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`High Performance Liquid Chromatography Principles and Methods
`
`in Biotechnology
`
`Volume
`
`Issue
`
`Date
`
`1996
`
`Pages 411E0A
`
`Author Benedek
`
`K
`
`Title The Application of HPLC for Proteins
`
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`

`

`High Performance Liquid
`Chromatography
`Principles and Methods
`in Biotechnology
`
`Edited by
`Elena D Katz
`Biotechnology Department PerkinElmer Corporation Norwalk CT USA
`
`JOHN WILEY
`
`Chichester
`
`SONS
`New York
`
`Brisbane
`
`Toronto
`
`Singapore
`
`

`

`Copyright C 1996 by
`
`Sons Ltd
`John Wiley
`Bans Lane Chichester
`West Sussex P019 IUD England
`
`Telephone
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`Ali rights reserved
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`International + 44 1243 779777
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`No part of this book may be reproduced by any means
`or transmitted or translated into a machine language
`without the written permission of the publisher
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`John Wiley
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`Library of Congress Cataloging inPublication Data
`
`principles and methods in
`
`High performance liquid chromatography
`edited by Elena D Katz
`biotechnology
`p
`cmSeparation science series
`references and index
`Includes bibliographical
`ISBN 0471934445 alk paper
`1 High performance liquid chromatography 2 Biotechnology
`I Katz Elena
`Technique
`II Series
`TP24825H54H544
`1996
`66063dc20
`
`9522111
`CIP
`
`British Library Cataloguing us Publication Data
`
`A catalogue record for this book is available from the British Library
`
`ISBN 0 471 93444 5
`
`Produced from cameraready
`copy prepared by the author
`Printed and bound in Great Britain by Bookcraft Bath Limited
`from sustainable forestation
`This book is printed on acid free paper responsibly manufactured
`for which at least two trees are planted for each one used for paper production
`
`

`

`CHAPTER 9
`
`THE APPLICATION OF HPLC FOR PROTEINS
`
`Kalman Benedek
`
`I
`
`Introduction
`
`The 1980s witnessed the growing pains and maturing of a new technology
`in pharmaceutical discovery and development
`the emergent biotechnology
`industry The spectrum of drugs has been extended from the traditional
`small molecules
`to peptide and protein based
`pharmaceuticals
`has become a practical and feasible possibility for medicine
`Biotechnology
`cornerstones of this new era are the three Cs cloning
`The technical
`and chromatography
`Chromatographic
`techniques
`implemented for the purification and characterization of all proteins from
`
`computer
`
`are
`
`ill High performance
`basic discovery research to full product development
`separations are required to isolate a given protein from the complex matrix
`of cell culture media and are involved in verifying the purity and identity
`of the product and in monitoring its stability
`
`so far because
`
`the
`
`HPLC is the most versatile separation technique
`acronym HPLC includes
`such mechanistically diverse chromatographic
`methods as hydrophobicinteraction chromatography HIC reversed phase
`liquid chromatography RPLC electrostatic
`chromatography
`EIC size exclusionchromatography SEC metal interaction chromat
`chromatography B IC or affinity The use
`ography MIC and biospecific
`of so many HPLC separation modes is required because of the diversity of
`proteins In order to apply successfully the techniques of modern HPLC
`
`interaction
`
`

