the support should play a completely passive role during the
separation while also being able to couple the desired afnity
ligand. These are not trivial requirements. For example, quick
access of solutes to the ligand will require small support
particles. But if these solutes are large biomolecules, the support
must also have large pores. For the support to play a passive role,
it must be chemically inert toward the chromatographic solvents and
solutes, have no ionic or hydrophobic groups, and be resistant
toward the mechanical strains exerted during the chromatographic
process. And yet, for the support to be used for ligand attachment,
it must be able to undergo chemical modication or somehow adsorb
the ligand. In reality, no true ideal support exists for afnity
chromatography. This is because many of these requirements are in
direct conict with each other. As a result, all current afnity
supports involve some compromise in these properties and are
generally geared toward a particular application. Some extreme
examples of this are micrometer-sized nonporous particles, which
are optimized for rapid analytical or micropreparative separa-
tions, and 0.2-mm expanded-bed particles or 0.4-mm Big Beads, which
are optimized for separating crude extracts in the early stages of
a purication process. This chapter examines the various properties
that should be considered when selecting a support for afnity
chromatography. By being familiar with these properties, one can
make an informed choice in selecting a support for a given
application. 2.2 PROPERTIES OF SUPPORT MATERIALS 2.2.1 Chemical
Inertness The rst important property for a successful afnity column
is that it should rmly and specically bind the desired solute while
leaving all other molecules in the sample or process stream
untouched. This requires that the support within the column contain
an afnity ligand that is capable of forming a suitably strong
complex with the solute of interest. Second, the support material
must be inert to other solutes to avoid the simulta- neous binding
of nondesired sample components. This requires that the support
have a chemical character that is very similar to that of the
medium in which it is operating. Since almost all afnity
separations occur in aqueous solutions, the support should thus be
as hydrophilic as possible. As a rule, the mobile phase used in
afnity separations has a low ionic strength. The support should
therefore contain as few charges as possible to prevent ionic
interactions. Many supports that are available today fulll these
requirements. This is because either their basic structure has the
desired properties or because they are provided with a hydrophilic
coating that gives them such properties. A well-known example is
the polysaccha- ride agarose, which was used in the rst modern
application of afnity chromatography [1]. This is sold under
several trade names, like Sepharose Fast Flow from Amersham Bio-
polymeric chains of the disaccharide agarobiose, which in turn is
made up of D-galactose in agarose are clustered together in
bundles. These form a porous and hydrophilic network, 2006 by
Taylor & Francis Group, LLC sciences or Af-Gel from Bio-Rad
Laboratories (see Table 2.1). Agarose consists of and
3,6-anhydro-L-galactose, as shown in Figure 2.1a. The individual
polymeric chains
30. SupportMaterialsforAffinityChromatography17 Table 2.1
Examples of Traditional Afnity Supports Trade Name Material Average
Particle Diameter (m) Examples of Available Ligands Preactivated
Form Available? Manufacturer/Supplier Sepharose HP Sepharose FF
Agarose Agarose 34 90 Protein A, heparin, Cibacron Blue Protein A,
heparin, Cibacron Blue Yes Yes Amersham Biosciences Amersham
Biosciences Mimetic series Agarose 105 Synthetic ligands No
Prometic Biosciences/ACL Af-Gel Agarose 150300, 75150 Protein A,
heparin, Cibacron Blue Yes Bio-Rad Laboratories Sepharose Big Beads
Agarose 200 IMACa No Amersham Biosciences Cellthru Big Beads
Agarose 400 Cibacron Blue, heparin, IMACa Yes Sterogene TSK-Gel
Polymethacrylate 10 Boronate, IMACa , heparin Yes TosoHaas/Supelco
Af-Prep Polymethacrylate 10, 50 Protein A, polymyxin No Bio-Rad
Laboratories SigmaChrom AF Polymethacrylate 20 Protein A, IMACa ,
Cibacron Blue No Supelco Fractogel Polymethacrylate 30, 65 IMACa ,
heparin Yes Merck ProteinPak Silica 40 No Yes Waters Bakerbond
Silica 40 No Yes J.T. Baker Trisacryl Polyacrylamide derivative 60,
200 Cibacron Blue, Basiline Blue No BioSepra/Pall Af-Gel 601
Polyacrylamide Not specied Boronate No Bio-Rad Laboratories Cellune
Cellulose 85, 90, 170 Heparin, IMACa , gelatin Yes
Chisso/Amicon/Millipore a IMAC, immobilized metal-ion afnity
chromatography.. 2006 by Taylor & Francis Group, LLC
31. 18 Gustavsson and Larsson where the groups facing the
solvent have a minimum tendency to attract sample compo- nents.
