Prediction of Protein Retention Yn Chromatography

8/7/2019 Prediction of Protein Retention Yn Chromatography

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Journal of Chromatography A, 978 (2002) 7179

www.elsevier.com/locate/chroma

Mathematical correlations for predicting protein retention times inq

hydrophobic interaction chromatography

*M. Elena Lienqueo , Andrea Mahn, Juan A. AsenjoCentre for Biochemical Engineering and Biotechnology, Millennium Institute for Advanced Studies in Cell Biology and Biotechnology ,

Department of Chemical Engineering, University of Chile, Beauchef 861, Santiago, Chile

Received 1 May 2002; received in revised form 14 August 2002; accepted 23 August 2002

Abstract

This paper reports how a novel method for determining the surface hydrophobicity of a protein can be used to predict the

dimensionless retention time (DRT) in hydrophobic interaction chromatography. The methodology has three steps. First, it is

necessary to know the three-dimensional structure of the target protein. Then, it is necessary to calculate the surface

hydrophobicity of the protein, considering that each amino acid has a relative contribution to the surface properties, using an

appropriate equation. This will depend largely on the relative scale used to evaluate the hydrophobicity of each amino acid.

Forty-two scales were investigated and two gave the best correlation, the MiyazawaJernigan and the CowanWhittaker2

scales. Finally, a quadratic model (DRT 5 a 1 bF 1 cF ) is used to predict the retention time of the targets urf ace su rface

protein. The methodology was able to correlate adequately the retention data for monomeric proteins not included in the

generation of the model. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Mathematical modelling; Hydrophobic interaction chromatography; Hydrophobicity; Retention time prediction;

Proteins; Amino acids

1. Introduction the hydrophobic surface of a chromatographic

medium. HIC is widely used in the downstream

Hydrophobic interaction chromatography (HIC) is processing of proteins as it provides an alternative

an important technique for protein purification; it basis for selectivity compared with ion-exchange and

exploits the hydrophobic nature of the solute (pro- other modes of adsorption. Additionally, HIC is anteins). HIC is based on the reversible interaction ideal next step after precipitation with ammonium

between the hydrophobic patches on a protein and sulphate or elution in high salt during ion-exchange

chromatography (IEC) [1]. The main variables of

HIC are protein hydrophobicity, protein size [2],

concentration and type of salt [3] and type of matrix

[4].q

Presented at the 21st International Symposium on the Sepa-There are more than 40 different scales that have

ration of Proteins, Peptides and Polynucleotides, Orlando, FL,been used to estimate the hydrophobicity of amino1114 November 2001.

*Corresponding author. Fax: 156-2-699-1084. acids. There is no clear correlation or study today

0021-9673/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.

P I I : S0021-9673(02)01358-4


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72 M.E. Lienqueo et al. / J. Chromatogr. A 978 (2002) 7179

that can be used to relate any of the theoretical 2. Theory

hydrophobicity determinations and the behavior of

the proteins in a HIC column. In this paper we

present a novel method for describing the surface 2.1. Evaluation of the surface hydrophobicity of

hydrophobicity of a protein that can be used to proteinspredict the dimensionless retention time in HIC.

The surface hydrophobicity of proteins was evalu-

ated by two methodologies proposed by Berggren et

1.1. The retention mechanism of hydrophobic al. [11] to evaluate protein behavior in aqueousinteraction chromatography two-phase systems (ATPSs). The first assumes that

each amino acid on the surface of a protein has a

The retention mechanism of proteins in HIC has relative contribution to the surface properties, then:

been widely studied, but none of the proposed

theories has enjoyed general acceptance [5]. Melan- F 5O(f r ) (1)surface aai aa ider and co-workers [6,7] proposed a thermodynamic

model based on the cavity theory for the effects ofwhere F is the calculated value of the surfacesurfaceneutral salt on retention in HIC. These investigators hydrophobicity for a given protein, i (i 5 1, . . . ,20)

considered that the most important parameters for indicates the 20 different amino acids, and f is theaa iretention in HIC are the salt molality and the molal value of the hydrophobicity assigned to amino acidsurface increment of the salt. Additionally, Staby i. r , the relative surface area exposed for eachaa iand Mollerup [8] proposed a model for the solute amino acid i on the surface r , is defined as:aa iretention behavior of a protein on HIC perfusion