`

`414
`
`should be
`
`understood
`
`and taken
`
`into
`
`science
`
`in
`
`phenomena
`the underlying
`consideration Protein chromatography
`is a multidisciplinary
`which protein biochemistry polymer adsorption surface chemistry and
`theories have to be simultaneously
`considered during the
`chromatographic
`development of each separation method In the following chapter some of
`the relevant phenomena associated with protein chromatography will be
`highlighted with special attention to adsorption conformational effects and
`The examples were selected according to
`criteria which could best help to illustrate the most
`frequently occurring
`accomplishments of protein
`
`their impact on chromatography
`
`problems and to focus on significant
`
`chromatography
`
`2 Protein Composition and Structure
`
`the technical
`
`Compared with the challenges of the analysis of small molecules most of
`of protein chromatography are the direct
`difficulties
`of the polymeric macromolecular nature of proteins Proteins
`consequence
`are linear polymers of up to 21 different amino acids with a molecular
`ranging from a few to a few hundred kilodaltons Each particular
`weight
`protein has its characteristic
`amino acid sequence called the primary
`forces between the amino acids of
`structure Intermolecular
`polymer generate local helices sheets and turns called secondary structure
`The folding of the secondary structure into a threedimensional entity
`produces a specific biological activity and is called the tertiary structure of
`can be assigned to the
`a protein In general only one folded conformation
`native protein A number of proteins have a quaternary structure which is
`the association of folded polymer units into a biologically relevant structure
`
`the linear
`
`Protein structures can further be divided into two major classes
`
`on the
`
`basis of their composition simple and conjugated Simple proteins are
`linear polymers of the common amino acids Conjugated proteins have
`other organic andor inorganic components attached through the side chains
`of particular amino acid residues The attached moieties are called prosthetic
`groups Conjugated proteins can be classified on the basis of the chemical
`nature of their prosthetic groups eg nucleoproteins lipoproteins phospho
`
`

`

`415
`
`asparagine
`
`proteins metalloproteins and glycoproteins Examples of such prosthetic
`modifications are phosphorylation of serine and threonine glycosylation of
`serine and threonine the attachment of heme the binding of
`and the attachment of lipids These prosthetic
`metals and other cofactors
`groups and co factors can be important determinants in all aspects of poly
`behavior
`secretion activity metabolism and
`including
`peptide
`chromatographic behavior
`
`3 Protein Adsorption
`
`31 Adsorption Isotherms
`
`During the chromatography of proteins the elution is based on two major
`effects One of them is size exclusion large proteins may sometimes be
`column The second
`sterically excluded from small pores of
`the packed
`effect is based on the complex interactions of an individual protein with the
`stationary phase surface The first effect may result in separation without
`energetic interactions whereas the second is based on energetic interactions
`The energetic interactions can be electrostatic hydrophobic biospecific and
`are usually the combination of various interactions Figure 1
`above
`described differentiation between the various chromatographic
`
`illustrates the
`
`modes 2
`
`No Energetic
`Interactions
`
`Interactions between the
`Energetic
`SampleStationary PhaseMobile Phase
`
`to
`
`Retention Time
`
`Figure 1
`
`Dual Retention Mechanism of Chromatography in Porous Materials l21
`
`

`

`416
`
`The adsorption of proteins at the solid—liquid interface is the fundamental
`phenomenon governing all interactive chromatography [3,4,5]. Adsorption
`and desorption isotherms represent the key physicochemical information to
`all separation optimization, Analytical separations are performed on the
`linear portion of the adsorption isotherms, which is easy to accomplish
`with small molecules, Preparative separations, by contrast, are typically
`performed at loadings corresponding to the non-linear portion of the
`adsorption isotherms. The polymeric, macromolecular nature of proteins
`requires a better understanding of the adsorption process and dynamic
`measurement of the isotherms, as is performed by frontal chromatography
`6,7) which provides a good description of the adsorption process
`[8,9,10,11]. Recent advances in protein HPLC highlighted two important
`problemsrelated to the traditional single-protein isotherm approach, First,
`the adsorption isotherm for a specific compound can vary significantly
`depending on the number, amount, and type of other components in the
`sample. Second,selectivity changes can occur due to the crossing of the
`isothermsat particular values of composition and concentration of the feed.
`Both of these problems should be accounted for
`in preparative
`chromatography, where protein solutions of high concentrations are loaded
`onto the columns and separations are performed outside the ideal
`chromatographic range (see Chapter 6). The increased feed concentration
`might also lead to protein-protein interactions resulting in aggregation
`and/or precipitation. Aggregate formation and multiple equilibrium between
`oligomers introduce new complexity and can alter the adsorption
`characteristics of the proteins. Since the adsorption isotherms of the
`associated and dissociated forms are different, they can also be used for
`protein characterization. The effects of other protein components on the
`adsorption isotherm can be studied by acquiring the composite isotherms to
`explore the intricate competition for adsorption sites on the surface.
`
`In displacement chromatography, high performance columns are operated
`outside the linear portion of the adsorption isotherm, allowing high loading
`capacities without loss in resolution [12], In displacement chromatography,
`instead of obtaining peaks during the elution process, the separated sample
`elutes in blocks in the order of increasing adsorption strength. The final
`component is the displacer.
`
`