Cellulose is another example of a polysaccharide support that is
used in afnity chromatography. Another example of a support used in
afnity chromatography is silica. This is used in the method of
high-performance liquid afnity chromatography (HPLAC) or high-
performance afnity chromatography (HPAC), which was rst reported in
1978 [2]. Although silica-based materials are certainly
hydrophilic, they are unsuitable for afnity chromatography unless
they have rst been modied at their surface. This is the case
because the native surface of silica is primarily covered with
silanol groups. These groups are weak acids that give silicas
surface a strong negative charge at neutral pH. These charges, in
combination with other binding forces, often result in the
irreversible adsorption of solutes like proteins to native silica.
However, several schemes can be used to render this surface inert
toward such solutes, including polymer coating techniques and
reactions between silica and alcohols or trialkoxysilanes [3, 4].
An example of such a scheme is the reaction of silica with
-glycidoxypropyltrimethoxysilane [5], followed by acid hydrolysis
of the resulting epoxy groups; this leaves the modied silica with a
hydrophilic, noncharged surface (shown in Figure 2.1b) that has
little or no binding to many biological compounds. The polymeric
support polystyrene (illustrated in Figure 2.1c) is also unsuitable
in its original form for afnity separations due to the highly
hydrophobic character of this material. Native polystyrene, which
is often used as a reversed-phase material, must rst Figure 2.1
Common materials used in the preparation of afnity supports. The
structure in (a) shows the repeating unit of agarose: D-galactose
and 3,6-anhydro-L-galactose. The structure in (b) shows diol-bonded
silica, and (c) is the structure of polystyrene/divinylbenzene.
CH2OH OH OH CH2 O H H O H HO H H H HO O OH HO H O Si CH2 CH2CH2
OCH2 CH2CH OH OH O O (a) (b) (c) 2006 by Taylor & Francis
Group, LLC
32. Support Materials for Affinity Chromatography 19 be
rendered hydrophilic by one of various surface-coating techniques
before it can be used in other chromatographic methods [6, 7].