smedia, using both isocratic and gradient elution. In aa i]]r 5 (2)aa ithis case the capacity factor k9 is a function of the Osaa i

protein activity coefficient in the mobile phase and

the protein activity coefficient on the stationary where s is the total exposed area of the amino acidaa iphase, expressed by a simple non-linear term. Jen- residue i in the protein and os is the total

aai

nissen [9] suggested that the adsorption of a protein surface of the protein.on a hydrophobic surface is a multistep reaction. The second methodology considers residue acces-Lin et al. [10] proposed a binding mechanism in sibility on the protein surface [12]. The residueHIC based on microcalorimetric studies. They con- accessibility or degree of exposure (DE) is the totalsidered five sequential subprocesses: (1) dehydra- exposed area of each residue divided by the solvent-tion or deionization of the protein; (2) dehydration accessible surface area of the residue in a GlyX

i

or deionization of the adsorbent; (3) hydrophobic Gly peptide in extended conformation (where Xi

interaction between proteins and hydrophobic ad- represents the individual residue). Then:sorbents; (4) the structural rearrangement of the

protein; (5) rearrangement of the excluded water inF 5O(f n ) (3)Residue accessibility aai aa ithe bulk solution.

There are almost no studies on the relationship where F is the calculated value of theresidue accessibilitybetween retention of proteins in HIC and thehydrophobicity for a given protein considering thephysicochemical properties of proteins [such asdegree of exposure for each amino acid and n isaa imolecular mass, surface hydrophobicity, isoelectricthe equivalent number of amino acid residue i inpoint (pI)]. If a quantitative relationship can bethe surface protein, defined as:established, it will allow us to predict the retention of

proteins with known properties in gradient elution. Insaa ithis paper we evaluate the surface hydrophobicity of ]]]]n 5 (4)aa i saa i(GlyX Gly)proteins to predict behavior in HIC. i


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M.E. Lienqueo et al. / J. Chromatogr. A 978 (2002) 7179 73

where s or ASA is the solvent-access- USA) and analytical-reagent grade ammonium sul-aa i(GlyX Gly) ii

phate were used in the preparation of the eluent.ible surface area of the residue in a GlyXGlyi

peptide in extended conformation, where X repre-i

sents the individual amino acid [13]. 3.2. Equipment

Both methodologies were investigated for evaluat-ing the surface hydrophobicity of a protein and its The high-performance liquid chromatography sys-behavior in HIC. tem employed consisted of a fast protein liquid

chromatography (FPLC) system (Pharmacia, Upp-

sala, Sweden) equipped with a 500-ml injection loop.2.2. Amino acid hydrophobicity The chromatographic columns were 1 ml Phenyl-

Sepharose Fast Flow (a gift from Amersham Phar-There are more than 40 hydrophobicity scales macia Biotech, Uppsala, Sweden). The experiments

proposed for amino acids. For example, the Tanford were performed at room temperature, using a flow-scale [14], which is based on the amino acid free rate of 0.75 ml/min and 10 column volumes. Finally,energy of transfer from ethanol to water; the Janin retention times (t ) were recorded.R

[15] and Rose et al. [16] scales, based on the aminoacids accessible surface; the Bigelow scale [17],3.3. Buffer

based on the fraction of the number of different

amino acids buried within proteins; the Meek [18]Elution was obtained by a decreasing gradient of

and CowanWhittaker [19] scales, based on theammonium sulphate. The initial eluent was 20 mM

peptide retention times in reversed-phase HPLC (RP-BisTris pH 7.0 plus 2 M ammonium sulphate. The

HPLC), and others [21,2446]. Therefore, it wasfinal eluent was 20 mM BisTris pH 7.0 (Buffer A).

necessary to study which scale is more useful as aThe gradient used was 7.5% B/min. All buffers were

design variable in HIC.filtered through 0.22 mm Millipore filters after

This paper describes how different amino acidpreparation and degassed with helium for 10 min.