`

`Ono aline Line
`
`417
`
`071aimponent 3
`
`or Displacer
`
`Component 2
`
`1
`
`Component
`
`I
`
`qd
`
`r
`
`I
`
`I
`
`L
`
`iv
`I If
`
`I
`
`cl
`
`I
`
`Mobile Phase Concentration
`
`£2
`
`The operating line connects
`the point on the isotherm corresponding to the displacer
`concentration cd with the origin et and c2 are the isotachic concentrations of the feed
`
`components
`
`Figure 2 Schematic Diagram of
`Components 951
`
`Individual Adsorption
`
`Isotherms of Three
`
`In Figure 2 diagrams of individual adsorption isotherms illustrate the
`relationships among the concentrations of the sample components and the
`train The operating line connects
`the point on
`in the displacement
`displacer
`concentration cd with the
`the isotherm corresponding
`to the displacer
`origin ci c2 and c3 are the isotachic
`concentrations of the sample
`components Displacement
`chromatography
`excellent option for both analytical and preparative chromatographies
`The trend
`is toward overloaded
`
`has been proven to be an
`
`1131
`
`in preparative protein separations
`chromatography However
`large samples can
`nonlinear or displacement
`the column
`irreversible adsorption which consequently
`cause
`changes
`characteristics and reduces capacity and column life time 141516
`
`The chromatographic
`behavior of protein aggregates was intensively
`studied under conditions of hydrophobic interaction
`chromatography
`
`

`

`418
`
`using 13lactoglobulin as model system 1719 As a result of adsorption a
`region of the protein interacts with the stationary phase to the exclusion of
`other regions The adsorption mechanism is ultimately based on multiple
`attachment points Taking a rigid model of a protein molecule it
`
`is easy to
`
`the binding of the stationary phase
`visualize that steric factors might limit
`to a surface on the protein that is complementary to the stationary phase For
`example a hydrophobic region might preferentially bind to a hydrophobic
`retention time
`stationary phase Studies establishing a relationship between
`
`and the hydrophobic amino acid content of large polypeptides or proteins
`shown very little
`correlation because of conformational effects
`have
`Efforts to make retention time predictions
`in reversed phase elution have
`failed for polypeptides with more than 20 residues 2021 demonstrating
`the size and threedimensional structure of proteins play a governing
`that
`role in the adsorption process and emphasizing the importance of a
`complementary surface The crucial
`role of surface complementarity has
`been shown in the HICEIC and metal affinity chromatography of lysozyme
`22 Because of multiple attachment points the type and strength of the
`forces involved in adsorption to a particular surface will depend
`physical
`on the amino acid composition of
`the complementary surface of the
`protein The distribution of the side chains located on the exterior of the
`threedimensional
`structure is manifested
`and
`in the great variability
`diversity of adsorption The polymeric nature of proteins is apparent in the
`cooperativity of folding and unfolding and as a result of this cooperativity
`force of interaction can be very strong
`
`the total
`
`32 Stoichiometric Displacement Model SDM
`
`Some characteristics of
`can
`be
`the protein complementary surface
`measured by using the stoichiometric displacement model 2324 The
`capacity factor V is related to the number of solvent molecules or ions
`Z that are displaced and the concentration of the solvent Do and is
`
`expressed by
`
`k =
`
`Do Z
`
`