Polymeric supports based on polymethacrylate [8, 9] are more
hydrophilic than polystyrene supports and can be used directly in
afnity chromatography. Examples of these and other polymeric afnity
supports, such as those A support material should be inert toward
solutes, but to make it suitable for afnity chromatography, it
should also be easy to couple to a ligand. Since most support
materials are rich in hydroxyl groups, the chemistries developed
for the attachment of ligands have focused mainly on using these
regions as anchoring points. Many such methods are available [10,
11], A support material with a tendency to adsorb solutes in a
nonspecic manner may still be useful, provided that the surrounding
medium is modied accordingly. For instance, if one changes the
buffer in an afnity column so that the buffers chemical character
(e.g., hydrophilicity and ion strength) matches that of the
support, there will be a minimum tendency toward nonspecic
adsorption. As an example, when a support has charged groups
present that can create undesired ionic interactions, a mobile
phase buffer with an ionic strength of about 0.15 M can be used to
suppress such binding [12, 13]. Even if the support is essentially
free of nonspecic interactions with sample com- ponents, the nal
afnity adsorbent obtained with this support could have a
dramatically different character. This occurs because the procedure
used to couple the ligand to the support may introduce undesired
groups that might then act as a new source of nonspecic binding. A
well-known example occurs during the activation of agarose and
other polysac- charide supports with cyanogen bromide. In one
standard protocol for this method, a large excess of cyanogen
bromide is used. This excess is necessary because the alkaline con-
ditions used for the reaction lead to a substantial breakdown of
the active cyanate ester groups (see Figure 2.2a). Several
breakdown products (including charged ones) may form when this
occurs, which later leads to nonspecic interactions between the
support and injected solutes. To avoid this problem, a modied
protocol can be utilized that involves activating the support at a
low temperature in the presence of a cyanogen transfer agent (e.g.,
triethylamine), which results in a less-altered support [14]. The
cyanogen transfer agent in this alternative technique increases the
electrophilicity of the cyanogen bromide by complex formation (as
shown in Figure 2.2b). This allows the activation to be carried out
at a neutral pH, which, in turn, minimizes hydrolysis of the
cyanate ester groups and increases the overall reaction yield.
Figure 2.2 Reaction schemes showing (a) the classical cyanogen
bromide (CNBr) activation tech- nique and (b) the modied cyanogen
bromide activation technique using a cyano transfer agent (CTA). OH
OH CTA-CN+ O OCN CNBr Overall reaction yield 20% of added CNBrBr +
pH 7-8 (a) (b) 2006 by Taylor & Francis Group, LLC based on
polyacrylamide, are listed in Table 2.1. with these being described
in greater detail in Chapter 3.
33. 20 Gustavsson and Larsson The ligand itself may be
responsible for unwanted interactions on the nal support. For
instance, suppose that some agarose beads contained an immobilized
derivative of adenosine triphosphate (ATP) and that the nal support
had a ligand concentration of 10 to 15 mol ATP per milliliter of
packed-bed volume. This is a normal degree of substitution and
would be suitable for retaining ATP-dependent proteins. However,
this modication has also made the agarose into a cation-exchange
resin through the presence of the phosphate groups on the ATP. At
pH 7, the charge concentration on this gel would be about 50 mol
per milliliter of packed bed. Taking into account the multivalency
of the charged triphos- phate groups, the mobile phase used with
this support would need to contain a 0.1 to 0.2 M buffer to cancel
the ion-exchange properties imposed by the ATP afnity ligand. Such
effects should be considered whenever a new afnity adsorbent is
being designed. To avoid these nonspecic effects and thereby
improve selectivity, it may also be wise to avoid working at
unnecessarily high ligand concentrations. A related source of
unwanted binding can occur via the ligand spacer. The spacer
molecule serves to make an immobilized ligand more accessible to an
injected macromol- ecule. Hexamethylenediamine is often used as
spacer for this purpose [10, 11], but this introduces both charged
groups and a patch of hydrophobicity to the supports surface.
Sometimes these extra functionalities may act with the ligand to
give even stronger binding for the desired sample components.
However, in most cases they will lower the selectivity of the
adsorbent, due to its retention of undesired sample components.
