IntroductionTherapeutic biomolecules often must be greater than99% pure prior to human use. This degree of purifica-tion can be achieved through use of three or four liquidchromatography processes such as ion exchange, reversedphase, size exclusion, affinity (eg. dye, metal, antibody,protein A), and hydrophobic interaction (HIC). Whilethe type and sequence of chromatographic processeschosen are based on physicochemical characteristics ofboth the target biomolecule and the contaminantspresent, the final step is usually size exclusion chro-matography because it removes protein aggregates andalso exchanges the purified protein into the final formu-lation buffer.

Size exclusion chromatography negatively affectsproductivity because of the limited sample volume that can be loaded. Column load volume greater thanabout 5% of the overall column volume results in diffu-sion-related band spreading and dilution as the soluteband moves through the column. The volume limita-tion can be circumvented through the use of an ultrafiltration concentration step prior to columnloading. Ultrafiltration concentration also introducesproductivity losses due to: nonspecific binding of theprotein to the membrane and other materials in the system; volume losses to tubing and pumps; and complications associated with equipment prepara-tion, operation, and cleaning. The purification processwould clearly be simpler if protein could be directlyeluted from the column immediately in front of the

size exclusion chromatography column in a concen-trated form.

GoalThe purpose of this work was to demonstrate that the displacement effect of liquid chromatography couldbe applied to an HIC system in order to collect aconcentrated target protein upon elution. The possi-bility of future scale-up was considered in the experi-mental design. A chromatographic gel of mediumparticle diameter having fairly low pressure rating was utilized in an isocratic elution process. These choices would simplify a manufacturing operation andlower its cost. The displacement-effect-promotingmolecule phenylalanine of molecular weight 165.15 was chosen because it is nontoxic and two orders ofmagnitude smaller than the target protein moleculelysozyme of molecular weight 14,600. The sizedifference allows for effective separation by sizeexclusion chromatography in a later step. Since thecommon philosophy for purifying biomolecules requires the removal of as many contaminants aspossible as soon as possible, it was reasonably assumedthat the target protein in this study (lysozyme) had been sufficiently purified prior to the HIC step suchthat an HIC elution window existed between it and the remaining bound contaminants. Elution windowdetermination could be performed by HIC “scouting”runs which would isolate buffer conditions that initiallyremoved the weaker binding contaminants withouteluting the target. The target protein could later be

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The displacement effect ofphenylalanine on lysozyme elution in hydrophobic interaction chromatographyR.J. Klimchak and S. Wang*Department of Chemical and Biochemical Engineering, Reuters University, New Brunswick, New Jersey,08855–1360 USA

The volume, retention time, and shape of the lysozyme peak eluted from a hydrophobic interaction chromatographycolumn (TosoHaas 650 M Phenyl) was influenced by the presence and concentration of phenylalanine in the elutionbuffer. Lysozyme peak retention time decreased by a factor of 2.5 with the addition of 86 mM phenylalanine to theelution buffer.

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eluted while the stronger binding contaminants wereretained on the column.

Materials and methodsChemicalsLysozyme, phenylalanine, (NH4)2SO4, acetone, mono-basic sodium phosphate (NaH2PO4), dibasic sodiumphosphate (Na2HPO4), deionized H2O.

EquipmentMillex-HV 0.45m syringe filters from MilliporeCorporation; 0.45m nylon filters; Pharmacia HR 5/10column; TosoHaas TSK Phenyl 650 M HIC media;HPLC system with UV/VIS detector.

Solution preparation2 M (NH4)2SO4/100 mM NaH2PO4Na2HPO4 (pH 7.0) load buffer and 1.4 M (NH4)2SO4/10 mMNaH2PO4Na2HPO4 (pH 7.0) elution buffer wereprepared and filtered through 0.45m nylon filters.Lysozyme was dissolved in load buffer and filteredthrough a 0.45m Millex low protein binding filter. Phenylalanine was dissolved in elution buffer and filtered through a 0.45m Millex filter. Lysozymeconcentration was verified by absorbance at A280 andphenylalanine concentration was verified by absorbanceat A257.

Column preparation and runningThe 0.5 cm internal diameter column was packed to abed height of 11 cm using a slurry of HIC media indeionized H2O pumped at 0.96 MPa. At the start ofeach run the column was equilibrated with load bufferfollowed by the loading of 10 mL of 1.38 mg/mLlysozyme at a flow rate of 0.5 mL/min. 1.4 M(NH4)2SO4/100 mM NaH2PO4Na2HPO4 (pH 7.0)containing the specified concentration of phenylalanineeluted the column at 0.5 mL/min. The elution concen-trations of lysozyme and phenylalanine were monitoredat A280 and A257. It had previously been determinedthat phenylalanine in the elution buffer did not appre-ciably interfere with the A280 absorption readings of lysozyme. Also, the differences in A280 absorptionreadings associated with changes in (NH4)2SO4 concen-tration were minimal – 0.041 absorption units over therange of 2 M (NH4)2SO4/100 mM NaH2PO4Na2HPO4

to 0 M (NH4)2SO4/100 mM NaH2PO4Na2HPO4.