`

`where 1 is a constant
`representing the combination of numerous
`characteristics eg the equilibrium constant phase ratio
`
`chromatographic
`
`419
`
`ligand density
`
`intercept
`
`solvent concentration
`
`The Z number can be determined by plotting log k values against
`the slope of the curve is the negative of Z and the
`is log 1
`The analysis of Z numbers derived under a variety of different conditions
`can elucidate the adsorption mechanism of a protein Typical Z values of
`proteins are much larger than those for traditional small molecules and
`thus are indicative of multiple attachment points as shown in Figure 3
`Protein desorption occurs in a very narrow range of eluent concentration
`between the individual attachment
`suggesting a high level of cooperativity
`points Since the solvent concentration range for protein elution is not only
`narrow but also different
`for each protein gradient elution is usually
`required to elute all of the components of a protein mixture
`
`Log 10
`
`Small
`Molecules
`
`B Solvent Concentration
`
`Figure 3 Determination of the Z Number of the Stoichlometric Displacement Model
`
`

`

`10
`
`6
`
`4
`
`2
`
`ZNumber
`
`420
`
`0
`
`10
`
`20
`
`30
`
`40
`
`$0
`
`60
`
`Temperature °C
`Figure 4 Temperature Dependence of the z Number for aLactalbumin under 111C
`Conditions 29
`
`useful
`
`for the
`
`states of
`
`displacement model has proven
`The stoichiometric
`of protein adsorption in ion exchange 2526 reversed
`characterization
`interaction chromatography 29 Although
`phase 2728 and hydrophobic
`in each chromatographic
`the displacement
`process is slightly different
`mode the same equation can be applied Different conformational
`alactalbumin were identified in HIC as a function of
`temperature as
`shown in Figure 4 The Z number
`increases with the unfolding of the
`surface area The results
`molecule which generates a larger complementary
`structure The
`=respond to the increased retention of the unfolded
`displacement model was used for characterizing interleukin
`stoichiometric
`2 muteins in RPLC Mazsaroff et al used the stoichiometric
`displacement
`to study the change in molecular orientation of IgG when bound to
`model
`the entire range of the adsorption isotherm
`an ion exchange surface over
`20 They found that Z decreases with an increase in the adsorbed
`amount of protein as shown in Figure 5 The data were interpreted
`the orientation of adsorbed IgG changes according to the
`
`indicate that
`
`protein concentration Consequently
`
`the complementary
`
`surface area and
`
`thus the affinity also vary which can lead to differences in selectivity
`
`to
`
`

`

`421
`
`35
`
`30
`
`25
`
`05
`
`15
`10
`IgG mg
`The decreasing Z number indicates a smaller complementary surface area 26
`Figure 5 Sample Loading Dependence of the Z Number
`
`20
`
`A feed concentration dependent
`change in selectivity implies that caution
`has to be exercised when a linear scale up procedure is applied
`
`4 Protein Conformation and Chromatography
`
`The main environmental
`effects which can cause changes
`in the protein
`1 Under chromatographic
`in solution are listed in Table
`conditions one must contend with the occurrence of almost all of
`these
`
`structure
`
`effects
`
`Table 1
`
`Physical Parameters Affecting Protein Structure
`
`Parameter
`
`Temperature
`Solvents
`PH
`Interfaces
`
`Effect
`
`>50°C Molecule Specific
`Organic Solvents Chaotropics
`Extreme
`LiquidGas SolidLiquid
`
`

