Separation & Purification Reviews

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CHEMICAL, PHYSICAL, AND BIOCHEMICAL CONCEPTS

*
IN ISOLATION AND PURIFICATION OF PROTEINS
a a
Milton T. W. Hearn & Birger Anspach
a
Monash University, Clayton, Victoria, Australia
Published online: 15 Feb 2007.
To cite this article: Milton T. W. Hearn & Birger Anspach (2001): CHEMICAL, PHYSICAL, AND BIOCHEMICAL CONCEPTS IN
*
ISOLATION AND PURIFICATION OF PROTEINS , Separation & Purification Reviews, 30:2, 221-263
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SEPARATION AND PURIFICATION METHODS, 30(2), 221–263 (2001)
Downloaded by [University of Chicago Library] at 05:05 10 June 2013
CHEMICAL, PHYSICAL, AND

BIOCHEMICAL CONCEPTS IN ISOLATION
AND PURIFICATION OF PROTEINS*
Milton T. W. Hearn and Birger Anspach
Monash University, Clayton, Victoria, Australia
CONTENTS
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
2. Chemical Structure of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
3. Basis of Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
4. Chromatographic Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
5. Physicochemical Basis of Liquid Chromatography . . . . . . . . . . . . 235
6. Mass-Transfer Resistances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
7. Scaling-Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
1. INTRODUCTION
Major advances have occurred in recent years in the development of prepar-

ative and analytical techniques for the separation of peptides, proteins, and nucleic
acid derivatives. With the availability of many proteins through the advent of ge-
netic engineering techniques, the criteria for establishing biorecovery throughput
*
Reprinted from Separation Processes in Biotechnology; Asenjo, J.A., Ed.; Bioprocess
Technology, Vol. 9; Marcel Dekker, Inc.; New York, 1990; 17–64.
221
Copyright © 2001 by Marcel Dekker, Inc. www.dekker.com

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222 HEARN AND ANSPACH
in large-scale purification strategies for protein purity are currently undergoing

substantial reexamination. Furthermore, the potential now exists, due to the emer-
gence of rapid, high-resolution chromatographic and electrophoretic techniques,
to address systematically the quality control of peptides and proteins at analytical
levels hitherto not feasible.
Central to these advances are the well-recognized requirements to develop
new strategems for the purification of a specific peptide or protein from complex
mixtures. The attainment of very high purities for peptides or proteins can be
achieved only by the application of several high-resolution separation techniques.

To purify a biopolymer to near-homogeneity and satisfy the stringent limits of con-
taminant detection, sophisticated analytical and preparative separation techniques
must be integrated by the biochemist, protein chemist, and biochemical engineer.
For these reasons it is not surprising that extensive multidisciplinary and multi-
faceted research is currently under way to refine and extend existing chromato-
graphic and electrophoretic procedures to allow improved resolution and recovery.
Table 1. Laboratory-Scale Purification of New Biologicals
Purified Biomacromolecule References
Inhibin Forage et al. (1986), Robertson et al. (1985),

Leversha et al. (1987)
Heparin binding growth factors Bertolini and Hearn (1987), Hearn et al. (1987),
Ensch et al. (1985), Shing et al. (1984)
Insulin receptor Ullrich et al. (1985), Pilch and Czech (1980)
Epidermal growth factor receptor Downward et al. (1984)
Hemopoietic growth factors Whetton and Dexler (1986)
Epidermal growth factor Das (1982)
Platelet derived growth factor Waterfield et al. (1983), Chesterman
et al. (1983)
Angiotensin Shapiro et al. (1986)
-Transforming growth factor Roberts et al. (1983), Frolik et al. (1983)
Insulin-like growth factor-1 Rinderknecht and Humbel (1978)
Interleukin 2 Robb et al. (1983)
Nerve growth factor James and Bradshaw (1984)
Colony stimulating factor 1 Nicola et al. (1983)
Interleukin 3 Clark-Lewis and Schrader (1981)
Colony stimulating factor 2 Metcalf (1985)
-Transforming growth factor Derynck (1986)
POMC peptides Bennett (1986)
Gondatropin-releasing hormone Seeburg and Adelman (1984)
Cholecystokinin Reeve et al. (1986)
FSH releasing protein Vale et al. (1986), Robertson and Hearn (1987)
Neurophysins Kanmena and Chaiken (1985)
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ISOLATION AND PURIFICATION OF PROTEINS 223
Nearly all modern high-performance chromatographic methods lend them-

selves to the requirements of either analytical separation or “scale-up” preparative
separation. By combining the potentials of the various separation techniques, it is
now feasible to achieve purification factors of 100,000 to 500,000-fold for bioac-
tive substances present in only trace amounts in biological fluids.
Numerous examples could be cited where such separation achievements
have been reached. Table 1 provides a representative sampling of selected appli-
cations where very large purification factors have been achieved in the small lab-
oratory-scale purifications of important new biologicals. What is not evident from

this table are the years of effort that the purification of a few micrograms of a par-
ticular polypeptide or protein from a biological extract can entail, nor the exquisite
resolving power of separation procedures that were originally designed as analyt-
ical systems but found application as micro- or ultramicropreparative approaches.
We can also ask what lessons have emerged from these research investigations
that are relevant to the large-scale or process-scale purification task.
The conventional approach to small-scale purification of biosolutes is
predicated on the ability of chromatographic scientists to design and develop an-
alytical systems that allow very high resolution. Although extremely good selec-
tivity and bandwidth may be achieved by these methods in so doing, biological
activity of the solutes may be lost. With preparative experimental approaches, it
is essential to address the issue of bioactivity recovery specifically. By proper at-
tention to the physicochemical and biological consequences of biopolymer dy-
namic behavior in bulk solution and at liquid-solid interfaces, some of these re-
quirements can be addressed systematically. Problem solving associated with
loss of bioactivity or mass is still largely based on empirical, or in a limited num-
ber of cases [e.g., large-scale purification of human albumin (Curling, 1980)],
more specific experience on the behavior of a particular biomacromolecule in a
chemically defined solution or within the macro- and microenvironment of a sta-
tionary-phase surface.
Improvement in separation performance according to more quantitatively
predictive approaches flows from data accumulated largely from nonchromato-
graphic measurements such as physicochemical studies on structure and function
and from the evaluation of biological/immunological activities in response to
changes in separation variables in static or batch experiments. Experimental meth-
ods that allow the kinetics of biopolymer adsorption/desorption behavior or the ki-
netics associated with conformational, ion-binding, or hydration phenomena of
proteins have special importance in these analyses. However, such approaches are
based essentially on chemical equilibrium concepts, the major challenge being
proper understanding of factors leading to the stabilization of biopolymer structure
so that the solutes manifest preferred orientations in the distribution process dur-
ing separation, and high mass and activity recovery following desorption. Atten-
tion to system residency and dwell effects, the nature of the heterogeneity of the
distribution process and minimization of large changes in entropy due to the solute
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binding or permeability in the stationary-phase surfaces are all important parame-

ters in this regard and find ample manifestation in preparative high-performance
liquid chromatographic (HPLC) separations. With recovery of bioactivity the ma-
jor goal, current limitations in instrument design, system engineering, or process
development may lead to inadequate resolution, lower purification factors, and the
potential for trace contaminants to adulterate the final product or confuse the data
analysis.
Obviously, analytical options based solely on chromatographic optimiza-
tion approaches have a number of features in common with the more preparative
approaches to biopolymer purification which have biological criteria as their end-
points (i.e., maximizing specific bioactivity, minimizing degradation, etc.). With
the emergence of modern biotechnologies for protein production, the need for pre-
cise deterministic models, which fuse the solely chromatographic behavior of
biopolymers with their biophysical/structural behavior is more pressing than ever
before. Recently, empirical and mechanistic models for the retention of biopoly-
mers to microparticulate stationary phases have been the subject of increasing
scrutiny to address in part the propensity of biomacromolecules to undergo slow
conformational equilibria in solution or at liquid/solid interfaces (Melander et al.,
1984b; Stadalius et al., 1984; Geng and Regnier, 1984; Hearn and Grego, 1983a;
Armstrong and Boehm, 1984; Hearn et al., 1985). The utility of modern chro-
matographic methods coupled with new detection methods (e.g., on-line photodi-
ode array detection, optical rotary dispersion-circular dichroism, and derivative
spectroscopy) to assess these phenomena has important ramifications for the fu-
ture way that biochemists and engineers deal with various biorecovery problems
with regard to alternative strategies in protein purification. Thus to carry out a ma-
trix experiment investigating the influence on protein recovery and resolution of
two different salts at three different pH values with 10 different gradient options,
a little over 45 hours of experimental time was required (Hearn et al., 1988) using
modern high-performance ion-exchange support materials, while comparable ex-
periments with soft-gel ion exchangers required more than 800 hours of experi-
mental time.
2. CHEMICAL STRUCTURE OF PROTEINS
The chemical structure (i.e., the primary amino acid sequence) and surface
topography (i.e., the three-dimensional structure) of a peptide and protein are the
two key parameters around which most separation skills must be developed. Table
2 lists factors known to control chromatographic stability and resolution of pep-
tides and proteins.
Several options are available for the manipulation of protein solubility, in-
cluding techniques based on salt precipitation, organic solvent precipitation, or-
ganic polymer precipitation, isoelectric precipitation, or extraction/partitioning
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Table 2. Factors Controlling Chromatographic Stability of

Proteins
Mobile Phase Stationary Phase
Organic solvents Ligand composition

pH Ligand density
Metal ions Surface heterogeneity
Chaotropic reagents Surface area
Oxidizing or reducing reagents Pore diameter
Temperature Pore diameter distribution