hydrophobicity scales and knowledge of the three-

dimensional structure of proteins can be used to

3.4. Sample preparationdetermine a proteins surface hydrophobicity andthus its behavior in HIC.

Protein solutions were prepared to contain approx-

imately 2.0 mg/ml dissolved in the initial eluent. All

protein solutions were filtered through 0.22 mm

Millipore filters.3. Experimental

3.5. Determination of hydrophobicity of proteins

3.1. Materials

The proteins were characterized based on their

amino acid sequences. The program Graphical Rep-Eleven proteins of known three-dimensional struc-

resentation and Analysis of Structural Propertiesture were used: conalbumin (1OVT), cytochrome c(Grasp) [20] was used to visualize protein surfaces(1HRC), ribonuclease A (1AFU), ovalbuminand to calculate the accessible surface area of single(1OVA), chymotrypsinogen A (2CHA), lysozymeresidues in a protein. The program takes as input achicken (2LYM), a-lactoalbumin (1A4V), myoglo-Protein Data Bank file (PDB, http://www.rcsb.org/bin (1YMB), a-chymotrypsin (4CHA), and b-lacto-pdb) and a probe radius value, the default value 1.4globulin (1CJ5) from Sigma (St. Louis, MO, USA).A representing a water molecule. Then, the hydro-Thaumatin (1THV) was a gift from 4F Nutrition

phobicity of each standard monomeric protein (F)(Northallerton, UK). Water prepared from a Milli-Q

was calculated using the superficial area of eachwater cleaning system (Millipore, Bedford, MA,
http://www.rcsb.org/pdbhttp://www.rcsb.org/pdbhttp://www.rcsb.org/pdbhttp://www.rcsb.org/pdbhttp://www.rcsb.org/pdbhttp://www.rcsb.org/pdbhttp://www.rcsb.org/pdbhttp://www.rcsb.org/pdb


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amino acid in the protein, and the different hydro- 4.2. Comparison of different amino acid

phobicity scales for the amino acids. hydrophobicity scales

In order to compare the amino acid scales, the3.6. Computation of the correlations and

amino acids were classified into four groups, avalidationrepresentative residue from each one being chosen:

aspartic acid (Asp) as a charged amino acid,The information obtained in the previous task, thetryptophan (Trp) as an aromatic amino acid, iso-hydrophobicity (F) and the dimensionless retentionleucine (Ile) as a long-chain aliphatic amino acid,time (DRT) of each standard monomeric protein,and glycine (Gly) as a short-chain aliphatic aminowas fitted. DRT was defined as:acid. As can be seen in Fig. 2 the various scales

t 2 tR 0 differ in residue hydrophobicities. In most of them,]]DRT 5 (5)t 2 t they are ordered in a similar position: Ile and Trp,f 0

generally, present the highest level of hydropho-where t is the time corresponding to the peak of theR bicity, Gly presents an intermediate level of hydro-chromatogram, t is the time corresponding to the0 phobicity and the lowest level was that of Asp. Then,start of the salt gradient, and t is the time corre-f the amino acid hydrophobicities could be ranked as:sponding to the end of the salt gradient.

long-chain aliphatic, aromatic.short-chainDRT is equal to 1 for an extremely hydrophobic

aliphatic.charged. This general order has beenprotein (in this case the protein ankirin, a membrane

reported previously [3,4,19].protein, which showed the maximum surface hydro-

phobicity).4.3. Computation of the correlations betweenComputation of the correlations was performeddimensionless retention time (DRT) and proteinusing CurvExpert 1.3 Software, evaluating over 30hydrophobicity (F)different mathematical models. Finally, the best

amino acid hydrophobicity scale and the best method4.3.1. Selection scales and definition of proteinfor evaluating the surface hydrophobicity to predicthydrophobicity

each protein retention time in HIC were determined. Forty-two hydrophobicity scales were used for

computing the protein hydrophobicities using the two

methods described by Eqs. (1) and (3) to evaluate4. Results and discussion

the surface hydrophobicity.