`

`422
`
`Some of the conformational changes upon adsorption
`are gentle and
`reversible and the protein retains thermodynamic properties close to those
`in the solution state yielding biologically active material Other changes
`might be extensive leading to significant alteration in the thermodynamic
`and biological properties of the protein molecule It must be realized that
`
`be facilitated under chromatographic
`protein denaturation may even
`to aqueous solutions due to the synergism of the
`conditions in contrast
`
`listed effects 30
`
`at the dawn of protein HPLC was that under reversed
`The general concept
`phase elution conditions all proteins were denatured However it has been
`reported that under RPLC conditions
`certain peptides and proteins elute
`anomalously and the chromatogram of pure samples displays multiple
`peaks andor odd peak shapes These phenomena were traced back
`conformational changes occurring during chromatography and it was
`states can be eluted in
`established that proteins in different conformational
`run Under
`the same chromatographic
`reversed phase and hydrophobic
`conditions the denatured protein elutes later
`than does the native protein 3135 However
`in the gradient
`exchange and affinity chromatography
`the opposite elution behavior
`occur 36 The elution time variation as a function of protein conformation
`surface In the case of
`can be explained by changes of the complementary
`
`to
`
`in ion
`
`can
`
`interaction
`
`chromatographic
`
`reversed phase and hydrophobicinteraction
`chromatography
`increases providing more attachment points and a stronger binding
`whereas in electrostaticinteraction
`the alteration of the
`
`this surface
`
`chromatography
`the specific ionic side chains weakening
`
`complementary
`
`surface decreases
`
`the binding to the stationary phase
`
`Measurement of events starting from the adsorption of protein on the
`adsorbent
`the adsorption
`to the elution is the key to understanding
`desorption mechanism Methods of studying proteins on surfaces include
`circular dichroism 38 total
`ellipsometry 1371
`fluorescence 3940 intrinsic fluorescence spectroscopy 4142 Raman
`spectroscopy 43 and Fouriertransformed infrared spectroscopy 4445
`
`reflectance
`
`internal
`
`

`

`41 stationary Phase Effects Separation Modes
`
`423
`
`The difficulties
`
`in early protein separations were largely related to
`unwanted protein adsorption the physical and chemical properties of the
`silica base material as well as imperfections
`in the bonding chemistry The
`separation of proteins has improved rapidly with the development of new
`bonded phase chemistries and a greater understanding
`mechanism
`
`of
`
`the retention
`
`Every chromatographic mode represents in reality a multimodal retention
`mechanism However conditions can be found where a particular retention
`mechanism is dominant The complexity of solute and stationary phase
`interaction is illustrated in Figure 6 where lysozyme elution is shown as a
`function of mobile phase salt concentration using different size exclusion
`columns
`
`2
`
`Figure 6
`
`Dependence of the Distribution Coefficient KD of Lysozyme on Mobile
`Phase Ionic Strength p Using Different Size Exclusion Columns 1481
`
`

`

`424
`
`The minimum of
`
`the curves
`
`indicates a noninteractive
`
`region for
`
`lysozyme which can be considered as the optimal
`ionic strength range for
`SEC Outside this range lysozyme is apparently retained on the column
`with electrostatic andor hydrophobic
`then contribute
`forces which
`significantly to the elution mechanism The descending and ascending
`columns
`regions of the curves in Figure 6 indicate that sizeexclusion
`under appropriate conditions can be used for electrostatic or hydrophobic
`interaction chromatography Alternatively electrostatic and hydrophobic
`columns can be used for SEC depending on
`chromatographic
`interaction
`the ionic strength and composition of the mobile phase The elution curves
`seem to be characteristic for all size exclusion stationary phases but their
`vary from protein to protein and column to column It
`shape can
`recommended that the real sizeexclusion domain should be determined
`
`is
`
`for a specific application Similar multimodal retention mechanisms can be
`observed with other separation modes such as electrostaticinteraction and
`
`affinity
`
`and even
`
`reversed phase chromatographies complicating the
`
`prediction of molecular structureretention relationships In such studies
`appropriate conditions for a pure retention mechanism should be
`established from the outset
`
`411 SizeExclusion Chromatography SEC
`
`In sizeexclusion chromatography SEC as was shown in Figure I no
`protein adsorption is presumed to occur the separation being based strictly
`on a simple sieving mechanism Column materials for SEC have improved
`significantly since the introduction of crosslinked dextran gels The most
`popular size exclusion materials are crosslinked dextran polyacrylamide
`and agarose polymethacrylate polyvinyl alcohol bare and bonded silica
`gel and porous glass Lately several new HPSEC column materials have
`resolution and recovery can now be achieved
`been developed and excellent
`with high performance sizeexclusion chromatography Calibration curves
`are usually linear in certain molecular weight
`ranges indicating an ideal
`size and shape dependent distribution mechanism 4647 Sizeexclusion
`ideally is a non interactive chromatographic
`chromatography
`separation
`mode However even
`in sizeexclusion
`
`the available
`
`stationary phases vary appreciably
`
`chromatography
`in performance and elution conditions
`
`