Buffer composition Particle size
Ionic strength
Loading concentration
in aqueous or nonaqueous two-phase or multiphase liquid/liquid systems. All of

these options take advantage of solute hydration effects and a bulk hydration
property of the solute, such as its ability to form a finite or infinite intermolecu-
lar network or aggregate under a particular set of ionic strength or solvent di-
electric conditions. All of these processes clearly involve the participation of so-
lution chemical equilibria. Hence the potential exists for rational modulation of
separation selectivity or zone-broadening processes. However, in terms of defi-
nition of the molecular forces, the interaction of solvent molecules or ions with
biopolymers has, until recently, been explored largely only by methods that dis-
tinguish bulk differences of the induced physical characteristics of the solutes
and not microenvironment changes in the physical state of the solutes. For ex-
ample, the differential migration of biopolymers in gravitational or thermal
fields, which is the basis of all centrifugation procedures, thermal denaturation
methods, or thermal field flow fractionation, largely reflects the physical charac-
teristics of the solute in terms of its molecular weight, hydrodynamic volume,
state of self-aggregation, or association with other biopolymers. Differences in
the bulk parameters of global size and shape of the biopolymer, which reflect
molecular weight and sequence differences, also form the basis of ultrafiltration,
sodium dodecyl sulfate polyacrylamide gel electrophoresis, and gel filtration
chromatographic separations. Such separation procedures largely reflect a bulk
property of a biopolymer in terms of its average molecular weight, average
Stokes radius, and so on. Although such bulk properties are often taken to imply
a fixed physical property of a biopolymer, in fact all biomacromolecules undergo
dynamic changes in shape, self-association, and mobility in response to varia-
tions in the surrounding liquid environment. In most cases these environmental
changes affect the chemical potential of the biopolymer and involve reversible
solution equilibria processes.
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Where such phenomena are rapid (i.e., within a time scale of nanosec-
onds to milliseconds) they have minimal influence on purification selectivities
other than for molecular “breathing” and the binding or dissociations of small
ions. However, when these phenomena have long relaxation times (i.e., sec-
onds) the consequences can be dramatic, as are often associated with denatura-
tion, subunit dissociation, and related adsorption hysteresis. Knowledge of so-
lute-solvent interactions and their optimization thus represents one of the
essential requirements behind the development of general strategies for
biopolymer separation, based on either noninteractive or interactive chromato-

graphic media.
3. BASIS OF INTERACTION
The attraction between hydrophilic biopolymers and stationary phases,

while immersed in aqueous media, can be described as weak physical bonds,
while covalent bonds are usually not encountered. The main bonds involved are
(1) coulombic bonds, (2) metal ion bridges, (3) hydrogen bonds, and (4) Lif-
shitz/van der Waals bonds (van Oss et al., 1986a).
Hydrogen bonds are of polar origin and are usually stronger than other non-
covalent bonds. Although the dissociation energy for these bonds is high, they do
not seem to play a significant role in primary interactions between the solute and
adsorptive stationary phases, due to a very short operative distance, on the order
of 1.5 to 5 Å. Furthermore, hydrogen bonds in aqueous media are strongly atten-
uated by hydrogen-bond formation between hydrogen donor and hydrogen recep-
tor moieties and the ambient water molecules.
Coulombic or electrostatic interactions are, like hydrogen bonds, of po-
lar origin. The interactions occur between charged groups on the stationary
phase and the solute and are effective over distances on the order of 10 to 100
Å. Unlike other types of interactions, coulombic forces can be attractive in case
of oppositely charged groups at the surface, or repulsive in case of identically
charged groups of the interacting molecules. In various chromatographic sys-
tems, attraction between negatively charged biomacromolecules is also af-
fected through cross-linking (chelate development) of plurivalent cations such
as Ca2 or Cu2.
Between all atoms that are brought into very close proximity, electrody-
namic attractions arise between fluctuating dipoles occurring in one atom, and
other dipoles induced by it in a neighboring atom, in addition to permanent
dipole-dipole and permanent dipole/induced dipole attractions (van der Waals in-
teractions). The existence of attractive forces between neutral atoms gives rise to
analogous forces between any two macroscopic bodies whose surfaces are sepa-
rated by very small distances (Dzyolashinskii et al., 1955; van Oss et al., 1986b).
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In the liquid state, the three van der Waals bonds (London, Debye, and Keesom
dipole interactions) can be treated from completely different and purely macro-
scopic points of view, in which the interacting bodies are considered as continu-
ous media. These bonds are called long-range or LW (Lifshitz/van der Waals) in-
teractions. Since the energy of LW interactions decreases monotonically to the
distance (in the configuration of two parallel slabs) they are operative up to 1000
Å compared to hydrogen bonds which are active only up to 1.5 to 5 Å, due to an
exponential decay of the energy (van Oss et al., 1986a). As can be seen in Fig. 1,
primary bonds (respectively, orientation based on LW interaction) occur at a very

early stage in the adsorption process and are usually present at any time during
adsorption. The short operative distance of hydrogen bonds is the reason that
mainly coulombic and LW bonds develop in primary interactions between bioso-
lutes and the stationary phase. Secondary bonds can evolve after a certain time
lapse at a closer distance and involve mainly LW and hydrophobic interactions.
In some cases secondary bonds develop after structural changes of the interact-
ing biosolute, generating very strong hydrogen bonds. Under these circumstances
it can be essential to let as little time as possible elapse between the adsorption
and elution steps, to minimize strengthening of interaction by secondary bond
formation.
Figure 1. Schematic representation of the energy of interaction between a protein and

any surface in relation to the distance between the interacting species.
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4. CHROMATOGRAPHIC MODES
In recent years, a large amount of developmental effort has been expended

to transfer knowledge gained with chemically modified soft polymeric gels such
as the cross-linked dextrans, agaroses, or acrylate copolymers to the selection
and chemical modification of chemically more robust stationary phases with nar-
row particle and pore size distributions (Unger and Anspach, 1987; Unger et al.,
1987; Chen et al., 1988; Rounds and Regnier, 1988). Many of the criteria listed
in Table 3 apply equally to analytical separations as they do to large-scale prepar-

ative separations. Clearly, in the latter case the issues of column productivity, in
terms of kilograms of product resolved at a defined purity level per unit time per
unit cost of operating the overall separation system, is of fundamental impor-
tance. The potential of the purification approach to satisfy good manufacturing
practice (GMP) scale-up procedures, and thus meet governmental regulatory
agency guidelines, is also of major relevance in industrial application of protein
purification methods.
It has been appreciated for many years that the so-called “noninteractive”
modes of separation cannot exhibit the same level of resolution as that of adsorp-
tion techniques. The most successful separation techniques are those capable of
probing the topography of the desired biopolymer in the feedstock mixture. A
number of separation techniques are capable of resolving biopolymers on the ba-
sis of differences in net charge. These include zone electrophoresis, isota-
chophoresis, and most important at the preparative level, ion-exchange chro-
matography. Under conditions in which a transient or static pH condition can be
generated, such that the pH at a point within the separation system corresponds to
the pI of the protein of interest, further extensions of the net charge separation ap-
proach are found in chromatofocusing and isoelectrofocusing.
Table 3. Criteria for the Selection of Chromato-

graphic Media
Chemical and physical stability

Particle uniformity
Mechanical strength and resistance to deformation
Hydrophilicity and wettability
Sterilizability
Cost
Reproducibility between batches
High capacity
High resolution or selectivity
High mass and biological recoveries
High product throughput
Potential for GMP scale-up
ORDER REPRINTS
Ion-exchange chromatography separations take advantage of the net charge

and charge distribution of the surface of the biopolymer (Velayudhan and Horvàth,
1988; Drager and Regnier, 1987; Hearn and Hodder, 1988) (see Fig. 2a). Hy-
drophobic interaction chromatography in comparison exploits the accessibility and
surface distribution of lipophilic or nonpolar residues (Fig. 2b). The term “hy-
drophobic interaction chromatography” is frequently attributed to separations af-
fected by a decreasing salt concentration (Miller et al., 1985; El Rassi and Horvàth,
1986b), while the term “reversed-phase chromatography” has become identified
with separations involving an increasing concentration of organic solvent in the

eluent (Hancock and Harding, 1984). The physicochemical basis for both these
separation methods is largely a result of incremental changes in the microscopic
Figure 2. Schemata representing the more common modes of interaction: (a) ion ex-
change: interaction between oppositely charged ionic groups at the surface of the protein and
the stationary phase; (b) reversed-phase and hydrophobic interaction: Interaction of mainly
hydrophobic moieties of proteins and stationary phases; (c) metal chelate: exposed func-
tional groups of amino acids (e.g., imidazole from histidine) are involved in an immobilized
metal-chelate complex; (d) group-specific interaction such as phenylboronic acid affinity:
cis-diol groups of carbohydrate side chains of proteins from complexes with boronic acid;
(e) group-selective interaction such as dye affinity: Multiple interaction of ionic and hy-
drophobic moieties of proteins with the dye matrix (e.g., Cibacron Blue F3GA).
(continued)
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Figure 2. Continued.
surface tension associated with the solute/solvent/stationary phase interaction

(Melander and Horvàth, 1977; Melander et al., 1984a; Hearn and Grego, 1981;
Fausnaugh and Regnier, 1986; Sinanoglu, 1968). Further examples of separation
techniques that exploit the asymmetric distribution of amino residues at the surface
of folded proteins, for example, access to exposed histidine residues or a coordi-
nation site of a metal-ion cofactor, include ligand-exchange chromatography and
ORDER REPRINTS