This phenomenon was observed early in the development of afnity
chromatography and eventually led to the development of a
separation mode known as hydrophobic interaction chromatography
(HIC) [15]. 2.2.2 Chemical Stability Obviously, the afnity
adsorbent should be chemically stable under the operating condi-
tions that will be used in the column. This includes stability
toward enzymes and microbes that might be present in the process
stream. This also means that the support must be stable in the
presence of the elution buffers, regenerating solvents, and
cleaning agents that will be used with the column. Agarose-based
supports are almost ideal in this respect, especially when they are
in their cross-linked form (e.g., Sepharose Fast Flow from Amersham
Biosciences). Such supports are not attacked by enzymes, can be
used between pH 3 and 12, and can withstand all commonly used
water-based eluants without shrinking or swelling [11]. An
additional attractive feature of cross-linked agarose is that it
easily withstands sanitation with 0.5 M sodium hydroxide. This last
feature is especially important in industry, where regular
sanitation is used to achieve reliable performance and good product
quality. A preferred way of doing this is cleaning in place (CIP)
with a strong sodium hydroxide solution. This strong alkaline
solution is an active bactericide and removes otherwise
irreversibly deposited material from the column, such as particles,
denatured proteins, lipids, and other compounds. If not removed,
such deposits can contaminate the desired product and lead to
column clogging. The fact that cross-linked agarose is stable at
high temperatures is also important, since this allows it to be
sterilized by autoclaving at 121C. Inorganic materials like porous
glass and silica are vulnerable to hydrolytic damage. These
supports should not be used above pH 8, and preferably not above pH
7, for any prolonged period of time, since these conditions can
lead to breakdown of their silica structure [11]. However, silica
materials are rarely used without the presence of a coating to make
them more inert toward solutes, as discussed in the previous
section. To a certain extent, these coatings also shield and
protect the silica from hydrolysis. Furthermore, certain brands of
silica supports are treated to incorporate zirconium or aluminum
into 2006 by Taylor & Francis Group, LLC
34. Support Materials for Affinity Chromatography 21 their
surfaces, which can considerably improve the stability of these
supports in an alkaline environment [16, 17]. Up to this point, it
may appear that afnity supports based on agarose will be satis-
factorily stable under all situations encountered. But this is not
the case, since often the weak point is not the matrix itself but
rather the ligand or the attachment between a ligand and the
support. For example, derivatives of adenosine monophosphate (AMP)
and nicotinamide adenine dinucleotide (NAD+ ) are both efcient
ligands for purifying NAD+ -dependent dehy- drogenases. But these
are also delicate molecules prone to breakdown, either
spontaneously or in the presence of hydrolytic enzymes. In this
case, less efcient but more stable ligands, such as the dye
Cibacron Blue, may be preferred, especially in large-scale
applications where the cost of frequently replacing the separation
material may otherwise be prohibitive. An example where the
anchoring between a ligand and matrix is the weak point on a
support can be observed in the cyanogen bromide method. Although
this method is convenient to use, it does lead to an isourea bridge
between the support and ligand, which will be slowly hydrolyzed at
an alkaline pH. This problem can be overcome by alternative
immobilization techniques that give a more stable product. Examples
include methods that couple through ether linkages using
bisepoxides [10, 11] or various other approaches, 2.2.3 Mechanical
Stability The mechanical stability of an afnity chromatographic
support should be sufcient to withstand the pressure drop across a
column when the column is run at an optimum speed and ow rate for a
separation. Most packing materials meet this requirement in
well-behaved systems. However, in preparative-scale work, the
sample or feed stream can contain many substances that may foul the
separation bed. Deposits of lipids, denatured proteins, and
particulate contaminants may restrict ow and quickly raise the
column backpressure to unacceptable levels. In the case of soft
gels like standard agarose beads, high backpressures will compress
the column bed, which will increase the pressure even further and
ultimately cause a collapse of this bed. Stronger supports such as
silica or heavily cross-linked polymers will not collapse at such
pressures, but even here very high backpressures are undesirable.
Analytical-scale chromatography is often performed using short- to
medium-sized columns with small- sized beads. In this situation,
supports like silica and polystyrene are preferred, since the
pressure drops in these columns can be high, often extending up to
several hundred bars [18, 19]. The higher pressures in these
applications are due, in part, to the desire for good mass-transfer
properties and fast analysis times, which are generally obtained
through the use of fast ow rates and small-diameter supports. 2.2.4
Pore Size It was stated earlier that the ideal afnity support
should allow unhindered access of a solute to the immobilized
ligand. For a macromolecular solute, this requires a support that
has large pores. But just how large must these pores be? An answer
to this question is given by the Renkin equation [20], which allows
one to estimate the effective diffusion coefcient (Deff) of a
solute in a porous material. (2.1) In this equation, Rs/Rp is the
ratio of the solutes radius (Rs) to the pore radius (Rp), p is the
particle porosity, is the tortuosity factor, KD is the distribution
coefcient for the solute, and D is the diffusion coefcient for the
solute in free solution. By inserting different D DK R R R R RD p s
p s peff = +1 2 10 2 09 0 953 . ( / ) . ( / ) . ( ss pR/ )5 / 2006
by Taylor & Francis Group, LLC as outlined in Chapter 3.