`

`481 This discrepancy is partially due to the undesired interaction between
`the protein molecules and the surface which is also affected by the mobile
`phase conditions 49
`
`425
`
`412 Electrostatic Interaction Chromatography EIC
`teMrs
`
`The two distinct
`
`and
`
`at
`
`is
`
`types of EIC are ion exchange chromatography
`The interactions between the charged stationary phase
`chromatofocusing
`and the hydrophilic and charged surface of amino acids of proteins are
`typically reversible and nondenaturing
`Because the majority of proteins
`have isoelectric points below pfl 7 anion exchange chromatography
`505152531
`is more often used than cation exchange chromatography
`neutral pH values 5456 Electrostaticinteraction
`chromatography
`between the protein surface and the
`based on the charge differences
`stationary phase The mobile phase conditions are mild and the majority of
`proteins do not unfold during chromatography Extensive literature is
`of electrostaticinteraction
`and practice
`available on the theory
`for protein separation Pa ers by Mazsaroff and co
`chromatography
`workers 5758 Regnier and Mazsaroff 591 Melander
`et al 601 and
`et al 61 are particularly valuable
`from a theoretical
`StMilberg
`practical point of view
`
`and
`
`Electrostaticinteraction
`
`chromatography
`
`is
`
`ideal
`
`for
`
`detecting
`
`dearnidation differences in sialylation and phosphorylation and other post
`
`translational processing of the primary sequence High performance
`because
`it has a resolving
`
`is also attractive
`
`exchange chromatography
`power which can in certain cases be comparable to that of electrophoresis
`The resolving power of ion exchange chromatography
`on closely related
`polypeptides is illustrated by the example given in Figure 7 The use of a
`strong anion exchanger Mono Q gave rapid baseline resolution 62 of
`three polypeptides differing only at their carboxy termini
`
`ion
`
`En chromatofocusing a complex mixture of buffers Ampholytes is used
`to establish a continuous pH gradient which in turn is used to elute proteins
`bound to an ion exchange matrix
`
`

`

`426
`
`006
`
`A
`
`004
`
`Absorbance
`280 rim 002
`
`45
`
`3 5
`
`25
`
`Acetonitrile
`Concentration
`
`vv
`
`16
`8
`Time minutes
`
`24
`
`Na CI
`niNt
`200
`
`0
`
`4
`8
`Time minutes
`
`006
`
`004
`
`002
`
`Absorbance
`280 nm
`
`03
`
`04
`Absorbance
`280 rim 02
`0 1
`
`41
`
`20
`
`40
`
`m
`
`60
`
`m
`
`80
`
`pH
`
`58
`
`54
`
`50
`
`46
`
`42
`
`Elution nil
`
`A RPLC B IEC C chromatofocusing 62
`
`Figure 7 Chromatography of Diphtheria Toxin Fragments
`
`chromatography The
`This can function as an alternative to ion exchange
`has been compared with that of
`resolving power of chromatofocusing
`isoelectric focusing 63 and with that of a weak anion exchanger Mono Q
`64 the latter is shown in Figure 7 Chromatofocusing has some advantages
`focusing in that the separation is rapid UV detection can
`over isoelectric
`be used at and above 280 nm and recovery of high molecular weight
`proteins can be more readily accomplished Chromatofocusing is an
`excellent separation method with good reproducibility and it also provides
`the isoelectric points of the separated peptides 65
`
`

`

`I 13 HydrophobicInteraction Chromatography HIC
`
`427
`
`HIC is
`
`the milder version of
`
`the hydrophobicinteraction based
`The
`on protein conformation
`in terms of
`chromatography
`its effect
`appropriate stationary phases for HIC are much less hydrophobic than the
`reversed phase nalkyl bonded phases Some of the traditional
`traditional
`columns can be used in the HIC mode as
`sizeexclusion or ion exchange
`well Hydrophobicinteraction
`is carried out on weakly
`chromatography
`hydrophobic columns in high salt concentrations of ammonium or sodium
`sulfate 6671 The high surface tension of a protein dissolved in these
`force for protein adsorption 72741
`the solvophobic
`solutions provides
`Proteins are then eluted with a descending salt gradient and the individual
`
`proteins elute according to their solubility
`
`100
`
`50 11
`
`0
`
`00
`
`cYT
`I X
`
`MY0
`RIROI
`
`CHYGN
`
`CHYM9
`CON
`
`CHYGN
`CHYMO
`
`1
`
`I
`
`CYT
`
`IMY0
`
`RNS
`I CON
`
`a
`
`A220
`
`50 B
`11111 CI
`
`0
`
`24
`12
`Time minutes
`a PROPYL Aspartamide b ETHYL Aspartamide CYT = cytochrome c RIBO =
`ribonuclease A MY0 = myoglobin CON = conalburnin CHYMO = chymotrypsin
`CHYGN = chymotrypsinogen 751
`
`36
`
`Figure 8 The Effect of Alkyl Chain Length on the Separation of Proteins in HIC
`Columns
`
`

`

`428
`
`1
`
`Retention of proteins in HIC is extremely sensitive to the hydrophobicity of
`the stationary phase which can easily be modified by both the length and
`the alkyl side chains of the bonded phase The ability of
`the density of
`synthesizing stationary phases with different hydrophobicity opens new
`possibilities in the optimization of separation Changing the attached ligand
`on the hydrophilic base group 75 affords control over retention and
`different selectivities as shown in Figure 8 Column regeneration is far
`more rapid than in RPLC Samples eluted from HIC columns contain high
`concentrations of salt which has to be removed in a consecutive separation
`step RPLC or SEC Other chromatographic
`parameters such as pH type
`of salt and temperature can be utilized in the optimization of separations
`7677
`
`414 Reversed Phase Chromatography
`
`In reversed phase chromatography proteins adsorb to a hydrophobic
`stationary phase usually made of alkyl chains The elution is accomplished
`with aqueous bufferorganic solvent based mobile phases in either isocrafic
`or gradient mode Protein adsorption to these types of hydrophobic
`surfaces can cause unfolding andor denaturation of proteins However
`apparently negative effect has certain advantages The larger complementary
`surface area of the unfolded state usually provides improved resolution In
`applications where the potential unfolding of proteins is not a limiting
`factor RPLC is frequently used The use of RPLC for analytical purposes
`has become a standard procedure for purity assessment of biotechnological
`products RPLC is widely used for peptide analysis and purification
`is carried out using RPLC for the separation of
`Proteolytic mapping
`
`this
`
`digested peptides
`
`RPLC is also frequently
`used in protein purification In many cases the
`protein of interest unfolds during the reversed phase step However when
`refolding of the protein is a fast process with excellent yield the use of
`RPLC is favored One of the major advantages of using RPLC is that
`as compared to sodium dodecyl
`an orthogonal
`
`it
`
`is
`
`purification technique
`
`sulfatepolyacrylamide
`
`gel electrophoresis or electrostaticinteraction
`
`chromatography Consequently
`
`the mechanisms of resolution are different
`
`