Figure 2. Continued.
chelate affinity chromatography (Porath, 1988; Hochuli, 1988) (Fig. 2c). Similar
regioselective discrimination is also observed with hydroxyapatite chromatogra-
phy (Bernardi, 1979), with group-specific affinity chromatography such as the tri-
azine dye affinity (Kopperschläger et al., 1982; Lowe and Pearson, 1984; Scopes,
1986) (Fig. 2d) and borate affinity chromatography (Glad et al., 1980; Ackerman
et al., 1979; Pace and Pace, 1980) (Fig. 2e), as well as other forms of ligand inter-
actions based on generic biological ligands [i.e., biotin-avidin system (Henrickson
et al., 1979), protein A/IgG system (Phillips et al., 1985), oligosaccharide-lectin
systems (Lis and Sharon, 1981; Renault et al., 1985)]. The final group of separa-
tion parameters, and the ones that potentially give the highest selectivity, represents
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methods that exploit functional properties of a biopolymer such as a specific lig-

and binding site, antigenicity (Phillips et al., 1984), or a structural element such as
a lipid binding amphipathic (nonpolar) domain in lipoproteins or subunit contact
regions of multimeric protein complexes. With appropriate immobilization
chemistries and choice of the ligand, biospecific affinity chromatography and im-
munoaffinity chromatography both have the potential to generate separation peak
capacities more than two orders of magnitude greater than observed with adsorp-
tion methods based on simple chemical ligands such as those typically employed
for ion-exchange or reversed-phase chromatography. Table 4 summarizes exam-

ples of the separation parameters used in protein purification and the ranges of pu-
rification factors that can be expected in typical single-stage procedures.
Proper utilization of the specificity inherent in biological phenomena can
form the basis of very elegant immunoaffinity separations with, for example,
Table 4. Separation Parameters Used in Large-Scale Protein Purification and Typical

Purification Factor Ranges
Typical
Purification
Parameter Process Factor Range
Temperature Heat denaturation 2–20

Solubility Salt precipitation 2–20
Solvent precipitation
Polymer precipitation
Aqueous partitioning two-phase systems
Size and shape Gel filtration 2–20
Ultrafiltration 2–5
Gel electrophoresis 2–10
Net charge Free electrophoresis 2–5
Ion-exchange chromatography 2–40
Isoelectric point Chromatofocusing 2–10
Hydrophobicity Hydrophobic interaction chromatography 2–30
Reversed-phase chromatography 2–200
Function Biospecific affinity chromatography 50–1000
Antigenicity Immunosorption (e.g., monoclonal antibodies) 20–100
Carbohydrate content Lectin affinity chromatography 2–10
Content of free —SH “Covalent” chromatography 2–10
Exposed histidine Metal chelate affinity chromatography 2–10
Exposed metal ion Chelate affinity chromatography 2–10
Group specific Hydroxyapatite chromatography 2–10
Dipolar chromatography 2–40
Dye affinity chromatography 2–40
Charge transfer chromatography 2–20
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monoclonal antibodies or biospecific affinity separation with the appropriate bio-

logical ligand. The recent purification of the murine transferrin receptor is illus-
trative of the latter case (van Driel et al., 1984). This receptor specifically inter-
acts with the iron-binding protein transferrin and is an integral membrane
glycoprotein located on the surface of all proliferating cells. The key step in the
purification hinged on the changing affinity of transferrin, when depleted by iron
by a pH step, for its receptor. In this sense the immobilized transferrin affinity
chromatography method functioned as a retrometal chelate support. In the pres-
ence of Fe(III) the immobilized transferrin bound strongly to its corresponding re-
ceptor at pH 5.0. Simply by changing the pH of the eluent to pH 2.0, the Fe(III)
dissociated from the transferrin, resulting in a conformational change in ligand
protein and the concomitant dissociation of the receptor protein from the immo-
bilized biological ligand. The ability to exploit the known cellular biochemistry of
a protein at different stages of a purification procedure can readily be appreciated
as a crucial component behind this example and many other successful biospecific
affinity chromatographic purification attempts. Other recent examples where in-
formation gained from studies on the cellular biology has proved fundamental to
the purification of a very potent bioactive protein include the heparin binding
growth factors, the insulin receptor, and the epidermal growth factor receptor.
Because of the inherent requirements for high resolution in biopolymer pu-
rification, it is routine to utilize combinations of all of the separation parameters
listed in Table 4 at different stages and with different objectives during the isolation
strategy. To allow fully predictive and integrated strategies to evolve, greatly ex-
panded databases on, for example, protein behavior in various physical or chemical
environments are required, related to mass-transport and other mechanistic issues.
However, full knowledge of the mechanistic processes underlying biopolymer
separation selectivity and the kinetics of transport for a defined solute under the sep-
aration conditions is an ideal scenario rarely attainable in practice. Much of the re-
search effort associated with the development of new chromatographic separation
media, the introduction of improved preparative electrophoretic methods, and the
application of additional principles such as magnetized bed extraction or field flow
fractionation has nevertheless addressed the same questions central to the physico-
chemical nature of biopolymer separation selectivity and biopolymer kinetics. Par-
ticularly with adsorptive chromatographic systems, the molecular dynamics associ-
ated with multisite interaction of biopolymers with the stationary phase controls not
only retention and zone broadening behavior but also mass and bioactivity recovery.
For several practical reasons (e.g., cost or difficulties with column regenera-
bility), high-resolution purification methods are usually not brought into play with
sub-10-m microparticulate adsorption media of narrow particle-diameter distribu-
tions and narrow pore-size distributions until clarification is complete and partial
fractionation has been carried out. Considerable research and development activity is
now under way at both academic and industrial centers, exploring different options
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for improving stationary-phase characteristics to allow enhanced separation selectiv-

ity and improved kinetics with mesoparticulate media (dp 30 m) in preparative
separations of biopolymers. Because interactive stationary phases have the potential
to probe the topography of a biopolymer, in particular surface-accessible regions or
binding sites unique to the protein of interest, purification stratagems based on a ra-
tional mix of ion-exchange, affinity chromatography, and hydrophobic interaction
chromatography represent the core methods for high-resolution separation.
Exploitation of the interplay between hydrophobic and coulombic interac-
tive phenomena forms the basis of so-called “mixed-mode” chromatographic pro-

cedures. For example, under appropriately chosen eluent conditions, proteins can
be efficiently separated on stationary phases with immobilized coulombic ligands
with hydrophobic selectivity (Hearn and Hodder, 1988; Hearn, in press;
McLaughlin and Bichoff, 1987). Under these conditions, proteins are eluted in or-
der of increasing hydrophobicity under conditions of decreasing displacing salt
concentration from high ionic strength, typically 3 to 5 down to 0.5. Sim-
ilarly, under appropriately chosen solvent conditions, hydrophobic supports such
as n-alkylsilicas can be induced to exhibit polar-phase selectivity with peptides
and proteins eluting in order of increasing polarity (Hearn, 1982; Hearn and
Grego, 1983b). As a consequence, retrogradients based on eluents of high solvent
content to lower solvent content (i.e., from 0.9 down to 0.5) can be used
(Hearn and Aguilar, 1986) to separate hydrophobic peptides and proteins on re-
versed-phase packing materials with polar-phase selectivity.
The composite interplay between size-exclusion phenomena and solvopho-
bic and coulombic interaction processes is a feature of all current chromatographic
stationary phases. Depending on the magnitudes of these retention dependencies,
the retention behavior of a biosolute in interactive systems can be formalized in
terms of the summations of the overall retention process. A common manner in
which this formalism is expressed is given by
ln k ln(
seck sec
esk es
vdwk vdw
solv k solv )
where
sec,
es,
vdw, and
solv correspond to the mole fraction of the solute un-
dergoing hydrodynamic permeation, electrostatic, van der Waals, or solvation in-
teractions, with corresponding capacity factors of k sec, k es, k vdw, and k solv.
In a recent study (Johnston et al., 1987a) on the simultaneous separation of
the isohormones of lutropin, follicotropin, and thyrotropin, size-exclusion chro-
matography, preparative isoelectric focusing and anion-exchange HPLC were
used sequentially. Two observations of general applicability to protein fractiona-
tion arose from this study. First, by employing ion-exchange HPLC after prepar-
ative isoelectric focusing, it was feasible to resolve components with the same pI
value. Second, by employing multidimensional approaches on the same stationary
phase via eluent mixture design optimization, considerable improvement in re-
covery could be achieved.
ORDER REPRINTS
Which of the above chromatographic terms make the greatest overall con-
tribution to retention of the biopolymers depends not only on the permeability, lig-
and composition, and ligand density of the stationary phase, but also on mobile-
phase characteristics in terms of water content, pH, ionic strength, organic solvent
content, buffer composition, and whether such additives as ion pairing reagents,
organic dissociating reagents, or surfactants-detergents are present in the eluent.
5. PHYSICOCHEMICAL BASIS OF LIQUID