35. 22 Gustavsson and Larsson values for the ratio Rs/Rp, one
nds that the pore diameter should be at least ve times the diameter
of the solute to avoid severely restricted rates of diffusion. For
a protein of normal size (i.e., a diameter around 60 ), a ratio of
ve for Rp/Rs means that the support pores should be in the range of
300 . Several common supports are available with such pore sizes.
For example, supports based on 4 and 6% agarose have pore sizes of
about 700 and 300 , respectively. Silica particles are available
with pores in the range of 40 to 4000 . Polymethacrylate particles
can be obtained with pores that are 100, 200, 500, or 1000 .
Support materials with very large pores give essentially unhindered
diffusion for most solutes, but they also have a smaller surface
area per milliliter of bed volume than supports with smaller pores.
This reduced surface area leads to a diminished binding capacity.
As a rule, a pore size of 300 to 700 is usually a good compromise
in most situations encoun- tered in afnity chromatography, since
this gives fairly unrestricted diffusion for most biomolecules
while also providing a relatively large surface area for retention.
2.2.5 Particle Size Afnity supports are available in a wide variety
of particle diameters. These range from HPLC-type materials with
diameters of 10 m or less [18, 19] to large particles for
preparative work that have diameters of 400 m. But which particle
size is preferred for a given application? The answer will depend
on the purpose of the separation, the mechan- ical properties of
the support, and the characteristics of the sample. From a
theoretical viewpoint, it is always advantageous to have a small
particle size, since this will promote fast mass transfer of a
solute between the outer ow stream and interior of a support
particle. Figure 2.3 gives a simplied view of the various steps
that are involved in this process. In this model, sample molecules
are transported down through the column by the ow of the mobile
phase in the spaces between the support particles. To reach the
afnity ligands, these molecules must diffuse through the stagnant
mobile- phase layer surrounding the particles (i.e., the lm model)
and proceed to the inside pore network. It is here that the sample
molecules will nally bind to the afnity ligand. When the retained
molecules are eluted, the same steps occur but in a reversed order.
In this model, smaller support particles mean shorter diffusion
distances, since they have shorter pores and a thinner stagnant
mobile phase layer around and in the support. This, in turn,
results in shorter times being needed for diffusion [21]. According
to the Einstein equation [21], the diffusion time (td) that is
required for a molecule to travel a given mean Figure 2.3 Transport
processes that occur in a chromatographic column. FLOW 2006 by
Taylor & Francis Group, LLC
36. Support Materials for Affinity Chromatography 23 distance
(d) will be proportional to the square of the molecules diffusion
distance, or td = d2 /2Deff (2.2) where Deff is the effective
diffusion coefcient of the molecule in its surrounding medium. In
preparative afnity chromatography, relatively large support
particles are often used, making intraparticle diffusion the main
factor limiting efciency. In this case, dimin- ishing the particle
size will increase the rate of movement of solutes between the
support and surrounding ow stream, giving an improved column
performance. It is this effect that was the original driving force
behind the use of smaller supports in afnity columns, thus giving
rise to the technique of HPLAC [2, 5]. Under such conditions, a
decrease in particle size by a factor of ve can make it possible to
increase the ow rate by up to 25-fold and still retain good
chromatographic performance. This results in a dramatic improvement
in the productivity of the system. However, a point is eventually
reached when a decrease in particle diameter no longer gives a
proportional improvement in an afnity columns performance. This has
been observed in many analytical-scale systems that use HPLC-type
supports with particle sizes less than 10 m in diameter. Under
these conditions, diffusion in the particle is now relatively fast,
and it is the adsorption/desorption of sample molecules to and from
the afnity ligand that becomes the limiting factor in speed and
efciency [19]. Although better efciency is always obtained with
small support particles, using a small particle size is not without
difculties. One problem is the much higher ow resistance of these
smaller particles. According to the Kozeny-Carman relationship, the
pressure drop over a column (p) is inversely proportional to the