The displacement effectThe displacement effect occurs when the movement ofone solute population through the column distorts the movement of a second solute population (Colin,

1991). The basis for the displacement effect is that thevelocity at which a particular solute population travelsthrough a chromatographic column is directly relatedto its concentration (Tiselius, 1943). A concentratedpopulation of strongly binding solute moves throughthe column faster than a dilute population of the samesolute because excess solute molecules are forced to movedown the column as available stationary phase bindingsites become saturated. When the concentration ofsolute molecules in the mobile phase is great enoughto saturate available stationary phase binding sites, thecolumn is operating in the non-linear region of theadsorption isotherm. This is very different from theoperation of an analytical column which is typically usedin the linear region of the adsorption isotherm. In thatcase, available binding sites outnumber mobile phasesolute molecules so that solute velocity is not a strongfunction of solute concentration.

When a binary mixture is separated on a column, thesolute population having lower affinity moves throughthe column more quickly. However, if the higheraffinity population is at a large enough concentration toincrease its velocity, its leading edge may move fastenough to overtake the rear boundary of the precedingweaker binding population. The ensuing competitionfor stationary phase binding sites results in the highaffinity population displacing the weaker binding mole-cules and forcing them further down the column to jointhe bulk of the other low affinity molecules. The resultis that the weaker binding solute elutes from the columnmore concentrated than it would have without thedisplacement effect, and so achieves a degree of com-pression. The compression observed in that peak’schromatogram and, therefore, the degree of increase ineluate concentration, is measureable. We suggest thatcompression (C) may be quantified by defining it as theratio of peak height (PH in absorbance units) to thepeak width measured at 10% of peak height (P10%Win units of volume). Then the relative compression(CREL) may be defined as the ratio between somecompressed peak (Cn) and the standard peak (C0).Therefore, CREL = Cn/C0, and the compressibility of the various peaks in an experimental series can benormalized and compared.

Hydrophobic interaction chromatographyHydrophobic Interaction Chromatography (HIC) is apurification method in which solute molecules are sepa-rated based on differences in surface hydrophobicity.The retention of solute molecules on the chromato-graphic stationary phase is affected by the hydropho-bicity and coverage density of ligands on the solid phase,

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the salt type and molarity, the temperature, and the pHof system buffer. Horvath et al. (1976) explained themechanism of HIC in terms of surface tension. Geng et al. (1990) presented a theoretical description of theHIC process as a stoichiometric displacement of thebound protein molecule from the solid phase by watermolecules. Another explanation focuses on the decreasein system entropy as kosmotropic salt (water structureformers such as ammonium sulfate) is added to water.When protein binds to the hydrophobic ligand a ther-modynamically favorable decrease in free energy occurs,resulting in a negative change of Gibbs free energy (DG < 0) (Fausnaugh et al., 1984). This occurs as system entropy increases (then DS > 0) due both tosolute leaving the bulk phase and water molecules,released from the binding sites, “diluting” the bulkwater (DG = DH – TDS).

The use of HIC as a purification method in the biotech-nology area has increased because it tends to be non-denaturing to biomolecules. A negative characteristic ofHIC is the presence of a long tailing peak indicative ofa large elution volume. These broad peaks are thoughtto be caused by solid phase adsorption site hetero-geneity, conformational variations in protein tertiarystructure, protein aggregation, and relatively moreadsorption/desorption steps during elution, compared toother chromatography modes (Chicz and Regnier,1990).

Chromatographers have added various chemicals to HICelution buffers in attempts to alter column selectivityor to change the retention of proteins. The additiveseither alter the surface tension of the solution or thehydrophobic portion of the additive competes for ligandbinding sites. Unwanted protein aggregation and/ordenaturation frequently occurs with isopropanol,ethanol, urea, and guanidine additives. The utilizationof detergent additives is difficult in that they are in-effective beyond the Critical Micelle Concentration(CMC), but bind to the protein and ligand below theCMC. The additive that has been the most successfulis ethylene glycol, which is used to remove veryhydrophobic proteins from the chromatographic support(Pharmacia, 1993). The use of sugars and glycine hasbeen shown not to effect selectivity, but to only shiftproteins to an increase in retention by increasing surfacetension of the solution (Gagnon and Grund, 1996).

Results and discussionFigure 1 displays a comparison of four experiments in which the same mass of lysozyme was loaded onto the HIC column in 2 M (NH4)2SO4/100 mM

NaH2PO4Na2HPO4. Elution of lysozyme was performedwith 1.4 M (NH4)2SO4/100 mM NaH2PO4Na2HPO4

containing various concentrations of phenylalanine.Phenylalanine was absent in the elution buffer in theexperiment that had the most tailing. The experimentin which the elution buffer contained 25 mM phenyl-alanine resulted in a peak that exhibited less tailing.The chromatogram from the experiment with 50 mMphenylalanine in the elution buffer had even less tailing.Finally, with 86 mM of phenylalanine in 1.4 M(NH4)2SO4/100 mM NaH2PO4Na2HPO4 (its approxi-mate solubility limit in this buffer), the sharpest peakand least tailing was obtained.