In addition, we defined a new methodology to4.1. Classification of amino acid hydrophobicity evaluate the surface hydrophobicity based on Eq.scales (3). We defined a relative number for each amino

acid i on the surface, m , which is defined as:aa iThe amino acid hydrophobicity scales were classi-

[s /s ]fied into three groups based on the following princi- naa i aa i(GlyX Gly)i aa i

]]]]]]] ]]m 5 5 (6)aa iples: (a) direct scale based on amino acid properties

O[s /s ]

Onaa i aa i(GlyX Gly ) aai

i

such as Gibbs free energy of transfer, retention timein HPLC, polarity or aqueous two-phase partitioning This will give a relative hydrophobicity valuecoefficient; (b) indirect scales based on protein normalized according to the total protein surface,properties such as antigenic regions in proteins, similar to Eq. (1).accessible and buried surfaces in proteins or contact Then, the surface hydrophobicity is defined as:energy; and (c) mixed scales, scales based on direct

F 5O(f m ) (7)and indirect scales. relative number aai aa iAll scales were classified: 59% were direct scales,

The retention times of nine monomeric proteins28% were indirect scales and 13% were mixed(conalbumin, cytochrome c, ribonuclease A, chymo-scales. The classifications are shown in Fig. 1.


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Fig. 1. Classification of amino acid hydrophobicity scales.

Fig. 2. Comparison of amino acid hydrophobicity using different hydrophobicity scales.


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trypsinogen A, chicken lysozyme, a-lactoalbumin,

myoglobin, a-chymotrypsin, thaumatin) were

studied. Dimensionless retention times (DRT) and

surface hydrophobicities of proteins (F) were corre-

lated with a linear relation (DRT 5AF1B). The2correlation coefficients (r ) were, for Eq. (1), be-

tween 0.88 and 0.01, for Eq. (3), between 0.08 and

0.01, and for Eq. (7), between 0.87 and 0.01. Table 1

shows that the best average correlation was that

obtained using Eq. (1), followed by that obtained

from Eq. (7). Eq. (3) gave an extremely poor2

correlation coefficient (r , 0.08).

The poor result obtained using Eq. (3) is analysed

in Fig. 3, where we compare the surface hydro-

phobicity of 12 proteins using Eqs. (1) and (3). This

same correlation [Eq. (3)] gave good results whennormalized over the total possible hydrophobicity forFig. 3. Comparison of the surface hydrophobicity using Eqs. (1)

each protein [Eq. (7)], as shown in Table 1. and (3).In addition, Table 1 shows that the four best scales

nigan, CowanWhittaker, Brown and Wilson scales)were the MiyazawaJernigan, CowanWhittaker,were used to compute the protein hydrophobicitiesBrown and Wilson scales. These four scales wereusing the methodology given by Eq. (1). More thanchosen to evaluate other mathematical models to fit30 different mathematical models were investigated,the experimental data.such as quadratic, polynomial, heat capacity and

sinusoidal models.4.3.2. Best correlationThe best correlations found are shown in Figs. 4The four hydrophobicity scales (MiyazawaJer-

Table 12 a

Correlation coefficients (r ) of the four best scales for predicting the dimensionless retention time (DRT) with a linear relation

Scale Methodology to calculate surface hydrophobicity of proteins

b c dSurface Residue accessibility Relative number

F5 o(f r ) F5 o(f n ) F5 o(f m )aa i aa i aa i aa i aa i aa i

MiyazawaJernigan [44] 0.88 0.083 0.87

CowanWhittaker [19] 0.88 0.038 0.88

Browne et al. [23] 0.83 0.058 0.78

Wilson et al. [22] 0.83 0.051 0.85

Average 0.855 0.058 0.845

a

Linear relation: DRT 5AF1B.b Eq. (1):

saa i]]r 5 .

aa i Osaa ic

Eq. (3):

saa i

]]]n 5 .aa i s

aa i( G l y X G l y )i

dEq. (7):

[s /s ] naa i aa i( G l y X G l y )i aa i]]]]] ]]m 5 5 .

aa i O[s /s ] Onaa i aa i( G l y X Gly ) aaii


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and 5. They correspond to a quadratic model. An

excellent correlation between the protein hydropho-

bicities, calculated based on the CowanWhittaker

scale or the MiyazawaJernigan scale, and retention

time of monomeric proteins was obtained. TheBrown and Wilson scales also gave good correla-

tions. The models were:

(i) CowanWhittaker scale

2 2DRT 5 2 18.22F 1 22.07F 2 5.56,Cowan Cowan

0 ,F , 1 (8)Cowan

Model deviation 6.3%;

(ii) MiyazawaJernigan scale

2DRT 5 2 12.14F 1 12.7F 2 1.74,Miyazawa Miyazawa

0 ,F , 1 (9)Miyazawa

Model deviation 7.3%.