square of the supports particle size (dp), p/L = Cu/(dp)2 (2.3)
where L is the bed height, is the mobile phase viscosity, u is the
linear ow velocity, and C is a constant that depends on the bed
porosity. This equation indicates that decreasing a supports
particle size by a factor of ve will increase the pressure drop
across this support by a factor of 25. This increased ow resistance
may lead to bed collapse when using soft gels such as agarose. And,
although supports like silica can tolerate the higher pressures
that result, these will require the use of more expensive pumps to
work at such pressure, as is generally done in HPLC. Another route
that could be taken with small afnity supports is to use a short
and wide column instead of a long and narrow one. The advantages of
this are that the shorter, wider column can be run at higher ow
rates without creating high-pressure drops. Another drawback with
small particle sizes, especially in preparative work, is the
increased danger of fouling that exists when particulate
contaminants are in the feed stream or sample. This occurs because
the interstitial spaces in a bed of small particles can be too
narrow for such agents to pass through. Such fouling will increase
the ow resistance and may lead to bed collapse if the support
material does not have sufcient mechanical strength, as discussed
earlier in Section 2.2.3. As a result of these various
requirements, the particle size to pick when designing a new afnity
adsorbent will be a compromise between the desired chromatographic
performance, properties of the feed stream, and the mechanical
strength of the support. Some common selections made in specic
cases will be described in the next few sections. 2.2.5.1 Zonal
Elution Chromatography Zonal elution chromatography (also known as
weak or dynamic afnity chromatography) is mainly used in analytical
work and in basic studies of afnity phenomena. It is seldom 2006 by
Taylor & Francis Group, LLC
37. 24 Gustavsson and Larsson used for preparative purposes,
due to its relatively low capacity compared with other methods. The
sample molecules in this case are injected as a narrow plug. These
are then monitored as they travel through the bed at speeds that
are dependent on the strengths of their respective interactions
with the ligand. To obtain a good separation in this type of
system, it is important to have a column with a good efciency, as
is true in HPLC-based afnity methods. Ideally, this requires that
the support be based on particles that are as small as reasonably
possible. The use of small particles will reduce diffusion times,
as discussed earlier, which also promotes the formation of narrow
solute peaks. This can be performed by using HPLC-grade silica that
has been modied for use in afnity chromatography, as discussed in
Section 2.2.1.Another alternative is to use ultrasmall, nonporous
particles based on micron-sized silica or poly- styrene [18];
however, these materials are not yet commercially available in the
afnity mode. More information on zonal elution chromatography and
its applications in afnity 2.2.5.2 Adsorption-Desorption Afnity
Purication The most common way of performing afnity chromatography
for preparative-scale work is to use this as an
adsorption-desorption process. In this strategy, the target
molecule alone is bound tightly by the ligand, and the adsorbent
has a high binding capacity. After performing a suitable wash to
release weakly bound substances from the sample, the conditions on
the column are drastically changed (e.g., by using a substantial pH
shift) so that the target molecule loses its afnity for the ligand
and is eluted. Such a protocol works well with large support
particles, since there is no absolute need for high chromatographic
efciency. Still, small support particles can certainly help speed
up the adsorption step, make the washing step more efcient, and
permit the target molecule to be eluted in a smaller volume (i.e.,
as a narrower peak). This is especially valuable in analytical
applications, where sharper peaks allow for more convenient quan-
titation, better limits of detection, and faster analysis times. In
preparative-scale work, the greater ease of handling larger
particles is often preferred in practice, with particle sizes in
the range 30 to 100 m being common in such work. 2.2.5.3 Partial
Loading in Adsorption-Desorption Afnity Purication Partial loading
is a method for improving the performance of large particles in
preparative- scale afnity chromatography. The trick in this
approach is to utilize only the outer shell of the support particle
and thus diminish the distances required for solute diffusion.