CHROMATOGRAPHY
The physicochemical basis of all modes in liquid chromatography can be

described by solvophobic theory (Melander and Horvàth, 1977; Melander et al.,
1984a; Horvàth et al., 1976), where the isocratic retention factor k can be ex-
pressed as
1 RT
ln k G° ln
RT PV
where G° are the overall difference between the mobile and stationary phases in
free energy and represents a functional relationship to the system phase ratio.
G° is represented by
G° G°cav G°es G°vdw G°assoc G°red
where G°cav, G°es, and G°vdw are the differences between the mobile and sta-
tionary phases in free energy, associated with cavity formation, electrostatic ef-
fects, and van der Waals interactions, respectively. G°assoc is the free-energy
change for ligate-eluite association in the absence of surrounding solvent, and
G°red expresses the reduction of the free energy due to solvent-ligand and solvent-
solute interactions not treated in the first three terms and which is actually a cor-
rection of the expression above for nonideal behavior in terms of this theory. V
and P are the mean molar volume of solvent and the operating pressure, respec-
tively. The constant is determined by the properties of the column and is related
to the concentration of accessible ligand in the column.
The magnitude of G°es varies with the salt concentration, m. The depen-
dence of the protein activity coefficient in the mobile phase can be expressed by
the extended Debye-Hückel equation as
Bmp(m0.5)
G°es Amp Dmp m
1 C(m0.5)
where is the dipole moment of the protein, and the coefficients Amp and Bmp are
both proportional to the net charge on the protein. The coefficients C and Dmp are
constants peculiar to each eluite because their magnitudes are functions of the di-
ORDER REPRINTS
mensions of the macromolecule. The quantities pertinent to the mobile phase are
represented by mp. The coefficient Amp is inversely dependent on protein size as
well. Since the relationship between salt concentration in the mobile phase and ac-
tivity coefficient of protein bound to the stationary phase is unknown, it is as-
sumed to be similar in form to that given by the equation above. The electrostatic
free-energy change associated with the chromatographic retention process can
then be expressed as
G°es G°es,sp G°es,mp
where sp refers to the stationary phase. Under ideal conditions (i.e., the presence
of electrostatic interactions only), the equations above represent the theoretical ba-
sis of protein surface interaction in the ion-exchange chromatographic mode. If
multiple interactions can take place at the stationary surface, appropriate solvent
conditions must be chosen to suppress nonideal behavior.
The energy of cavity formation in the mobile phase is related to the surface
tension and surface area of the molecule, As, as
G°cav [NAs 4.8N1/3 (e 1)V2/3]
where e is a constant that corrects for the curvature of the cavity and N is Avo-
gadro’s number. The surface tension of aqueous salt solutions is a function of the
molal salt concentration, m, and is given by
° m
where ° is the surface tension of neat water and is a constant characteristic of
each salt, which is called the molal surface tension increment. Assuming that the
magnitude of the salt concentration has no effect on e, As, or V, the free-energy
change associated with cavity formation in the mobile phase, G°cav,mp, is found
to be
G°cav,mp [NAs 4.8N1/3(e 1)V2/3]°
[NAs 4.8N1/3(e 1)V2/3]m
which can be expressed in a simplified form as
G°cav As m constant
where As is the difference in surface area of ligate and protein exposed to mo-
bile phase between the bound and unbound states (i.e., equivalent to the molecu-
lar contact area upon binding).
G°vdw is assumed to be unaffected by exogenous salts, and therefore the net
free-energy change due to van der Waals interactions is expected to be nearly lin-
ear in salt concentration:
G°vdw G°vdw,sp G°vdw,mp
ORDER REPRINTS
Combining the equations for the different changes in free energy for
changes in the salt concentration leads to the following expression:
k Bm1/2
ln Dm Asm m constant
k0 1 Cm1/2
where k0 is the retention factor at zero salt concentration.
At sufficiently high ionic strength the leading term on the right-hand side
approaches a constant value and then the logarithmic retention factor becomes lin-
ear in salt molality:

k
ln m
k0
where is a parameter that measures the retentive strength of the salt and is sim-
ilar to the salting-out constant. When ionic interactions can be excluded in the pu-
rification of proteins, cavity formation and Lifschitz/van der Waals interaction are
the basis of hydrophobic interaction and reversed-phase chromatography.
Differences in the free energy between mobile and stationary phases asso-
ciated by cavity formation (G°cav), electrostatic charge (G°es), and van der Waals
interaction (G°vdw) correspond to solute specific parameters and are related to the
slope of the plots of the total free-energy change (G°) for a particular biopoly-
mer versus the reciprocal logarithmic concentration of organic solvent modifier in
the case of reversed-phase separations, or versus reciprocal logarithmic concen-
tration of displacing ion in the case of hydrophobic interaction and coulombic sep-
arations.
Figure 3 illustrates the manner in which the relative retention of two pro-
teins can change as a function of the concentration of the displacing reagent in the
hydrophobic interaction chromatographic mode. When analyzed in terms of the
framework of the solvophobic theory above, increased retention of proteins in hy-
drophobic chromatography is associated with an increase in salt molality in the
mobile phase or change of the salt to another with a greater molal surface tension
increment. Inherent in the solvophobic theory are the assumptions that the surface
area As, the cavity curvature e, the net charge coefficients Amp and Bmp, and the
topographic coefficients Dmp and C are not time dependent and show monotonous
changes with changing eluent pH or eluent dielectric properties. However, transi-
tions in conformation or ion bridges that may occur as the mobile-phase compo-
sition is manipulated clearly are manifested as time-domain dependencies. The
consequences of this behavior are condition-dependent changes in As, e, Amp,
Bmp, and C which are translated experimentally into discontinuitues or changes in
the log k versus log 1/[displacer] plots or changes in band-broadening relation-
ships again as a function of log 1/[displacer].
In addition to these mobile-phase effects, the stationary phase itself affects
the magnitude of the retention in a major way (i.e., through the Nernst equation
ORDER REPRINTS

Figure 3. Plots of logarithmic retention volume of eight polypeptides and proteins

against (NH4)2SO4 concentration in the starting eluent with gradient elution. The samples
are insulin ( ), insulin B-chain (), trypsin inhibitor (), trypsinogen (), insulin A-chain
(), ribonuclease (), myoglobin (), and cytochrome c (). [From Melander et al.
(1984a).]
k Cs/Cm). Besides various effects arising from the nature of the sorbent, both
the chemistry of the stationary phase and the density of applied functional groups
affect retention. In view of this dual interdependency, there are many ways to
modulate retention behavior of a given mixture of biosolutes if one of the phase
conditions is unsuited to preservation of biorecovery or recovery.
Depending on the magnitude of the different free-energy changes, a variety
of solute retention versus mobile-phase elutropic strength scenarios can be calcu-
lated. Figure 4 represents four limiting cases of such retention dependencies. Case
b is typified by shallow dependencies of free energy versus elution strength with
low free energies at (or [c] 0) and represents a commonly observed situation
with small polar peptides separated under reversed-phase or ion-exchange HPLC
conditions (O’Hare and Nice, 1979; Molnàr and Horvàth, 1977; Aguilar et al.,
ORDER REPRINTS

Figure 4. Schematic representation of the retention dependencies for peptides or proteins

chromatographed on mixed-mode support media. The figure illustrates four case histories
for the dependency of the free energy G° on the mole fraction of the displacing species.
As the contact area associated with the solute-ligand interaction increases, the slopes of the
G° versus plots increase, resulting in a narrowing of the elution window over which the
solute will desorb. Cases (a) and (b) are typically observed for the RP-HPLC of polar pep-
tides and small, polar globular proteins while cases (c) and (d) are more representative of
the RP-HPLC behavior of highly hydrophobic polypeptides and nonpolar globular pro-
teins, respectively.
ORDER REPRINTS
1985). Case c, which again exhibits shallow dependencies in terms of the free en-
ergy versus eluant strength dependency but with large values of G°0 values, is
more representative of situations found with middle molecular weight but very
high hydrophobic peptides under some reversed-phase conditions; in affinity dis-
placement ion-exchange or substrate analog displacement elution in affinity chro-
matography where the substrate/analog or displacing species is again typically of
low molecular weight (Stout et al., 1986; Kopaciewicz et al., 1983; Gooding and
Schmuck, 1984). Some examples of peptide displacement chromatography also
correspond to this case. Case a represents a typical scenario for polypeptide and
globular protein purification in reversed-phase and hydrophobic interaction tech-
niques and with most polymer- and silica-based anion and cation HPLC support
media (Benedek et al., 1984; Kennedy et al., 1986; Janzen et al., 1987; Lau et al.,
1984; Glajch et al., 1986; Hearn and Hodder, submitted; Mant and Hodges, 1985).
From practical considerations the limiting chromatographic conditions are
frequently chosen such that the minima of the plot of the free energy versus elu-
ent strength corresponds to k values equal or less than unity. Typically, this cri-
terion is easier to achieve in ion-exchange than in reversed-phase separations. In
situations associated with the purification of large globular proteins or hydropho-
bic proteins, such retention behavior is not usually observed with reversed-phase
or ion-exchange HPLC. In these cases retention dependencies approaching case d
are experienced. From the point of view of a generalized purification strategy, it
is obviously desirable to select chromatographic conditions in which the retention
dependencies approximate case 1 or case 3 rather than cases 2 and 4, where clearly
the affinities of the solute for the stationary phase are too high, the elution window
for desorption too narrow, the solute solubility parameters of the protein too low,
and the mass (or bioactivity) recovery potentially impaired. However, from a se-
lectivity point of view such situations should not necessarily be excluded. Ex-
ploitation of the potential offered by the case 2 and case 4 scenarios has proved
very useful for the removal of undesirable contaminants during the purification of
a number of therapeutic proteins, for example, the removal of trace components
of Hageman factor and associated plasminogen activator/prekallikrein proteins
from therapeutic-grade human immunoglobulins based on a tandem dye affin-
ity/anion-exchange chromatographic method (Hearn et al., 1986). Examples of
the plots of experimental G° against organic solvent strength for the several hor-
monal polypeptides are shown in Fig. 5.
Because of the pronounced dependencies of retention and zone broadening
phenomena on chromatographic conditions, behavior that reflects the magnitude
of the distribution coefficient and the complexity of the retention kinetics estab-
lished between the biopolymer and the stationary phase, the most commonly
adopted method for elution of biopolymers from adsorptive media involves gra-
dient or step elution procedures. Such conditions take advantage of the depen-
dency of free energy G° and eluent strength but do not necessarily address the
ORDER REPRINTS