Both scales, the CowanWhittaker and

Fig. 5. Relation between dimensionless retention time (DRT) on

Phenyl-Sepharose2 M ammonium sulphate and the protein

hydrophobicity (F) based on the MiyazawaJernigan hydropho-

bicity scale. (s) Experimental data, () model, (d) protein

used for model validation.

MiyazawaJernigan scales, have very good correla-2

tion coefficients (r ) of 0.96 and 0.94, respectively.

Therefore, we suggest that both scales can be used

for predicting the protein dimensionless residence

time in HIC. The Brown and Wilson scales had

correlation coefficients of 0.89 and 0.92, respective-

ly.

The CowanWhittaker scale, shown in Table 2, is

a direct scale that was obtained using hydrophobicity

indices determined by the amino acid retention time

in RP-HPLC at pH 3.0 [19].The MiyazawaJernigan scale, shown in Table 2,

is an indirect scale that corresponds to the ability of

the amino acids to be hidden in the proteins core

and it was obtained using the contact energy derived

from three-dimensional protein data [44].Fig. 4. Relation between dimensionless retention time (DRT) on The surface hydrophobicity was evaluated usingPhenyl-Sepharose2 M ammonium sulphate and the protein

Eq. (1), which considers that each amino acid on thehydrophobicity (F) based on the CowanWhittaker hydropho-

surface of a protein has a relative contribution to thebicity scale. (s) Experimental data, () model, (d) proteinused for model validation. surface properties.


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Table 2

Best amino acid hydrophobicity scales used for predicting the dimensionless retention time in HIC

Amino Scale

acidCowanWhittaker, pH 3.0 MiyazawaJernigen

Original [19] Normalized Original [44] Normalized

Ala 0.42 0.660 5.33 0.391

Arg 21.56 0.176 4.18 0.202

Asn 21.03 0.306 3.71 0.125

Asp 20.51 0.433 3.56 0.105

Cys 0.84 0.763 7.93 0.819

Gln 20.96 0.323 3.87 0.151

Glu 20.37 0.467 3.65 0.115

Gly 0.00 0.557 4.48 0.252

His 22.28 0.000 5.10 0.354

Ile 1.81 1.000 8.83 0.967

Leu 1.80 0.998 8.47 0.908

Lys 22.03 0.061 2.95 0.000

Met 1.18 0.846 8.95 0.987

Phe 1.74 0.983 9.03 1.000

Pro 0.86 0.768 3.87 0.151

Ser 20.64 0.401 4.09 0.188

Thr 20.26 0.494 4.49 0.253

Trp 1.46 0.914 7.66 0.775

Tyr 0.51 0.682 5.89 0.484

Val 1.34 0.885 7.63 0.770

4.3.3. Validation tested. As shown in Table 3, the MiyazawaJernigan

To validate the previous correlations [Eqs. (8) and prediction (average error 5%) was better than the

(9)], the retention times of two monomeric standard CowanWhittaker prediction (average error 15%) for

proteins (ovalbumin and a-amylase) were estimated. the two proteins tested.

Figs. 4 and 5 show that the CowanWhittaker and

MiyazawaJernigan correlations are valid for mono-

meric proteins, with a global average error of less 5. Conclusion

than 10% (see Table 3). Thus, we consider that the

correlations are adequate for the monomeric proteins The methodology described in this paper is a first

Table 3

Comparison between estimated and experimental dimensionless retention time in Phenyl-Sepharose2 M ammonium sulphate for

monomeric proteins

a bScale Protein DRT DRT Error Average Global

experimental predicted (%) error averagec

(%) error (%)

aCowan a-Amylase 0.749 0.774 3.4 15.2

Ovalbumin 0.567 0.720 27.0 9.9

dMiyazawa a-Amylase 0.749 0.681 9.2 4.7

Ovalbumin 0.567 0.566 0.09

a 2DRT 5 2 18.22F 1 22.07F 2 5.56.

Cow an Cowanb

Error 5 uDRT 2 DRT u ? 100/DRT .experimental predicted experimental

cTotal average error: average error considering both proteins and both scales.

d 2DRT 5 2 12.14F 1 12.7F 2 1.74.

Miyazawa Miyazawa


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