Binding to a thin outer layer of the support takes only a fraction
of the time needed to saturate the entire particle. However, this
outer shell still represents a substantial fraction of the supports
total binding capacity. determined based on the following equation
[21], tshell/ttotal = 1 3(1 a/r)2 + 2(1 a/r)3 (2.4) where ttotal is
the time needed to saturate the whole particle, tshell is the time
needed to saturate a layer with thickness of a in the particle, and
r is the particle radius. According to Figure 2.4, an outer shell
with a depth equal to one fth the total support-particle radius
(a/r = 0.2) will contain half the particle volume (Vshell/Vtotal =
0.5). But this gure also shows that such a shell will take only one
tenth the time needed to ll the whole particle (tshell/ttotal =
0.1). 2006 by Taylor & Francis Group, LLC Figure 2.4
illustrates results for the partial loading technique. These
results were separations can be found in Chapter 4 and in Chapters
18 to 24.
38. Support Materials for Affinity Chromatography 25
Admittedly, special arrangements have to be made to make full use
of this principle. For instance, fast recirculation through the bed
is needed to ensure that all the support particles experience the
same loading concentration. Also, the washing and elution steps are
not substantially improved by this approach, since the released
substances (impurities or the desired compound) will still have
access to the entire particle. 2.2.5.4 Particulate Contaminants:
Large Beads/Expanded Beds In large-scale purication schemes,
particulate contaminants such as cells and cell debris are a
problem, since they tend to clog afnity columns. This problem
becomes worse as the size of the packing materials decreases, as
noted earlier. One solution to this problem is to use extra-large
particles. This strategy has been adopted in products like
Sepharose Big Beads from Amersham Biosciences and Cellthru Big
Beads from Sterogene, which have particle sizes of 100 to 300 m and
300 to 500 m, respectively. These very large particles should be
run in the partial-loading mode described in the previous section.
If this is not done, their performance will suffer from their long
diffusion distances. Another solution to particulate contaminants
is to use the expanded-bed principle. In this technique, the
distance between the adsorbent particles is increased to allow
contaminants to pass through freely. The adsorbents necessary for
this mode of operation will be described later in Section 2.3.
2.2.6 Standard Commercially Available Afnity Supports The previous
sections of this chapter described the properties that an afnity
support should have, such as a hydrophilic character, good chemical
and physical stability, and an appropriate pore size for the target
solute. Most of these criteria are presently met by Figure 2.4 The
shrinking-core model for protein adsorption. It is assumed in this
model that the protein binds strongly to the adsorbent (step
adsorption) and that the binding kinetics are fast compared with
diffusion (i.e., the normal situation in preparative afnity
chromatography operated according to the adsorption-desorption
principle). The radius of the entire support particle is given by
r, and the thickness of the outer absorbed layer or shell is given
by a. The relative volume of the shell is given by the ratio
Vshell/Vtotal, where Vtotal is the total volume of the support
particle and Vshell is the volume occupied by the outer layer
occupied by the adsorbed molecule. The relative time needed to ll
the exterior shell is tshell/ttotal, where tshell is the time need
to ll the outer layer and ttotal is the time needed to completely
saturate the support particle. 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8
0.2 0.4 0.6 0.8 1.0 1.0 1.0 a/r ttotal tshellVshell Vtotal r a 2006
by Taylor & Francis Group, LLC
39. 26 Gustavsson and Larsson commercial adsorbents based on
porous beads. Several examples of these supports are The adsorbents
shown in Table 2.1 are available from a number of manufacturers and
are supplied either in bulk or as prepacked columns. In addition,
some of these materials can be obtained in forms that already
contain one of several common afnity ligands, such as protein A,
Cibacron Blue, heparin, or metal chelating groups. If other ligands
are required, it may be necessary to rst nd a suitable activation
and coupling procedure for the ligand (see preactivated supports,
which greatly simplies the preparation of new afnity columns. 2.3
AFFINITY SUPPORTS WITH SPECIAL PROPERTIES Porous supports like
agarose, polymethacrylate, or silica beads are the main workhorses
in most current applications of afnity chromatography. However, in
the past several years described for use in afnity chromatography.