Figure 5. Plots of the logarithmic capacity factors for hen lysozyme and several hor-
monal polypeptides against the volume fraction of the organic solvent in water-acetoni-
trile isocratic mobile phases. Conditions: Column, -Bondapak C18; flow rate, 2.0 mL/min;
primary mobile phases: (a) water/4 mM sodium sulfate/15 mM orthophosphoric acid, pH
2.2, and (b) water/4 mM sulfuric acid/15 mM orthophosphoric acid/15 mM triethylamine
with the acetonitrile content adjusted over the range 0.0–0.8. Polypeptide key: 1, hen
lysozyme; 2, porcine glucagon; 3, bovine insulin; 4, bovine insulin -chain; 5, arginine va-
sopressin; 6, lysine vasopressin. [From Hearn and Grego (1983a).]
ORDER REPRINTS
important requirements of desorption kinetics and conformational dynamics of the

solute.
Although optimization of chromatographic resolution with low-molecular-
weight solutes is now a mature area of the separation sciences (Schoenmakers,
1986; Kirkland and Glajch, 1983), similar endeavors with biomacromolecules are
still relatively in their infancy. However, important progress has recently been
made in the application of gradient elution theory which allows gradient retention
data with a particular solute to be interrelated to isocratic elution following the se-
lection of suitable systems, and vice versa (Stadalius et al., 1985; Quarry et al.,
1984; Hearn and Aguilar, 1987). Furthermore, it is feasible in circumstances of
regular retention and recovery behavior with, for example, peptides and small
globular proteins to apply data derived from small-scale or analytical experiments
as normalized integrals of the elution volume, column performance, and so on, to
the scale-up of the chromatographic bed configuration and the choice of the phys-
ical characteristics of the separation media (Eble et al., 1987; Poppe and Kraak,
1983; de Vos et al., 1987; Frenz and Horvàth, 1985).
With low-molecular-weight solutes, the conventional approach to purifica-
tion has been based on scale-up extensions of analytical column systems which al-
low very high resolution through optimization of chromatographic selectivity and
zone bandwidth. When similar methods are applied to proteins, it is often the case
that their biological activity may be lost. Inherent in all biopolymer purification
stratagems is the question to what end use the purified biopolymer will be re-
quired. If the task involves purification solely for the purpose of subsequent pri-
mary structure determination (i.e., essentially analytical), the requirements of ad-
equate control over bioactivity are not necessarily relevant. Obviously, in the case
of a new or partially characterized protein the recovery of the component with
high mass and bioactivity balance is essential. Similarly, in preparative ap-
proaches where subsequent biological uses are contemplated, it is mandatory that
the design of the separation system specifically addresses the issue of recovery of
bioactivity. By proper attention to the physicochemical and biological conse-
quences of the dynamic behavior of the solute in bulk solution and at liquid-solid
interfaces, the criterion of high recovery of bioactivity can usually be satisfied.
Where conformational requirements impinge on a purification strategy, other
data, gained from nonchromatographic measurements (Katzenstein et al., 1986;
Grego et al., 1986), and from evaluation of biological/immunological activity pro-
files (Johnston et al., 1987b; Janzen et al., 1987a) in response to changes in sepa-
ration variables in batch experiments, are essential prequisites. The major chal-
lenge here is to obtain sufficient information to allow proper understanding of the
factors that control the stability of the biopolymer structure during the chromato-
graphic distribution process so that high mass and high bioactivity recovery can be
achieved on elution. System residency effects, the nature of the binding hetero-
geneity associated with the overall distribution process, and the participation of en-
ORDER REPRINTS
tropic effects associated with solute binding or permeation through the stationary-
phase internal surfaces are all important parameters in preparative HPLC separa-
tions if proteins are to be recovered in bioactive form. Clearly, if these parameters
are to be included properly in the chromatographic optimization process, quantita-
tive structure-retention relationships must be developed. Such mechanistic ap-
proaches based on stochastic prediction models require an extensive data base be-
fore adequate response function and factor design analyses can be carried out.
Ultimate selection of the most optimal chromatographic strategy will hinge
very strongly on the ability of response function approaches to iterate structure-

function data into the overall chromatographic optimization scheme. From a chro-
matographic point of view the assessment of the quality of the separation in response
to a change in a chromatographic variable, such as mobile-phase composition, or
alternatively, a solute variable such as net charge or charge distribution, can be based
on evaluation of the system peak capacity and system productivity in terms of bioac-
tive mass throughput per unit time. Since the peak capacity (PC) depends on both
the relative selectivity and bandwidth, that is, for a chromatographic system with an
average resolution of Rs 1, peak capacity can be defined as PC (t g t0)/4t,
where t g is the solute elution time, t 0 the column dead time, and t the average stan-
dard deviation of the peak, optimization of peak capacity must of necessity take into
account knowledge of kinetic behavior associated with conformational or secondary
chemical equilibria mediated by the stationary-phase surface or, alternatively, com-
ponents in the mobile phase.
Biopolymer interconversion associated with unfolding and refolding path-
ways represents a unique set of resolution challenges, from both theoretical and
experimental aspects, not experienced with low-molecular-weight, conformation-
ally rigid solutes. Although at present the empirical “recipe,” for reasons of prac-
tical expediency, dominates most purification studies with peptides and proteins
using HPLC or electrophoretic systems, the trend is already evident for more sys-
tematic approaches based on computer-aided analysis of retention and kinetic data
in terms of different mechanistic models for biopolymer retention in high-resolu-
tion adsorption chromatography. Preliminary approaches to the classification of
retention and kinetic data in terms of different mechanistic pathways have already
been described for reversed-phase and ion-exchange HPLC of a number of en-
zymes and globular proteins (Hearn, 1986b; Wetlaufer and Koenigbauer, 1986).
The ability of modern HPLC techniques to yield quantitative data on rate con-
stants for protein folding and unfolding transitions as well as to resolve conform-
ers with relaxation half-times t 10 s has important ramifications in the selection
of a purification procedure. For example, if a protein were to undergo a two-stage
interconversion in both the mobile phase and at the stationary-phase surface, the
retention cycle representing a process of distribution of the protein in its native
form, Pn, and two unfolded forms, P*u and Pu, between the two chromatographic
phases can be written as follows:
ORDER REPRINTS

Based on such a distribution cycle, a number of different retention mech-

anistic pathways can be envisaged. Table 5 identifies some of these intercon-
version possibilities. Whether the retention mechanism of a particular biosolute
can be described in terms of this or other retention models will depend on the
respective rate constants for the various distribution and interconversion path-
ways. If the rate-determining step for the pathway is associated with the confor-
mational unfolding P*u,m D Pu,m, the distribution can be approximated by a four-
component cycle. Thus if the fraction of time the protein, P, spends in one form
is represented by t, the probability that this fraction is in the range t dt is given
by Pi(t), where i 1, 2, 3, . . ., representing each of the forms. If the intercon-
version cycle is represented by only four forms, the probability that P starts as
Pn,m can be given by
r1r2(1 t)
P11,(t) dt exp[ r1(1 t) r2t)I[4r1r2t(1 t)]1/2t
t
where
(k12k23 k14k21)t*
r1
k21
and
(k32k43 k34k41)t*
r2
k34
where t* is the separation time and
exp(2[r1r2t(1 t)]1/2)
I
4 1/2[r1r2t(1 t)]3/4
If Gaussian distributions for the concentration profiles of Pn,m and P*u,m cen-
tered around t 0 and t 1 are assumed, by allowing a single diffusion coeffi-
cient to describe the combined effects of the chromatographic process for each
conformational form, both P(t 0) and P(t 1) can be made discrete functions
ORDER REPRINTS
Table 5. Classification of Several Different Retention Pathways in Biopolymer

Adsorption Chromatography
This classification is based on the Pn → P*u → Pu interconversion. More complex branched

interconversions can be described similarly using the notation (isobi)br . The classification
of the pathways when the solute in its various conformational forms binds to the same class
of ligand can be noted as an “ordered pathway.” When binding of the solute to a heteroge-
neous stationary phase surface occurs, the notation “random pathway” is used. In this man-
ner, an N-dimensional retention network can be identified for the various conforma-
tional/secondary equilibrium processes that proteins and other bipolymers undergo at

adsorptive interfaces.
1. Uni-uni pathway: typified by a simple distribution process and kinetics, and a narrow
bandwidth with high mass and biological recovery; may be characterized by very rapid
interconversion kinetics as the solute traverses the chromatographic bed as a single, av-
eraged structure
2. Isouni-uni pathway: typified by solvent-induced solute unfolding-refolding phenomena
in the mobile phase with both solute species binding to the stationary phase with the
same distribution coefficient; single elution zone with high mass recovery possible but
time-dependent loss of biological activity in the mobile phase evident
3. Uni-isouni pathway: typified by ligand-induced solute unfolding-refolding phenomena
at the stationary-phase surface; single elution zone but time-dependent loss of mass and
bioactivity possible when k23 k32
4. Isouni-isouni pathway: typified by solvent- and ligand-induced solute unfolding-refold-
ing phenomena in both phases; may be characterized by impaired mass recovery of two
species (i.e., native and nonnative) with time-dependent loss of bioactivity when k14, k23
k41, k32
5. Isobi-uni pathway: typified by solvent-induced, time-dependent biphasic unfolding-re-
folding of solute in the mobile phase but stabilization of structure by the ligand surface
if k12 k14; characterized by high mass recovery and elution of a single zone with ap-
parent half-life for loss of bioactivity larger than in the mobile phase alone
6. Uni-isobi pathway: typified by ligand-induced, time-dependent biphasic unfolding-re-
folding of the solute at the stationary-phase surface with further destabilization of struc-
ture; characterized by time-dependent loss of mass and activity
7. Isobi-uni pathway: typified by mobile- and ligand-induced biphasic unfolding-refolding
of the solute; characterized by the time-dependent loss of mass and bioactivity with the
emergence of a second, often later-eluting, inactive zone of nonactive solute if k12, k43
k21, k34
and the probability distributions of the interconverting species, separated by re-

tention time differences of tR,1 and tR,2, can readily be computed. The calcu-
lated concentration profile for bovine trypsin undergoing a two-stage dynamic in-
terconversion during reversed-phase HPLC is shown in Fig. 6. Equivalent
expressions can also be derived for cooperative and noncooperative transitions in-
volving multistep conformational perturbations with several intermediate states,
ORDER REPRINTS