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Figure 1 Chromatograms of lysozyme eluted from phenylHIC column using various concentrations of phenylalaninein the elution buffer. Plots indicate absorption at A280 versustime.

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Figure 2 displays chromatograms from four experi-ments in which the same mass of lysozyme in 2 M(NH4)2SO4/100 mM NaH2PO4Na2HPO4 was loadedonto an HIC column. The overall wider peaks seen in the second experimental series are a function of a less efficient column – the column had been repackedafter the first experimental series. Elution of lysozyme

was performed with 1.4 M (NH4)2SO4/100 mMNaH2PO4Na2HPO4 containing various concentrationsof phenylalanine or, in the case of one experiment, 1.3 M (NH4)2SO4/100 mM NaH2PO4Na2HPO4 and no phenylalanine. As in the first series above, the elution buffer with 1.4 M (NH4)2SO4/100 mMNaH2PO4Na2HPO4 and no phenylalanine showed the most tailing. The experiment utilizing 1.3 M(NH4)2SO4/100 mM NaH2PO4Na2HPO4 and no phenyl-alanine showed the second most tailing. This result was expected due to the decreased salt in this elutionbuffer. The last two experiments followed the resultsobtained in the first experimental series – the elutionbuffer with the highest concentration of phenylalanine(82 mM) displayed the least tailing. An important point to note is that even though this elution buffer(82 mM phenylalanine) and the buffer utilizing 1.3 M(NH4)2SO4/100 mM NaH2PO4Na2HPO4 and no pheny-lalanine had similar conductivities (160–161 mS), theeluted peaks associated with them were very different.This implies that the decrease in tailing observed withincreases in phenylalanine concentration is not simplya salt affect.

Another experimental observation was that lysozymepeak retention time decreased with increasing phenyl-alanine concentration. Increasing the elution bufferconcentration of phenylalanine from 0 mM to 86 mMdecreased lysozyme peak retention time 2.5 fold.

Table 1 compares maximum peak height, elutionvolume at 10% peak height, and peak compression inthese sets of experiments. Relative compression increasesand seems to be directly related to the concentration ofphenylalanine in the elution buffer. In the current exper-imental design, the results suggest that the utilizationof phenylalanine in the HIC elution buffer can enablepeak compression by almost one order of magnitude.

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Figure 2 Chromatograms of lysozyme eluted from phenylHIC column using different concentrations of either phenyl-alanine or (NH4)2SO4in the elution buffer. Absorption at A280plotted against time.

Table 1 Peak compression increases with phenylalanine concentration in elution buffer

Phenylalanine Peak Peak10% Relativeconcentration in height width Compression compression1.4 M (NH4)2SO4 elution buffer (A280 units) (10% height; mL) C = PH/P10%W) (CREL = Cn/C0)

Experimental series I:0 mM 2.7 30.22 0.09 1.0025 mM 4.42 24.41 0.18 2.0350 mM 6.67 13.56 0.49 5.5186 mM 8.79 10.14 0.87 9.70Experimental series II:0 mM 1.66 44.54 0.04 1.000 mM, 1.3 M (NH4)2(SO4 2.74 25.85 0.11 2.6550 mM 3.32 29.16 0.11 2.8582 mM 5.36 16.21 0.33 8.25

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Compression is a complex function that is affected bychemical and physical properties of the system. Protein,additive, buffer salt, pH, water, and solid phase ligandinteract chemically. Physical properties such as bufferflow rate (v), surface tension (s), viscosity (m), temper-ature (T), chromatographic gel size (dp) and porosity (p)affect the chemical reactions.

C = fnc(solutes, pH, v, m, s, T, dp, p)

The result of these experiments suggest that thedisplacement effect can be used to sharpen peaks anddecrease both processing time and elution volume inHIC.

ReferencesChicz, R. and Regnier, F. (1990). In: Methods in Enzymology: Guide

to Protein Purification, M. Deutscher, ed., vol. 182, p. 411,New York: Academic Press, Inc.

Colin, H. (1991) Industrial Chromatography News 3, 29.Fausnaugh, J.L., Kennedy, L.A., and Regnier, F.E. (1984)

J. Chromatog. 317, 141–155.Gagnon, P. and Grund, E. (1996). BioPharm, 9, 5, 59–61.Geng, X., Guo, L., and Chang, J. (1990) J. Chromatog. 507, 1–23.Horvath, C., Melander, W., and Molnar, I. (1976) J. Chromatog.

125, 129–156.Pharmacia (1993). Hydrophobic Interaction Chromatography,

Uppsala, Sweden: Pharmacia BioProcess Technology AB.Tiselius, A. (1943) Ark. Kemi., Mineral. Geol. 16(A), 1.

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Received 24 March 1997; Revisions requested 7 April 1997;

Revisions received 6 May 1997; Accepted 6 May 1997

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