Many of these newer materials have prop- erties that give them
superior performance in certain applications. Materials that fall
in this category include nonporous supports, membranes, ow-through
beads, continuous beds, and expanded-bed particles. 2.3.1 Nonporous
Supports Nonporous beads with diameters of 1 to 3 m can be an
optimum choice for fast analytical or micropreparative separations,
since the limiting factor of pore diffusion is virtually eliminated
in these materials. Such beads may also be the best choice for
fundamental or quantitative studies of afnity interactions [18],
since the binding and dissociation behavior seen with these
materials should be more directly linked with the interactions
occurring between solutes and the afnity ligand. However, there is
a substantial loss of surface area and binding capacity that occurs
through the elimination of internal pores. For instance, 1.0-m
nonporous particles have a surface area of about 5 m2 per
milliliter of packed bed, but the corresponding value for porous
silica with 300- pores is about ten times higher. This difference
becomes even more accentuated when comparing larger beads. In
addition, as was discussed earlier, a smaller particle size leads
to larger column backpressures. Thus, micron-sized particles are
usually used in shallow beds to avoid high system pressures.
Difculties with back- pressure can also be minimized by using
monodisperse particles, which will create fewer problems than
polydisperse supports [23]. Nonporous bers are another category of
materials that have been used as supports for afnity
chromatography. These can have very high dynamic capacities [24].
Another advantage of these supports is that, in spite of the fact
that the bers are submicron in diameter, their backpressures are
low due to the low packing density of the overall ber bed. 2.3.2
Membranes Membranes have been used for afnity chromatography in
various formats, such as stacked sheets, in rolled geometries, or
as hollow bers [25, 26]. Materials that are commonly used for these
membranes are cellulose, polysulfone, and polyamide [25, 26].
Because of their lack of diffusion pores, the surface area in these
materials is as low as it is in nonporous beads. However, the at
geometry and shallow bed depth of membranes keep the pressure drop
across them to a minimum. This means that high ow rates can be
used, which makes 2006 by Taylor & Francis Group, LLC shown in
Table 2.1. For more information on the preparation of such support
materials, other types of supports have also become available
commercially (Table 2.2) or have been see the reviews in the
literature [4, 22]. Chapter 3 or the literature [10, 11]). Many of
the commercial suppliers in Table 2.1 also offer
40. SupportMaterialsforAffinityChromatography27 Table 2.2
Examples of Commercial Afnity Supports with Special Properties
Trade Name Format Material Examples of Available Ligands
Preactivated Form Available? Manufacturer/Supplier Sartobind
Stacked membranes Regenerated cellulose Protein A, IMAC Yes
Sartorius Poros Flow-through beads (20- or 50-m diameter)
Polystyrene/Divinylbenzene Protein A, protein G, heparin, IMACa Yes
Applied Biosystems CIM Monolithic disk or tubes Polymethacrylate
Protein A, protein G Yes BiaSeparations Streamline Composite beads
(120- or 200-m diameter) Agarose plus quartz or stainless s