Figure 6. Calculated concentration profile for bovine trypsin undergoing dynamic inter-
conversion while chromatographed on a reversed-phase column. It is assumed that the in-
terconversion processes occur by a two-state reversible transition and reverse conversion
rates r1/t* 1.25 10 3 s 1, that t* 0, and that an equilibrium mixture of two inter-
converting components of equivalent mole fractions is initially loaded onto the column. It
is further assumed that the migrating zone for each component generates a Gaussian distri-
bution profile with 0.1 and that the effective diffusion coefficients of both forms are
the same. [From Hearn et al. (1985).]
or non-Gaussian concentration profiles due to heterogeneous interactions of the

stationary-phase surface. A major criticism of the stochastic probability approach
is that relatively slow secondary reactions, for which the “near-equilibrium” as-
sumption does not apply, cannot be accommodated. In this situation it is necessary
to derive and solve simultaneous partial differential equations for mass conserva-
tion and obtain expressions for the first and second moments of the elution profile
and the concomitant plate height arising from slow kinetics of secondary equilib-
rium.
If the process Pn → P*u in the mobile phase and stationary phase can be rep-
resented as the reversible binding of two forms with first-order or pseudo-first-or-
der interconversion kinetics, the resolution of the interconverting species can be
given by
t0(k12/k21 k43k34)(1 e Da)
Rs
4Da(1 2)
ORDER REPRINTS
where
L(k12k23 k14k21)(k14 k41)
Da
(k14k21)u0
where is the phase ratio (Vs /Vm); ki,j are the respective rate constants for ad-
sorption, desorption, unfolding, or refolding; 1 is the peak width of component i;
L is the column length; and u0 is the linear velocity. The plate height increment
due to slow kinetics of interconversion, Hse, can be determined by
e Da 1

2A
Hse 1
Da Da
where
L(k0 k1)2/Km
A 2
[1 k1 (1 k0)/Km]
and k0 and k1 are the capacity factors for the two interconverting forms. The
Damkohler number (Da) represents the ratio of the time taken by the protein to
pass along the column in the mobile phase to the overall relaxation time for all
conformation interconversions in the chromatographic system. The influence of
the Damkohler number on the chromatographic profile is shown in Fig. 7. When
this ratio is small, and in the limit approaches zero, the chromatogram for a four-
component cycle will reflect the average macroscopic behavior of a fully unfolded
form, or alternatively, two peaks separated by a time interval tm(k12/k21
k43/k34). Conversely, when Da is very large, kinetic effects associated with con-
Figure 7. Computer simulations of the influence of the magnitude of the Damkohler

number on the elution profile of a peptide undergoing a four-cycle interconversion. Such
behavior has been observed in this laboratory for the dipeptides L-leucyl-L-proline, L-
valyl-L-proline, and L-alanyl-L-proline.
ORDER REPRINTS
formational interconversion essentially vanish. However, at intermediate reaction

rates where 1 Da 10, complex elution profiles that are dependent on the equi-
librium distribution of the two species, the rate of interconversion, the chromato-
graphic conditions, and the intrinsic column efficiency will be obtained. The in-
fluence of the Damkohler number on the dependence of the bandwidth is
demonstrated in Fig. 8. Relatively slow kinetics of interconversion, which is char-
acterized by a small Damkohler number, causes the overall bandwidth to go
through a maximum at intermediate retention times, and significantly high Hse can
be obtained at relatively small k values.

The impact of these kinetic processes escalates rapidly in multistep purifi-
cation procedures, where the disastrous effect of low repetitive yields can result in
unacceptable purification productivities (see Fig. 9). For example, if the average
yield per step in a 10-step purification method were 60% (a relatively favorable
situation), the overall yield would be only 0.6%. Should the average yield per step
drop to 30% due to partial denaturation, the overall recovery would reach the dis-
astrous value of 0.0006%; that is, 1,000 times as much raw material in the feed-
stock would have to be processed to yield the same mass of purified protein.
As is evident from Table 2, a large variety of mobile phase and stationary
factors can influence the chromatographic stability and recovery of proteins. The
effect of most of the mobile-phase characteristics, such as the nature and concen-
tration of organic solvent or ionic additives, the temperature, or the pH, can be as-
certained very readily from batch “test tube” pilot experiments. However, more
subtle mobile-phase effects, such as the influence of loading concentration on the
stability of the protein, or the influence of other protein components at the
solid/liquid interface, are much harder to assess. Similarly, the influence of many
Figure 8. Plots of bandwidth versus capacity factor k for different values of the
Damkohler number, Da.
ORDER REPRINTS

Figure 9. Decrease in yield in multistep purifications.
stationary-phase variables, such as ligand composition, ligand density, surface

heterogeneity, surface area, and pore diameter distribution, can be ascertained
from small batch experiments. However, it is clear that the behavior of many pro-
teins in such static batch systems can vary significantly from that observed in the
dynamic systems usually employed in column chromatography. This behavior is
not only related to issues of different accessibility of the biosolute to the station-
ary-phase surface area and hence different loading capacities but also involves the
complex relationships between diffusion kinetics and adsorption kinetics in the
overall mass transport phenomenon.
Conformational reordering of a protein structure can occur in both mobile
and stationary phases and leads to multizoning of a component into active and/or
inactive zones. Other phenomena can also lead to multizoning of a biopolymer
with adsorptive chromatographic stationary phases. Probably the easiest of these
phenomena to remedy is the so-called “split peak breakthrough effect,” seen very
often in affinity chromatography (Hage et al., 1986; Hearn, 1986a) and to a lesser
extent in ion-exchange and hydrophobic interaction chromatography (Hogg and
Winzor, 1985). This effect is manifested as a nonretained (or weakly retained)
peak and as a retained peak with the bound/free ratio dependent on the solute’s dif-
fusion kinetics and adsorption kinetics. The amount of protein in the breakthrough
zone is influenced by the flow rate, stationary-phase nominal pore diameter and
ligand density, and the injection volume. This effect can be circumvented by the
choice of a lower flow rate, the selection of stationary phases with better surface
ORDER REPRINTS
area/ligand accessibility characteristics for the particular protein of interest, and

more appropriate loading volumes and concentrations. A second type of multi-
zoning phenomenon is associated with nonlinear isotherm behavior due to matrix
heterogeneity and nonuniformity of the ligand distribution over the stationary-
phase surface. This effect is most noticeable between “virgin” and “conditioned”
columns and is problematic during the first few cycles of use of a particular col-
umn at preparative loadings. At the micropreparative level this effect can lead to
catastrophic results wherein irreproducible recoveries may occur. Other forms of
multizoning, associated with slow equilibria between the monomeric form and
higher oligomeric forms of the protein, also are known to affect resolution and re-
covery. Finally, the different times required for specific and nonspecific binding
phenomena to reach thermodynamic equilibria have an important consequence on
adsorption behavior.
Since multiple chromatographic steps are the norm in protein purification
strategies, the stage at which a particular chromatographic selectivity mode is em-
ployed requires careful planning. Previous experience with the systematic opti-
mization of resolution for low-molecular-weight solutes based on solvent selec-
tivity triangle can be used as a basis for multistep chromatographic optimization
involved with the purification of peptides and proteins. Resolution contour plots
for each of the peak zones can be obtained from the experimental data obtained
with different binary/ternary mobile-phase combinations under either isocratic or
gradient elution conditions. By integrating this information with data on bioactive
contour profiles, derived, for example, from on-line manipulation of spectro-
scopic data accumulated with multichannel or photodiode array spectrometers
such as second derivative spectra (Katzenstein et al., 1986; Grego et al., 1986;
Johnston et al., 1987b; Wu et al., 1986), it is feasible to explore rapidly a variety
of separation variables with single-column or multicolumn systems. Importantly,
these approaches offer considerable potential for the optimization of resolution of
very complex mixtures of proteins using the same stationary phase operating un-
der different elution conditions.
Such methods have been widely used as multidimensional techniques in re-
versed-phase HPLC of peptides and proteins for a number of years. Integral to this
approach has been the application of mobile phases of different composition, no-
tably different ion-pairing systems. Similar procedures are equally pertinent to
ion-exchange HPLC separations with ions of different solvated radius and elec-
tronegativity (Hearn et al., 1989). By employing ion-exchange supports after a
preparative isoelectrofocusing stage, protein components with the same pI value
can be resolved by taking advantage of the Donnan effect on ionization equilibria
within the microenvironment of the stationary phase and the ability of the coulom-
bic ligand to act as a molecular probe for the asymmetric distribution of charge on
the protein surface. Furthermore, by utilizing mobile phases of different ion com-
positions, multidimensional separation strategies can be automated readily and
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carried out efficiently with tandem columns packed with the same ion exchanger.
The potential for resolution optimization exploiting ions of different electronega-
tivity and solvation state along the Hoffmeister series has been utilized exten-
sively in salting-in and salting-out phenomena with biopolymers. The availability
of rapid, high-resolution ion exchangers and hydrophobic interaction media will
lead to further development of this potential into much more predictive capabili-
ties for purification of specific proteins in complex mixtures.
One part of current research that offers considerable versatility with tandem
columns is the so-called “positive-negative” affinity or adsorption mode. In this