`

`429
`
`surface area is
`
`molecular variants
`
`An important advantage of RPLC is that
`the commonly used solvent
`systems are volatile and can therefore be easily removed prior to amino
`acid analysis Edman sequencing or mass spectrometry As mentioned
`a number of
`earlier genetically engineered proteins usually contain
`the same molecular weight Those minor
`variants with the same or almost
`often cannot be separated by other means RPLC
`analyses of those samples usually allows the side chain modified variants
`and muteins 787980 to be separated Becker
`et al showed the
`identification of sulfoxide and desamido derivative of hGH using RPLC
`79 Both products eluted prior to the intact human growth hormone
`hGH molecule which indicates that
`the complementary
`less hydrophobic due to the degradation process Similar observations have
`been made by Kunitani et al during RPLC analysis of interleukin2
`rnuteins 80 The importance of an understanding
`of
`the retention
`mechanism was illustrated through the analysis of recombinant malaria
`antigens where certain degradation products were not detectable by RPLC
`the complementary surface area 81
`because the degradation did not affect
`SDSPAGE revealed more than 20 components co eluted under a single
`RPLC peak However components generated by a degradation mechanism
`the complementary surface area were detected by the same
`
`affecting
`method
`
`constant
`
`Various alkyl and aryl chains have been thoroughly studied for their use as
`bonded phases Experiments showed that the retention as a function of n
`for both npropanol and acetonitrile
`alkyl chain length appeared
`8284 However it will be shown later the butyl C4 bonded phase has
`become the preferred stationary phase because a better recovery can be
`achieved for protein separations Comparison of columns from various
`vendors shown in Figure 9 indicates that column to column differences
`can be observed as variations in chromatographic profile resolution and
`recovery 85 The effects of the type of the silica material and its pore size
`on the separation of proteins have been also investigated 86 Lately a
`number of excellent polymerbased reversed phase stationary phases have
`been introduced Their performance is comparable with that of the silica
`based bonded phases
`
`

`

`430
`
`INS
`CYT
`
`0
`
`CHYGN
`
`BSA
`01
`
`OVALA
`
`AkAA
`AittiviA
`
`0
`
`5
`
`10
`
`15
`
`20
`
`Time minutes
`
`RIBO = ribonuclease A INS = insulin CYT = cytochrome c BSA = bovine serum
`albumin CHYGN = chymotrypsinogen OVA = ovalbumin 851
`
`Figure 9 Comparison of Retention Behavior of Standard Proteins on C8 RPLC
`Columns from Various Vendors
`
`The stability at high pH of these polymeric phases allows
`the pH range for all applications see also Chapter 3 The characteristics of
`such a phase were studied by Swadesh
`in highspeed tryptic fingerprinting
`8788 The polymer phase exhibited a lower peak capacity but shorter run
`to run re equilibration time and better cleaning ability than is usually
`observed with bonded silica phases Also at high temperature and flow
`rate peak capacity was improved
`
`the extension of
`
`characterization
`
`The application of RPLC to the area of protein and peptide separation and
`has proven to be extremely useful The substitution of a
`single amino acid residue can generate resolvable species 8990 Some of
`the amino acid side chains can be oxidized either during the fermentation
`
`

`

`431
`
`process or owing to storage conditions Oxidized methionines has been
`found in most Chinese hamster ovary derived protein products which can
`be separated by reversed phase chromatography 91 The oxidized
`methioninecontaining species elute prior to the main protein peak
`
`Other potentially resolvable species are glycosylation variants and species
`differing in the state of oxidation of the amino acid side chains 92 The
`glycosylation sites are usually ide