strategy, the chemical characteristics of the immobilized ligand are selected such
that components of interest are either absorbed or not absorbed by sequential or
tandem columns (Hearn et al., 1986b; Scopes, 1986). Such approaches are partic-
ularly suited to group specific approaches. Typical of this approach has been the
integration of dye affinity and ion-exchange methods as positive/negative modes
into automated protocols for the preparation of immunoglobulins from Cohn frac-
tions, which has led to the development of general stratagems for the purification
of other plasma proteins (Hearn et al., 1986).
With batch adsorption methods the effective peak capacity may be only be-
tween 2 and 10 per stage. Intercalation of batch methods with gradient elution
techniques where peak capacities in excess of 200 can be realized has many de-
sirable features for biopolymer fractionation. When used at an early stage of a pu-
rification scheme, tandem batch methods frequently improve overall recoveries.
Because greatly decreased protein masses are loaded onto chromatographic beds
following batch fractionation, their use allows substantially higher peak capacities
to be achieved at subsequent chromatographic stages. Such batch methods are
readily incorporated into a purification strategy using both chemically modified
silica-based media and polymer-based media. In the case of dye affinity systems
the participation of multiple retention mechanisms is again evident, with the fea-
tures of both cation exchange and hydrophobic interaction particularly noticeable
under neutral or weakly acidic pH conditions. For the purification of hormonal
peptides from endocrine tissue a number of tandem column approaches have been
proposed based on cartridges or columns packed with chemically modified silicas
(see also Table 1).
Whether a particular stationary phase functions in a single elution mode is
clearly not an essential requirement for its successful application in high-resolu-
tion purification of biomacromolecules. The logical extension of mixed-mode in-
teractions is, of course, biospecific chromatography and its immunological coun-
terpart, immunoaffinity chromatography, where the composite interplay of
coulombic, hydrogen bonding, and hydrophobic forces determines the magnitude
and nature of the association and dissociation phenomena. As currently manufac-
tured, all micro- and mesoparticulate chromatographic media, irrespective of the
nature of the ligand or the chemical functionality of the matrix, exhibit separation
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features characterized by these composite phenomena. Exploitation of secondary

retention capabilities of a stationary phase should thus not be discounted out of
hand since it may potentially provide the solution to difficult separation tasks. For
example, mixed chromatographic beds containing hydrophobic interaction and
ion-exchange media have been used successfully (El Rassi and Horvàth, 1986a;
Porath et al., 1983; Bollin and Sulkowski, 1981) for the purification of a variety
of proteins under conditions where the relative selectivity was significantly dif-
ferent from that observed with a single type of stationary phase. Extensions of this
mixed-bed approach underlie the recent further development of multimodal or

mixed ligand separation media and salt-promoted adsorption media (El Rassi and
Horvàth, 1984; Kennedy et al., 1986; Alpert, 1986; Chang et al., 1985; Maisans
et al., 1985; Porath et al., 1985). In the case of hydrophobic adsorption phenom-
ena, the salt-mediated changes in retention are largely entropically driven, with
changes in the associated water structure or bound ions providing a mechanism to
either stabilize or destabilize the three-dimensional structures of proteins. Al-
though there is useful information at hand on the effects of different salt species
on protein conformation in bulk solution, as noted before (Porath, 1986; Arakawa
and Timasheff, 1982), the systematic extension of these studies to adsorption
chromatography with micro- and mesoparticulate high-performance chromato-
graphic media requires substantial investigation before it reaches a similar level
of predictive maturity. For example, the salting-in or salting-out behavior of
chaotropic salts can be described quantitatively in terms of the empirical
Setchenow equation, such that
log(solubility) C S* (concentration of salt)
where S*, the Setchenow constant, is a characteristic of the salt and protein in
question, and C is a system constant. Although the form of the empirical
Setchenow equation is remarkedly similar to the empirical retention equation used
to describe reversed-phase, hydrophobic interaction, and ion-exchange chro-
matography (i.e., log k log k 0 Sx), demonstration of a direct physical rela-
tionship in terms of mechanistic pathways between precipitation and adsorption
parameters S and S* has yet to be firmly made. Such studies will certainly be a
fruitful avenue of research over the next several years because of their impact on
preparative biopolymer separation by chromatographic methods.
6. MASS-TRANSFER RESISTANCES
Large-scale purification of commercially important biomacromolecules to

the degrees of purity necessary for them to be used as pharmaceutical products re-
quires not only the successful equipment and engineering scale-up and integration
of each purification stage, which inevitably results in a complex manufacturing
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plant dedicated almost exclusively to one particular product but also procedures,
protocols, and research-based information which permit accurate prediction of the
biosolute’s mass-transport and bioactivity behavior under various operational con-
ditions (Wankat, 1974; Arve and Liapis, 1987a; Arnold et al., 1985; Chase, 1984a).
Recovery and purification of biological substances and pharmaceuticals from fer-
mentation broths or biological fluids in which the concentration of a substance of
interest is very low frequently involve the use of a creative combination of differ-
ent chromatographic separation techniques operated under nonlinear adsorption
conditions approaching column saturation. Mass transfer resistances associated

with the chromatographic separation of biomacromolecules, which from practical
experience are known not to be comparable to low-molecular-weight compounds,
currently results in very expensive scale-up procedures and does not necessarily
lead to a fully optimized preparative procedure even when the stationary-phase sur-
face is totally accessible (Chase, 1984b). The performance of the purification pro-
cess under variable conditions (flow rate, particle and pore size, etc.) is dependent
on the rates of mass transfer, adsorption, and desorption, and on axial dispersion.
Much of the information needed to evaluate column performance is con-
tained in plots of effluent concentration as a function of time or throughput vol-
ume. The shape of such a breakthrough curve is a complex mix of equilibrium and
nonequilibrium processes (see Fig. 10). The general position of the breakthrough
curve depends on the capacity of the column with respect to the feed concentra-
tion, which is set by the equilibrium. The spreading observed in actual break-
through curves is due to the existence of flow nonidealities, mixing, and finite
Figure 10. Breakthrough curve for adsorption of proteins. In case of an adsorption step
terminated at effluent concentration CT, a small amount of feed has been wasted, and a por-
tion of the column capacity remains unused. In the upper part of the breakthrough curve,
nonequilibrium effects can occur.
ORDER REPRINTS
mass-transfer and sorption rates. These nonequilibrium effects depend on the op-
erating conditions and system configuration. Scale-up and optimization require an
understanding of these nonequilibrium interactions as well as of the equilibrium
between the adsorbent and the solute.
The solute must proceed through a series of diffusion and reaction steps in
both the adsorption and elution operations. In the adsorption process, solute in the
feed must diffuse through a liquid film surrounding the particles and then diffuse
through the pores in the particles before interacting with the immobilized ligand.
Each of these mass-transfer or reaction steps can be explicitly included in a model,

but the resulting solutions are cumbersome and require detailed information on
mass-transfer coefficients and rate constants and complex numerical methods
(Chen and Hsu, 1987; Graham and Fook, 1982; Hossain et al., 1986).
Based on the isothermal sorption of a single solute in plug flow through a
packed bed of monodisperse porous particles, the mass balance over a section of
the column yields the continuity relation for the packed bed:
c c c 2c
u0 (1 ) E z 2
z t t z
where c(z,t) is the solute concentration, with a volumetric flow rate Q through a bed
of length L, cross-sectional area A, and void fraction , and s is the sorbate concen-
tration in the particle, which includes the solute in the pores. The particles are as-
sumed to be spherical with radius R, porosity , and density p. The sorbate concen-
tration in the particle is qi(r,z,t) and the solute concentration in the pores is ci(r,z,t).
The different terms in this equation account for convective transport of solute, accu-
mulation in the interstitial spaces, solute uptake in particles, and axial dispersion.
The rate of change in the average particle concentration is equal to the flux
of solute into the pores:
s 3(1 ) c
(1 ) Di i
t R r rR
where Di is the effective particle diffusion coefficients based on the entire particle
volume, and the quantity 3(1 )/R is the surface area per unit bed volume of the
spherical particles.
For a given particle, the following equation describes the diffusion of the so-
lute into the pores with adsorption at the pore surface:
2ci 2 ci ci qi
Di p 0
r2 r r t t
The concentrations ci and c in the pores and in the bulk liquid surrounding the par-
ticles, respectively, are coupled by the rate of mass transfer through the fluid film:
ci
kf (c ci)
rR
Di
r rR
ORDER REPRINTS
At this stage most fixed-bed adsorption models assume that film mass-trans-
fer resistance is small compared with the other transport resistances in the system
and that equilibrium is reached instantaneously between the solute in the pore liq-
uid and the sorbate. However, with the assumption of a homogeneous affinity ma-
trix, it can be shown that both film and pore diffusion mass transfer resistances
cannot be ignored (Kopaciewicz et al., 1987; Hsu and Chen, 1987) and that the dy-
namic behavior of the adsorption stage is greatly dependent on the rate of the ad-
sorbate-ligand interaction (Arve and Liapis, 1987a,b). Breakthrough of adsorbates
may occur at a much shorter time in a system where the adsorbate-ligand interac-
tion rate is finite compared to systems where local equilibrium exists between the
adsorbate and the adsorbate-ligand complex. The occurrence of early break-
through may result in a very low utilization of immobilized ligands.
An alternative approach is the use of high-performance nonporous packing
materials similar to those introduced by us in 1984 (Anspach et al., 1984) for the
design of improved stationary phases. Our investigations have demonstrated
much more favorable mass-transport and adsorption/desorption kinetic behavior
with these nonporous supports. Breakthrough curves obtained from nonporous sil-
icas are much steeper than those of porous silicas using the same ligand function-
alities and column configurations (see Fig. 11). Utilizing nonporous stationary
phases with such favorable mass-transport behavior avoids the loss of immobi-
Figure 11. Shape of the breakthrough curve at similar elution volumes of the substrate
for the affinity system Cibacron Blue F3GA-lysozyme, where the dye is immobilized on
(a) a nonporous silica matrix with 1.5 m particle size, and (b) a porous silica matrix with
25 to 40 m particle size and 30 nm pore size. The shallower breakthrough curve experi-
enced on the porous affinity matrix has its origin in diffusion-controlled adsorption pro-
cesses, which are, in addition, sterically hindered in the upper part of the breakthrough
curve.
ORDER REPRINTS
lized ligand, which is not used by the adsorbate due to an early breakthrough, or
of protein containing feed solution if the adsorption is not interrupted when break-
through of the adsorbate occurs. Furthermore, nonporous matrices exhibit im-
proved mass and biological recoveries and higher accessibilities of immobilized
ligands compared to equivalent porous affinity supports.
7. SCALING-UP
In all chromatographic work the first and arguably the most important step
is the choice of an appropriate bed material. The result of a chromatographic pro-
cess can never be better than selectivity of the bed material allows. The choice of
appropriate bed materials and optimization of the basic operating conditions is
made by laboratory trials prior to scale-up. It is very important that the mass trans-
port between the eluent and the bed material must be maintained on a large scale.
According to theory, optimum resolution is obtained with the smallest pos-
sible average particle size, and especially in large-scale chromatography, the
choice of particle size is governed by the necessity of high, steady flow rates
through often-soft and deformable bed materials at low hydrodynamic pressures.
For rigid particles only, such as silica, the relationship between the flow rate and
pressure drop obtained is linear and inversely proportional to the square of the par-
ticle size:
unL
p 2
k0dp
where p is the pressure drop over the bed, u the linear velocity of the eluent, n
the eluent viscosity, L the bed height, k0 the specific permeability of the column
(1 10 3 1.3 10 3), and dp the average particle diameter.
For nonrigid or deformable particles such as the soft gels, the observed re-
lationship is more logarithmic than linear and a function of both the bed height and
diameter as well as the water regain of the gel. The following empirical equation
was found to fit best with the experimental data (Joustra et al., 1978; Janson and
Hedman, 1982)
L k0
p ln
a k
where a is a function of the bed height and diameter and the gel solvent regain.
Theoretically, when using rigid particles, one should use the finest grade
possible considering the pressure limits of the entire chromatography system. In
practice, however, there is often very little choice for economic reasons. When us-
ing nonrigid particles the limiting parameter is the particle size, giving an inlet
pressure which should not exceed that giving the maximum obtainable flow ve-
locity.
ORDER REPRINTS
In any chromatographic column, the force that is exerted on the lowest part
of the bed is the sum of the weight of the packing material and the drag force act-
ing on it, minus the friction force of the column wall. The tendency for deforma-
tion of the nonrigid gel materials most often used in protein chromatography un-
til now decreases with decreasing bed height of the column. An increase in
hydrostatic pressure leads to bed compression and a drop in flow rate which is re-
versible within limits but is usually combined with hysteresis. This effect is ap-
parent even with very short columns.
There are several criteria that have to be taken into account when choosing
the most suitable column packing material for a specific chromatographic process.
In Table 6 are listed some of the common characteristics of soft gels and silica-
based rigid gels used for column packing materials. Since the aim for a cost-ef-
fective preparative separation of complex biological mixtures with all chromato-
graphic procedures is high mass recovery, maintenance of biological activity and
contemporary high resolution (Mazsaroff and Regnier, 1986; Katoh et al., 1987),
high peak capacity and short preparation time, a number of research groups have
devoted considerable effort to the preparation of tailor-made support media based
either on soft gels such as agarose or dextrans, (polyacrylamide or trishydroxy-
methylpolyacrylamide) or on rigid silicas, as well as the new polymer-based sup-
ports. The new supports have enlarged pore diameters that freely accommodate
Table 6. Comparison of Soft Gels and Silica-Based Matrices
Silica-Based
Property Soft Gels Matrices
Buffer compatibility Medium-high Low-medium

Mechanical rigidity Low High
Limiting pressure drop 2–10 bar 250 bar
Separation speed Low-high Low-high
Deformation properties Poor-good Good-excellent
Particle-size distribution Narrow-medium Narrow
Pore-size distribution Narrow-medium Narrow
Thermal stability Low High
Chemical stability Good-high Low-good
pH limitation Good-high Low-good
Specific surface area (m2/g) 10–100 20–400
Resolution Low-high Moderate-high
Capacity Low-high Low-high
Mass recovery Good-excellent Low-moderate
Biological recovery Low-moderate Low-moderate
Regeneration in base Good Poor
Cost (dollars per gram) Low-high Moderate-high
(10–200) (25–400)
ORDER REPRINTS
biomacromolecules with molecular weights of 200 kD and more, representing av-

erage pore diameters of 30 to 600 nm and particle sizes between 10 and 150 m
suitable for large-scale purification.
8. SUMMARY
In this article the physicochemical basis of protein retention in adsorption

chromatography has been examined. It is evident from this treatment that signifi-
cant developments are in process with regard to both the theory and practice of
high-resolution chromatographic methods, particularly at the preparative level
with adsorptive stationary phases. The recognition that most purification strate-
gies must be based on multidimensional multistage procedures presents numerous
challenges for the protein chemist, the chromatographic scientist, and the bio-
chemical engineer. The urgency for these developments has been stressed by the
potential of modern biotechnology to produce large quantities of new proteins that
must be obtained in highly purified form. The capabilities that these advances pre-
sent will prove catalytic in the materials sciences as new solid phases are exam-
ined with particular properties for large-scale utilization. Similarly, new initiatives
in protein engineering will lead to greater utilization of the fusion handle approach
for selective protein isolation and recovery. The realization of these capabilities
will represent an enthusiastic and innovative method that will revolutionize the
role of bioprocess development over the next decade.
ACKNOWLEDGMENTS
Support by the Australian Research Grants Committee and Monash Uni-

versity Special Research Grants is gratefully acknowledged. B. A. is a recipient of
a Monash University Postdoctoral Fellowship.
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CHEMICAL, PHYSICAL, AND

Milton T. W. Hearn and Birger Anspach

Monash University, Clayton, Victoria, Australia

Major advances have occurred in recent years in the development of prepar-

Copyright © 2001 by Marcel Dekker, Inc. www.dekker.com

222 HEARN AND ANSPACH

in large-scale purification strategies for protein purity are currently undergoing

achieved only by the application of several high-resolution separation techniques.

Table 1. Laboratory-Scale Purification of New Biologicals

Purified Biomacromolecule References

Inhibin Forage et al. (1986), Robertson et al. (1985),

ISOLATION AND PURIFICATION OF PROTEINS 223

Nearly all modern high-performance chromatographic methods lend them-

oratory-scale purifications of important new biologicals. What is not evident from

224 HEARN AND ANSPACH

binding or permeability in the stationary-phase surfaces are all important parame-

2. CHEMICAL STRUCTURE OF PROTEINS

ISOLATION AND PURIFICATION OF PROTEINS 225

Table 2. Factors Controlling Chromatographic Stability of

Mobile Phase Stationary Phase

Organic solvents Ligand composition

Temperature Pore diameter distribution

in aqueous or nonaqueous two-phase or multiphase liquid/liquid systems. All of

226 HEARN AND ANSPACH

biopolymer separation, based on either noninteractive or interactive chromato-

The attraction between hydrophilic biopolymers and stationary phases,

ISOLATION AND PURIFICATION OF PROTEINS 227

primary bonds (respectively, orientation based on LW interaction) occur at a very

Figure 1. Schematic representation of the energy of interaction between a protein and

228 HEARN AND ANSPACH

In recent years, a large amount of developmental effort has been expended

in Table 3 apply equally to analytical separations as they do to large-scale prepar-

Table 3. Criteria for the Selection of Chromato-

Chemical and physical stability

ISOLATION AND PURIFICATION OF PROTEINS 229

Ion-exchange chromatography separations take advantage of the net charge

with separations involving an increasing concentration of organic solvent in the

230 HEARN AND ANSPACH

surface tension associated with the solute/solvent/stationary phase interaction

ISOLATION AND PURIFICATION OF PROTEINS 231

232 HEARN AND ANSPACH

methods that exploit functional properties of a biopolymer such as a specific lig-

for ion-exchange or reversed-phase chromatography. Table 4 summarizes exam-

Table 4. Separation Parameters Used in Large-Scale Protein Purification and Typical

Temperature Heat denaturation 2–20

ISOLATION AND PURIFICATION OF PROTEINS 233

monoclonal antibodies or biospecific affinity separation with the appropriate bio-

234 HEARN AND ANSPACH

for improving stationary-phase characteristics to allow enhanced separation selectiv-

tive phenomena forms the basis of so-called “mixed-mode” chromatographic pro-

ISOLATION AND PURIFICATION OF PROTEINS 235

5. PHYSICOCHEMICAL BASIS OF LIQUID

The physicochemical basis of all modes in liquid chromatography can be

236 HEARN AND ANSPACH

ISOLATION AND PURIFICATION OF PROTEINS 237

ear in salt molality: