Effects of Tween 20 and Tween 80 on the Stability

Effects of Tween 201 and Tween 801 on the Stabilityof Albutropin During Agitation

DANNY K. CHOU,1 RAJESH KRISHNAMURTHY,3 THEODORE W. RANDOLPH,2 JOHN F. CARPENTER,1

MARK CORNELL MANNING1

1Department of Pharmaceutical Sciences, School of Pharmacy, Center for Pharmaceutical Biotechnology,University of Colorado Health Sciences Center, Denver, Colorado 80262

2Department of Chemical Engineering, Center for Pharmaceutical Biotechnology, ECCH 111, Campus Box 424,University of Colorado, Boulder, Colorado 80309

3Human Genome Sciences, Rockville, Maryland 20850

Received 2 November 2004; revised 2 March 2005; accepted 13 March 2005

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20365

ABSTRACT: The objectives of this work were to determine the effects of nonionicsurfactants (Tween 201 and Tween 801) on agitation-induced aggregation of therecombinant fusion protein, AlbutropinTM (human growth hormone genetically fused tohuman albumin), and to characterize the binding interactions between the surfactantsand the protein. Knowing the binding stoichiometry would allow a rational choice ofsurfactant concentration to protect the protein from surface-induced aggregation.Fluorescence spectroscopy and isothermal titration calorimetry (ITC) were employed tostudy Albutropin surfactant binding. Albutropin was agitated at 25� 28C to induceaggregation, and samples were taken during a 96-h incubation. Size-exclusion chroma-tography (SEC-HPLC) (HPLC, high-performance liquid chromatography) was used todetect and quantify the extent of protein aggregation. The effect of surfactants on theprotein’s free energy of unfolding was determined using guanidine HCl as a denaturant.Tween 20 and Tween 80 had saturable binding to Albutropin with a molar bindingstoichiometry of 10:1and9:1 (surfactant:protein), respectively. Binding of thesurfactantsto Albutropin increased the free energy of unfolding by over 1 and 0.6 kcal/mol,respectively. In protein samples that were agitated in the absence of surfactant, solubleaggregates were detected within 24 h, and there was almost complete loss of monomer tosoluble aggregates by the end of the 96-h experiment. At the molar binding stoichiometry,Tween 20 and Tween 80 prevented the formation of soluble aggregates, even though theconcentrations of surfactants were well below their critical micelle concentrations(CMC). Tween 20 and Tween 80 protected Albutropin against agitation-induced aggre-gation, even at concentrations below the CMC. Equilibrium unfolding data indicate thatTween confer protection by increasing the free energy of unfolding of Albutropin. � 2005

Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1368–1381, 2005

Keywords: protein aggregation; Tween 20; Tween 80; nonionic surfactant; fluore-scence; isothermal titration calorimetry

INTRODUCTION

Because most pharmaceutical proteins are sur-face-active and only marginally stable, interac-tions with interfaces (e.g., air-water and solid-water interfaces) often lead to loss of nativestructure and aggregation.1 Aggregation cancompromise biological activity and/or induce an

1368 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005

Mark Cornell Manning’s present address is Legacy BioDe-sign LLC, Loveland, CO 80538.

Correspondence to: Mark Cornell Manning (Telephone: 970-231-9744; Fax: 970-663-6006.;E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 94, 1368–1381 (2005)� 2005 Wiley-Liss, Inc. and the American Pharmacists Association

immunological reaction in the patient.2,3 Thus,the formulation must be designed to protect theprotein against surface-induced damage. Surfac-tants, particularly nonionic types, are often addedto prevent and/or minimize protein aggregationduring fermentation, purification, freeze-drying,shipping, and/or storage.4

There are several known mechanisms for pro-tein stabilization by surfactants. First, nonionicsurfactants can protect proteins against surface-induced damage by competing with proteins foradsorption sites on surfaces.4 In addition, as hasbeen demonstrated with human growth hormone(hGH), nonionic surfactants can protect proteinagainst surface-induced aggregation by binding tohydrophobic regions of the surface of the proteinmolecule, and thus decrease intermolecular in-teractions.5,6 In these cases, the concentration ofsurfactant required to protect the protein maxi-mally from aggregation does not correlate with thecritical micelle concentration (CMC) of the sur-factant. Rather, the degree of protection can bemaximized at the molar binding stoichiometrybetween the surfactant and protein.5,6 Binding ofsurfactant to the native state of the protein canalso minimize surface-induced aggregation byincreasing the free energy of protein unfolding,as has also been found with hGH.7 Finally, nonio-nic surfactants may act as a chemical chaperone,favoring refolding over aggregation by bindingtransiently with partially folded protein moleculesand sterically hindering intermolecular interac-tions that result in aggregation.8,9

Currently, the most common strategy employedfor stabilizing protein pharmaceuticals againstsurface-induced aggregation is to include in theformulation a sufficient concentration of surfac-tant to minimize protein adsorption to interfaces.Typically, the final surfactant concentration is justabove the CMC, where there is a monolayer ofsurfactant molecules oriented about the interface(e.g., the air-water interface).10–12 Although thisapproach can minimize the interaction of theprotein with denaturing interfaces (air-water,solid-water interfaces, etc.), it does not take intoaccount the binding between the surfactant andthe protein. For example, studies have shown thata protein can change the CMC of a surfactant bybinding to surfactant molecules.5,13 Because onegoal of formulation development is to utilize thelowest effective concentration of excipient, a sys-tematic approach to surfactant formulation de-velopment is needed. Through such an approach,potential surfactant binding to the protein as well

as the competition between protein and surfactantfor surfaces can be evaluated.

AlbutropinTM is a novel form of hGH, geneti-cally fused to human serum albumin (HSA). Al-butropin is expressed and purified to yield a singlepolypeptide with a substantially longer half-life invivo than that for hGH alone.14 Human serumalbumin is the most prevalent naturally occurringprotein in the human circulatory system, persist-ing in circulation in the body for over 20 days.15

Albumin fusion proteins may thus provide pa-tients with long-acting treatment options that mayoffer a more convenient dosing regimen, withsimilar or improved efficacy and safety. To date,there has not been published work on agitation-induced aggregation of albumin fusion proteins,nor on the effects of nonionic surfactants on thisprocess. Furthermore, although previous studiesinvolving surfactant formulation of hGH de-monstrated the importance of a specific protein-surfactant stoichiometry, the concentration ofsurfactants utilized were well above their CMC.6

Thus, another goal of this study is to determinewhether sub-CMC concentration of a surfactant issufficient to protect a protein against agitation-induced aggregation.

HSA is a major transporter of fatty acids in theblood stream, and the affinity of HSA for bothmedium and long chain fatty acids is high (106–107 M�1).16,17 In fact, the organic fatty acid,octanoic acid, is commonly added to the proteinto protect it from denaturation during heat treat-ment of commercial HSA products.16 Given thisbehavior and the known binding of surfactants tohGH, we hypothesize that nonionic surfactantswill bind to Albutropin as well. Using the informa-tion gathered from surfactant binding studies, thehypothesis that a sub-CMC amount of Tween 201

and Tween 801 is sufficient to protect Albutropinmaximally against agitation-induced aggregationwas tested. The requisite concentration of surfac-tant should correspond to or exceed the bindingstoichiometry between the surfactant and theprotein. In addition, studies were conducted totest the effects of the surfactants on the protein’sfree energy of unfolding.

MATERIALS AND METHODS

Protein and Reagents

Purified Albutropin (88.6 kD), expressed in yeast(Saccharomyces cerevisiae) was supplied byHuman Genome Sciences (Rockville, Maryland)

EFFECTS OF NONIONIC SURFACTANTS 1369

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005

at pH 7.2 and was stored at �808C until needed.After thawing and dialysis against 10 mMsodium phosphate buffer, size-exclusion high-performance liquid chromatography (SE-HPLC)analysis documented that the protein samplecontained approximately 99% monomer and 1%dimer. Sodium phosphate monobasic, sodiumphosphate dibasic, potassium phosphate mono-basic, guanidine hydrochloride (GdnHCl), poly-oxyethylene sorbitan monolaurate (Tween 20),and polyoxyethylene sorbitan monooleate (Tween80) (Sigma Ultra, low peroxide) were purchasedfrom Sigma Chemical Company. All protein andexcipient solutions were prepared using distil-led deionized water. Buffer solutions were filteredthrough 0.22-mm nylon filters.

Fluorescence Titration Studies

The protein concentration was determined on aHP model 8452 spectrophotometer using anextinction coefficient (e280) of 0.6 mL �mg�1 � cm�1

1 for Albutropin (data from HGSI). The e280 were0.81 and 0.58 mL �mg�1 � cm�1 for hGH and HSA,respectively.16,43 Measurements of steady-statefluorescence were performed on a 0.05 mg/mL pro-tein solution (258C) in sodium phosphate buffer(10 mM at pH 7.2), with excitation wavelength at295 nm in an Aviv ATF 105 automatic titratingspectrofluorimeter (Lakewood, NJ). Excitationbandwidth was set at 0.15 nm and emission band-width was set at 8 nm. For titration of the proteinwith surfactant, each injection of surfactant solu-tion was 2.6 mL in volume and the sample waskept at a constant volume of 2 mL throughout theexperiment and stirred with a magnetic stir bar.After each injection, the sample was allowed toequilibrate for 1 min prior to spectral acquisition.The fluorescence intensity of the protein wasmonitored at the wavelength of maximum emis-sion for each protein (340 nm for Albutropin,339 nm for rhGH, and 345 nm for HSA). All fluor-escence experiments were run in duplicate,intensity was corrected for dilution, and averagevalues are reported. Data were imported intoMicrosoft ExcelTM software for further proces-sing, fluorescence quenching data were analyzedwith the Stern–Volmer equation, and the effec-tive quenching constant was derived from theslope of the binding isotherm.

Isothermal Titration Calorimetry (ITC)

Microcalorimetry experiments were performed ona Microcal, Inc. Omega titration calorimeter. Both

the ligand and protein solutions were filteredthrough a 0.22-mm filter then degassed for at least15 min using a magnetically stirred vacuum ap-paratus. The injection syringe was filled with astock solution containing Tween 20 or Tween 80at 10 mM. Twenty five injections of 10 mL eachwere made into a 1.3 mL calorimeter cell, whichcontained either 10 mM phosphate buffer pH 7.2or the same buffer containing 16 mg/mL of Al-butropin. The cell was stirred at 375 rpm and wasthermally equilibrated at 258C for 30–40 minuntil a flat baseline was obtained, then injectionswere initiated. Analysis of protein samples aftercompletion of the titration by SEC documentedthat the protein has not aggregated (data notshown). The time between injections was set to240 s. Control experiments were conducted bytitrating Tween into buffer solution and the re-sulting heat of dilution was subtracted from theactual binding isotherm prior to data analysis.The resulting data were integrated using Originsoftware from Microcal1 to arrive at the enthalpygenerated for each Tween injection.

Operative CMC Determination UsingSurface Tensiometry

The effective CMC of both Tween 20 and Tween80 solutions containing protein was determinedusing a manually operated Fisher surface tensi-ometer. This instrument employs the du Nouyring method which utilizes a platinum-iridiumtest ring and two stainless-steel torsion wires.The manufacturer’s manual defines the apparentsurface tension as ‘‘the force necessary to separateplatinum-iridium ring from a liquid, and in thiscase it is measured and read directly to�0.25 dyn/cm’’.18 Direct results were obtained with solutionsthat contained increasing concentrations of sur-factant, and the breakpoint in the surfactantconcentration versus surface tension curve isthe apparent critical concentration of surfactantnecessary to achieve the minimum surface ten-sion, and thus corresponds to the effective CMC ofthe surfactant.19

SE-HPLC

SE-HPLC was utilized to quantify levels of mono-meric protein and soluble aggregates. A TosohaasTSK 3000SWxL gel filtration column connected toBeckman Gold chromatography system (Fuller-ton, CA) was used. The mobile phase was 104 mMsodium phosphate, 104 mM potassium phosphate,

1370 CHOU ET AL.


and 100 mM sodium sulfate (pH 6.7), and the flowrate was 1.0 mL/min. At neutral pH, Albutropinexists as a monomer with a molecular mass of88.6 kDa. The amounts of native monomer, non-native dimer, and larger soluble aggregates werequantified using UV absorbance at a detectionwavelength of 280 nm. The samples, chromato-graphy system, column, and buffer were all main-tained at 258C throughout the course of theexperiment. Albutropin that had not been agi-tated was used as a control. Data collected wereimported into Grams software program, and peakareas were analyzed using Beckman’s chromato-gram integrator software.

Equilibrium Unfolding Experiments

Albutropin stock solution was dialyzed against10 mM sodium phosphate buffer at pH 7.2 withPierce Slide-A-Lyzer dialysis cassette prior touse. Guanidine hydrochloride (GdnHCl)-inducedunfolding of Albutropin at 0.1 mg/mL was con-ducted with and without Tween 20 or Tween 80.Stock solutions of 8M GdnHCl in buffer, with andwithout Tween 20 or Tween 80 at concentrationsthat correspond to stoichiometric molar ratio of

surfactant:protein, were prepared as described byPace et al.20 Appropriate amounts of Tween 20and Tween 80 solutions were added to the bufferand protein solutions and then combined withGdnHCl stock solutions to prepare protein sam-ples with various GdnHCl concentrations. A totalof 30 samples with various concentrations ofGdnHCl, ranging from 0M to 6.4M were preparedand equilibrated overnight at 48C prior to datacollection. All samples were performed in tripli-cate and the results are the average of threeseparate experiments. Denaturant induced un-folding of Albutropin in 10 mM sodium phosphate(pH 7.2) was followed using an Aviv circularDichroism (CD) spectrometer (model 62 DS). A1-mm pathlength sample cell was used for all CDspectroscopic measurements. Averages (60 s) offar UV CD signals at 222 nm were collected.Samples without Albutropin were used as blanksto correct for non-Albutropin contributions to farUV CD signal. The unfolding curves were ana-

lyzed using the method devised by Hung andChang and it is briefly described below.21 In athree-state unfolding transition, the equilibriumbetween the states can be described by the fol-lowing equation:

N ��! ��KN ! I

I ��! ��KI ! D

D ð1Þ

Assuming linear free energy changes theory,which states that each unfolding step has thefollowing relationship of the standard free energychanges:

DG0i ¼ DGi þmi½D� ð2Þ

in which DGi is the free energy change of eachstep, m represents the dependence of the DG ondenaturant concentration [D]. The following rela-tionship holds true in terms of equilibrium con-stants between each state

Ki ¼ exp�ðDG0

i �mi½D�ÞRT

� �ð3Þ

in which R is gas constant and T is the absolutetemperature in kelvin. The method for analysis ofthree-state transition unfolding curves is shownin Equation 4.

Albutropin unfolding curves were fitted to thismodel with Igor Pro software using Equation 4,and the results were chosen based on a combina-tion of parameters that achieved the best fit to thedata.

Agitation Studies

Triplicate samples containing 0.3 mg/mL Albu-tropin (1 mL) of each formulation were placed into2.0 mL polypropylene microcentrifuge tubes (dia-meter¼ 10 mm), which provided sufficient headspace for bubble entrainment into the solutionfrom the air-liquid interface during agitation.Albutropin was formulated without surfactantand with Tween 20 or Tween 80 slightly above thecritical micelle concentration (CMC) of each sur-factant, at an amount of Tween 20 or Tween 80that corresponds to the stoichiometric ratio of 10:1and 9:1, respectively, as well as an amount thatcorresponds to one-half of stoichiometric ratio

Yobs ¼

YN þ YI exp½�ðDGðH2OÞ;N ! I �mN ! I½D�Þ=RT� þ YU exp½�ðDGðH2OÞ;N ! I �mN ! I½D�Þ=RT� � exp½�ðDGðH2OÞ; I ! U �mI ! U½D�Þ=RT�

1þ exp½�ðDGðH2OÞ;N ! I �mN ! I½D�Þ=RT� þ exp½�ðDGðH2OÞ;N ! I �mN

! I½D�Þ=RT� � exp½�ðDGðH2OÞ; I ! U �mI ! U½D�Þ=RT� ð4Þ



(5:1) as determined by ITC and fluorescencetitration. A set of unagitated samples were alsoprepared and served as controls for surface-induced aggregation. When the amount of Tween20 added corresponded to its stoichiometric bind-ing ratio with Albutropin, the concentration ofTween 20 was approximately 30 mM. This con-centration was more than three times lower thanthe operative CMC for Tween 20 (100 mM) deter-mined by surface tensiometry. For Tween 80, theoperative CMC was also found to be �100 mM.Samples were oriented horizontally and agitatedat 600 RPM on a Labnet Orbit 300 orbital shaker(Woodbridge, NJ) at room temperature (25� 28C)for 96 h. Sample tubes were removed for testing at0, 24, 48, 72, and 96 h. In addition, nonagitatedcontrol samples were also assayed at each timepoint. Prior to testing by SE-HPLC, vials werecentrifuged at 13000 g for 15 min using a refri-gerated (48C) Savant centrifuge (Holbrook, NY),and the level of native monomer and the forma-tion of soluble aggregates were determined usingSE-HPLC.

RESULTS AND DISCUSSION

Fluorescence Quenching

Fluorescence spectroscopy often has been used tothe study of various protein–ligand interactions.It is a sensitive technique that is used to monitorchanges in protein tertiary structure upon ligandbinding and may complement ITC, given itshigh sensitivity. The intrinsic fluorescence ofthe aromatic residues in a protein, particularlytryptophan and tyrosine, is very sensitive totheir microenvironment. Because ligand bindingusually results in a perturbation of this micro-environment, the fluorescence may be quenched,providing an effective method to monitor ligandbinding. There are several mechanisms by whicha ligand may induce changes in a protein’s intrin-sic fluorescence intensity. First, ligand bindingmay cause a protein to undergo conformationalchanges that results in loss of resonance energytransfer between fluorophores and, thus, de-creased fluorescence intensity. Second, a ligandmay directly interact or ‘‘collide’’ with the fluor-ophore and absorb its intrinsic fluorescence afterexcitation. This process is termed collisionalquenching. Moreover, a ligand may complex withthe protein to form a nonfluorescent ‘‘dark’’complex. Such a phenomenon is called static

quenching. Importantly, the magnitude of thisfluorescence quenching process has been found tocorrelate with the binding affinity between aprotein and its ligand.20

A number of papers have been publishedrecently that utilized the intrinsic fluorescence ofthe single tryptophan residue in HSA as a way tomonitor its binding affinity for fatty acid ligandsand pharmaceutical compounds. For example,Johansson et al. 23 successfully used fluorescencequenching to investigate the nature of interactionbetween HSA and the volatile anesthetic, chloro-form. Gelamo and Tabak 24 in 2000 also employedfluorescence quenching to arrive at associationconstants between BSA, HSA, and a number ofionic surfactants.

The binding to Albutropin by both Tween 20 andTween 80 was measured by excitation of the twotryptophan residues in the protein at 295 nm.There is a shift in the wavelength of maximumemission (lmax) from 342 to 340 nm when asurfactant is added (data not shown), whichindicates that the tryptophan residues are in arelatively more hydrophobic environment afterbinding to the surfactant. Increasing concentra-tion of surfactant did not result in further shift oflmax. In all the experiments, the addition of Tweento the protein solution decreased the intensity ofthe intrinsic tryptophan fluorescence. The extentof fluorescence quenching is different for hGH,HSA, and Albutropin, but all reach a plateau atrelatively low surfactant concentrations. Paststudies have found that a plateau in the ligand-induced spectrofluorometric changes, such assurfactant induced quenching of tryptophanintrinsic fluorescence, are highly correlated withsaturation of available binding sites on theprotein.25,26 It appears that the break point inthe fluorescence-quenching curve occurs at theapproximate stoichiometry between ligand andprotein.23–26 Interestingly, the steady-state fluor-escence quenching data appear to support theexistence of two saturation points on the bindingisotherm (Figure 1A,B). The first inflectionpoints observed in the fluorescence quenchingisotherms between Tween 20 and Tween 80 withAlbutropin are at approximately 2:1 ratio (Tween20:Albutropin) and 1:1 ratio (Tween 80:Albu-tropin), respectively, which corresponds to thesaturation point for the high affinity binding site(Figure 1A,B). Tween appears to bind to loweraffinity sites on Albutropin after occupying thestronger binding sites. This observation corrobo-rates with recent findings by other researchers

1372 CHOU ET AL.


that multi-domain proteins possess specific anddistinctive binding sites for small ligands withdifferent affinities.27–29 The binding isothermsbetween the two Tweens and each of the twodomains of Albutropin and the intact fusionprotein are shown on Table 1. Our data show thatthe Tween 20-protein binding stoichiometry is 11:1(Tween 20:HSA) for HSA, 9:1 (Tween 20:hGH) forhGH, and 10:1 (Tween 20:Albutropin) for Albu-tropin. The binding stoichiometry between Tween80 and Albutropin was found to be 9:1 (Tween T

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Figure 1. (A) Fluorescence quenching isothermshowing progressive reduction of Albutropin trptophanintrinsic fluorescence intensity with increasing concen-trations of Tween 20. Error bars represent standarderror of the mean for duplicate samples. Where errorbars are not apparent, the bars were smaller than thesymbol. (B) Fluorescence quenching isotherm showingprogressive reduction of Albutropin trptophan intrinsicfluorescence intensity with increasing concentrations ofTween 80. Error bars represent standard error of themean for duplicate samples. Where error bars are notapparent, the bars were smaller than the symbol.



80:Albutropin). The fact that the stoichiometricbinding ratio between Albutropin and Tween doesnot correspond to the sum of its two domains isprobably due to changes in the number and/orconfiguration of available binding sites upon theformation of the chimeric protein. Stoichiometricvalues derived from a recent crystallographicanalysis found in the literature show excellentagreement with data obtained in the present study(Table 1); Bhattacharya et al. 30 found a total of11 binding sites on HSA for fatty acids with 10–14carbon aliphatic tails (note that the lauric acidmoiety found in Tween 20 has a 12 carbonhydrophobic group) and up to 7 sites for fatty acidswith longer hydrocarbon chains (Tween 80 con-tains a 18 carbon hydrophobic tail).

In order to compare the binding affinities forTween for each protein, it is necessary to calculatethe dependence of F0/F (F0 and F are the fluor-escence intensities in the absence and presence ofsurfactant, respectively) as a function of Tweenconcentration. The Stern–Volmer equation char-acterizes the interaction in terms of a quenchingconstant derived from the slope of the Stern–Volmer plot.

F0=F � 1 ¼ KSVQ

F0 and F are the fluorescence intensities beforeand after addition of quencher; Q is the concentra-tion of quencher; Ksv is the effective quenchingconstant of the binding reaction and it is a productof rate constant for quenchingkqand lifetime of thefluorophore t0.20

The Stern–Volmer quenching constants (Ksv)obtained for HSA: Tween binding resemble valuespublished in the literature for HSA and othersurfactants. For example, for HSA binding withhexadecyl-N,N-dimethyl-3-ammonium-1-propan-esulfonate (HPS) and cationic cethyltrimethylam-monium chloride (CTAC) the Ksv were found to be(3.5–3.6)� 103 and (2.9–3.1)� 103 M�1, respec-tively.24 By comparison, the Ksv for Tween 20 andHSA was determined to be (1.7–1.8)� 105 M�1.Ksv

has a strong correlation with binding affinitybetween the quencher (surfactant in this case)and a given protein. In fact, when the quenchingis completely static in nature due to formation ofnonfluorescent complex, Ksv is the affinity con-stant.22

Until this work, there has been no attempt tomeasure the binding of surfactants to hGH usingfluorescence quenching. Table 1 lists the Stern–Volmer quenching constant obtained for Albutro-pin and each of its two domain proteins. It appears

that Tween 20 has a much higher affinity for HSAthan hGH. The difference in association constants(Ksv) between HSA and hGH is approximately twoorders of magnitude. For Albutropin, the Stern–Volmer quenching constant of the high-affinitysite was (2.0–2.1)� 105 for Tween 20 and (1.8–2.0)� 105 M�1 for Tween 80.

ITC Binding Studies

Although its sensitivity is less than that of fluor-escence titration, ITC provides direct thermo-dynamic information about ligand binding toprotein.31 ITC measures the heat associated withbinding of a ligand to macromolecule. In a singleexperiment, the values of binding constant (Ka),the stoichiometry (n), the enthalpy of binding(DHb), and the entropy of binding (DS) can bedetermined.31,32

ITC experiments with hGH, HSA, and Albutro-pin were performed to determine whether bindingparameters obtained by this method agree withthose obtained from fluorescence titrations. Theassociation parameters derived from fluorescencetitration differs from those determined by ITC(Table 1). Since two completely different phenom-ena are being measured, it is not surprising thatthere are differences in the calculated bindingaffinity. However, it is important to note that therank ordering of the binding affinity is the same forboth methods, with Albutropin having the highestbinding affinity for Tween 20, followed by HSA,then hGH. Due to the weak interaction betweenTween 20 and hGH, it is not possible to determinethe binding constant using ITC. Although previousstudies have shown that nonionic surfactantsbinds with hGH via hydrophobic interaction,Tween appears to exhibit higher affinity for theHSA domain of Albutropin as evidenced by theassociation constants derived from these experi-ments. A comparison of the ITC binding isothermobserved in this study with hGH shows that ourresult matches previously published data by Bamet al.6 In fact, the binding stoichiometry betweenhGH and Tween 20 we found is identical to thevalue obtained by Bam et al. 5 via refractive indexstudies.

Importantly, ITC yields the same protein-surfactant stoichiometry as the fluorescence titra-tion experiments (Table 1). Analysis of the bindingisotherm by established models for each methodindicate that there are specific sites to whichsurfactants bind that can be saturated (Figures 1and 2B). This is demonstrated by both the plateau

1374 CHOU ET AL.


in the isotherm generated by fluorescence quench-ing as well as the progressively smaller enthalpicsignal from ITC titration (Figures 1 and 2A).Moreover, it was found that ITC and fluorescencetitration data were best fit using a binding sitemodel with two sites of different affinities. As onecan see in Table 1, for Tween 20, the associationconstant for the high affinity and the low affinitysite of Albutropin as determined by ITC is (2.1–2.2)� 104 and (1.1–1.2)� 103 M�1, respectively.Essentially, the high affinity site of Albutropin is

bound by Tween 20 over 20 times more avidly thanthe low affinity site. For Tween 80, the associationconstant is (1.4–1.5)� 104 M�1 for the high affinitysite and (1.2–1.3)� 103 M�1 for the low affinitysite. As early as the 1970s, researchers haverecognized the ability of HSA to assume differenttransient solution conformations upon fatty acidbinding. Given its natural biological role, HSA isknown to exhibit much flexibility in solution andthis property is believed to be responsible for itsability to bind a diverse array of ligands.16 Spectortermed this property of HSA ‘‘configurationaladaptability’’ and it was found that fatty acidswith 12 carbon hydrophobic tails exhibit thehighest affinity for HSA.17 Since the hydrophobictail of Tween 20 contains 12 carbons versus 18in Tween 80, Tween 20 probably has a greaternumber of productive interactions with bindingpockets on Albutropin. The overall lower affinity ofTween 80 for Albutropin may be due to the longerhydrophobic tail of Tween 80, which may causesteric hindrance at the high affinity binding site.

Agitation Study

Agitation is a frequently encountered source ofstress for protein pharmaceuticals, especially forliquid formulations. During vial filling, shipment,reconstitution, and administration, agitation ofthe solution could repeatedly expose the protein todenaturing surfaces, such as air-liquid and solid-liquid interface. If a sub-CMC concentration ofTween is sufficient to prevent agitation-inducedaggregation under the strongly destabilizing con-ditions employed in this study, it is a good indi-cator that a protein-specific, rational approach tosurfactant formulation could be accomplished.

The ability of Tween to protect Albutropinagainst agitation-induced damage is an importantformulation consideration. If it does protect, is thiseffect due to specific binding to the protein or bysaturating the air-water interface or both mechan-isms? The stability of Albutropin during agitationwas monitored using size-exclusion chromatogra-phy (SE-HPLC). After Albutropin (at pH 7) wasagitated for 24 h, there was detectable conversionof the monomeric protein into soluble aggregates(Figure 3). The loss of monomer occurs concur-rently with an increase in the amount of dimer,trimer, and large oligomer (that elutes with thevoid volume). After 96 h, the level of monomericprotein decreased from 99% to 30% (Figure 5A). Insamples with Tween 20: Albutropin molar ratios of5:1, the loss of monomer is delayed relative to the

0 10 20 30 40 50 60 70 80

-10

-8

-6

-4

-2

Time (min)

µca

l/sec

0 5 1 0 1 5 2 0 2 5

-1 .2

-1 .0

-0 .8

-0 .6

-0 .4

-0 .2

0 .0

Molar Ratio

kc

al/

mo

le o

f in

jec

tan

t

A

B

Figure 2. (A) Raw isothermal titration calorimetry(ITC) data for Albutropin titration with Tween 80.(B) ITC isotherm for the binding of Tween 80 toAlbutropin. After integration of the raw signal, enthalpyof binding of each injection was obtained and plottedversus molar ratio of Tween 80: Albutropin.



samples with no Tween. However, aggregationstill proceeds to a significant extent by the end ofthe study. By contrast, at a stoichiometric ratioof 10:1 Tween 20: Albutropin, aggregation wasessentially completely inhibited, even after 96 hof continuous agitation (Figure 5A). In order toensure that aggregation was not simply due tostorage of samples at 258C, nonagitated controlswithout Tween 20 were stored at 258C for theduration of the agitation study. After 96 h ofstorage at the aforementioned temperature, therewas no loss of monomeric Albutropin in the controlsamples.

It is important to note that surfactant CMC ishighly dependent on solution conditions, such asionic strength, temperature, pH, and other solu-tion components such as excipients, as well asthe presence of protein.1 The implication of thisfinding is that even when a CMC level of surfactantis desired, the true CMC of the protein solutionmay not be known unless effort is undertaken todetect the existence of surfactant-protein interac-tion and its effect on the operative CMC. When adecision must be made regarding how much sur-factant is to be added to a formulation, it may behazardous to utilize the published CMC values fora particular surfactant, as the CMC value may beunderestimated in these cases. To ensure that,under the conditions of this study, the concen-

tration of surfactant is below the operative CMCwhen added at a stoichiometric ratio with Al-butropin, surface tensiometry was employed tomeasure the effective CMC with the proteinpresent in solution. Bam et al.5 using electronparamagnetic resonance (EPR) found that appar-ent surfactant CMC in the presence of hGHwas shifted to lower Tween concentrations. Thisphenomenon was attributed to the formation ofmixed-surfactant protein aggregates at a surfac-tant concentration below the reported CMC. Thereported CMC values of Tween 20 and Tween 80are 59 and 12 mM, respectively, in pure water at258C.33 In the present study, the apparent CMC ofTween 20 and Tween 80 were both shifted to over100 mM (data not shown). It is possible that in thepresence of Albutropin, the concentration ofTween at the air/water interface decreases as thesurfactant is bound onto the protein and drawninto the bulk solution; as a result, the amount ofsurfactant needed to reach CMC is increased.Barreiro-Iglesias et al.34 recently observed thesame phenomenon when they studied the interac-tion between Tween 80 and carbopol1 polymers.The authors attributed the increase in CMC intheir system to direct polymer-surfactant binding.This finding further supports our hypothesis thatTween binds Albutropin. At the concentration ofprotein used (0.3 mg/mL) a 10:1 molar ratio ofTween 20 corresponds to a concentration of about30 mM, which is about one-third of the effectiveCMC measured by surface tensiometry. Increas-ing the concentration of Tween 20 to >30:1 molarratio (above the CMC) confers the same degree ofprotection against agitation-induced aggregationas at 10:1. This demonstrates that a stoichiometricamount of Tween 20 is sufficient to protect Al-butropin against agitation-induced aggregation.Similar results were obtained using Tween 80as the surfactant (Figure 4). As can be seenon Figure 5B, both samples that contained Tween80 at 9:1 molar ratio (surfactant: protein) andsamples with Tween 80 just above CMC did notform soluble aggregates throughout the study’sduration.

One potential advantage of minimizing theamount of surfactant used is that it is well knownthat Tween surfactants can contain a low level ofresidual peroxide, which can potentially affect thestability of oxidation-sensitive proteins. Surfac-tants are also known to be susceptible to degrada-tion during storage; and the resulting formationof oxidizing free radicals may compromise bothphysical and chemical stability of the protein

0 hours

24 hours

48 hours

72 hours

4 5 6 7 8 9 10 11 6 7 8 9

Abs

orba

nce

(280

nm

)

Elution time (minutes)

soluble aggregates

monomer

fragment

Figure 3. Size-exclusion chromatography (SEC) dataindicating time-dependent loss of monomer and forma-tion of soluble aggregates during agitation in theabsence of Tween.

1376 CHOU ET AL.


during long term storage.35 The results of thecurrent study demonstrate that it is not alwaysnecessary to obtain a complete coverage of theair-water interface with above-CMC concentra-tion of surfactants to maximally inhibit agitation-induced aggregation, if direct binding of the sur-factant to the protein occurs. Binding of surfactantcan inhibit aggregation either through a reductionin intermolecular contact between protein mole-cules and/or increased protein conformationalstability.

Equilibrium Unfolding Study

To gain further insight into the mechanism forinhibition of agitation-induced aggregation byTweens, the free energy of unfolding was mea-sured in the absence and presence of surfactant.Since the first step in surface-mediated proteinaggregation is the adsorption and unfolding of theprotein at the interface, we hypothesize that oneof the mechanisms by which a sub-CMC level ofTween stabilizes Albutropin against agitation in-duced aggregation is by increasing the free energyof protein unfolding. The ability of Tweens toretard aggregation may be due to thermodynamicstabilization of the native state, thereby decreas-ing the population of partially unfolded, aggrega-tion-competent species in solution.36,37 As can beseen in Figures 6 and 7, the addition of Tween 20

and Tween 80 at stoichiometric ratio (10:1) and(9:1), respectively, resulted in a shift in unfold-ing curves to higher GdnHCl concentrations.These results suggest that binding of Tween tothe native state of Albutropin increases the freeenergy of unfolding.

The unfolding curve displays two distinct un-folding events, presumably due to unfolding of the

Time (hours)

0 20 40 60 80 100 120

Per

cent

Rec

over

y of

Sol

uble

Mon

omer

0

20

40

60

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0 20 40 60 80 100 120

Per

cent

Rec

over

y of

Sol

uble

Mon

om

er

0

20

40

60

80

100

A

B

Figure 5. (A) Percent recovery of soluble monomericprotein as a function of duration of agitation. Filledcircle, control (no Tween); open circle, 5:1 molar ratio[Tween 20:Albutropin]; filled triangle, 10:1 molar ratio[Tween 20:Albutropin]; open triangle, Tween 20 at cri-tical micelle concentrations (CMC). Each point repre-sents the mean of three vials� standard deviation.Where error bars are not apparent, the bars weresmaller than the symbol. (B) Percent recovery of solublemonomeric protein as a function of duration of agitation.Filled circle, control (no Tween); open circle, 9:1 molarratio [Tween 80:Albutropin]; filled triangle, Tween 80 atCMC. Each point represents the mean of three vials�standard deviation. Where error bars are not apparent,the bars were smaller than the symbol.

Figure 4. SEC data indicating no time-dependentloss of monomer and formation of soluble aggregatesduring agitation in the presence of Tween 80 at 9:1 molarratio [Tween 80:Albutropin].



two individual domains (the HSA and hGH por-tions). Thermodynamic analysis of these curvesrequires that each transition behaves as a two-state model, whereby the unfolding is reversible.In order to demonstrate the reversibility of theunfolding process, far UV CD scans were takenafter exhaustive dialysis against buffer was per-formed to remove GdnHCl from samples thatcontained 6M of the denaturant. It was found thatunfolding of Albutropin was>93% reversible (datanot shown). In their 1996 paper Bam et al.,7 usingelectron paramagnetic resonance (EPR) spectro-scopy found that Tween could stabilize and popu-late a partially unfolded intermediate of hGH at4.5M guanidine hydrochloride. In the currentstudy, such a folding intermediate of the hGHdomain was not observed with the addition ofTween. This discrepancy is most likely due to thefact that the chosen spectroscopic method (Far UVCD spectroscopy) is primarily used to monitorchanges in protein secondary structure. Since EPRspectroscopy is used to monitor changes in bothsecondary and tertiary structure of a protein, itmay explain why such a stable intermediate wasobserved in the earlier study.7 It should be notedthat the most significant spectroscopic transitionsfound by Bam et al. occurs over a narrow range ofguanidine concentrations, just as we have ob-served with Albutropin.

A comparison of the denaturation midpoint aswell as the free energy of unfolding values with theliterature suggests that the first part of the curvecorresponds to the unfolding of the HSA domain,while the second transition event agrees withthe values derived from unfolding of the hGHdomain.7,38,39 Observation of multiple-unfoldingevents has been observed for proteins with sepa-rate domains. Since the unfolding curves ofAlbutropin do not follow the traditional singletwo-state model and appears to have a significantplateau between two separate unfolding events,thermodynamic parameters could not be obtainedby the linear extrapolation approach often used forprotein that have a two-state unfolding behavior.Analysis of unfolding curves with multiple transi-tions has been a great challenge until recently. In2001, Hung and Chang reported a formal deriva-tion of a mathematical model that describes thefolding-unfolding process involving multiple inter-mediates (Equation 4).21 The model was developedto analyze the unfolding data for human placentalalkaline phosphatase and could be applied to anyother multi-state unfolding data. The Hung andChang model takes into account the equilibrium

[Guanidine HCI] (M)

0 2 4 6

Far

UV

CD

Sig

nal a

t 222

nm

-20

-15

-10

-5

0

Figure 6. Unfolding curves for Albutropin at 0.1 mg/mL in the absence and presence of Tween 20. Unfoldingcurves were measured using Far UV CD spectroscopymonitoring the loss of protein helical structure at222 nm. Open circle, control (no Tween 20); filled circle,Tween 20 10:1 molar ratio [Tween 20: Albutropin]. Eachpoint represents the mean of three samples� standarddeviation. Where error bars are not apparent, the barswere smaller than the symbol.

[Guanidine HCI] (M)

0 2 4 6

Far

UV

CD

Sig

nal a

t 222

nm

-20

-15

-10

-5

0

Figure 7. Unfolding curves for Albutropin at 0.1 mg/mL in the absence and presence of Tween 80. Unfoldingcurves were measured using Far UV CD spectroscopymonitoring the loss of protein helical structure at222 nm. Open circle, control (no Tween 80); filled circle,Tween 80 9:1 molar ratio [Tween 80: Albutropin]. Eachpoint represents the mean of three samples � standarddeviation. Where error bars are not apparent, the barswere smaller than the symbol.

1378 CHOU ET AL.


constants between the various folding intermedi-ates and bases its assumptions on the linear freeenergy changes theory developed by Santoro andBolen.20,40

Using this model, our analysis shows that thedenaturation midpoint (D0.5) in GdnHCl solutionsof the first unfolding event was increased from1.8� 0.1 M GdnHCl in the absence of Tween 20 to2.1� 0.2 M in the presence of Tween 20 (Table 2).It was determined that the addition of stoichio-metric amount of Tween 20 results in an increasein thermodynamic conformational stability of theHSA domain of Albutropin by 1.3� 0.5 kcal/mol.By comparison, a stoichiometric amount of Tween80 conferred about 0.6� 0.2 kcal/mol of stabiliza-tion to Albutropin (Table 2). In both cases, the freeenergy of unfolding of the second unfolding event,presumably involving the hGH domain, was notsignificantly altered. This observation may be dueto the fact that both Tween 20 and Tween 80appear to bind more strongly to the HSA domain ofAlbutropin, thus reduce the effect of these surfac-tants have on stability of the hGH domain of thefusion molecule.

These results, along with the Tween bindingstudies, indicate that Tween stabilizes Albutropinagainst agitation-induced aggregation by prefer-entially binding to the native state of the protein,mostly to the HSA domain. This effect can be bestexplained by the Wyman linkage function, whichstates that differential binding affinity of a ligandin a two-state equilibrium will shift the equili-brium in favor of the state with the greater bindingaffinity.37,41,42 Spectroscopic studies of Albutropinboth in the presence and absence of Tween 20 andTween 80 in buffer showed complete retention ofnative protein secondary and tertiary structure(data not shown). Thus, Tween stabilizes Al-

butropin by preferentially binding to the nativemolecule, which results in greater protein con-formational stability; this means that as a popula-tion, fewer Albutropin molecules are in expanded,aggregation-competent conformations that canaggregate upon exposure to agitation.

Previous reports have shown that Tween couldprotect protein against aggregation via severalmechanisms simultaneously. In addition to stabi-lizing the native state of Albutropin and competingfor occupation of the air-water interface, Tween 20Tween 80 may prevent protein aggregation bybinding to hydrophobic sites on the protein’ssurface. The result of this interaction is that lessaggregation prone-hydrophobic areas on the pro-tein are exposed to the solvent and thus theprobability of protein–protein interaction isdecreased. Tween could inhibit intermolecularcontact by occupying the hydrophobic regions ofthe protein with its nonpolar aliphatic tail. Thedriving force for this type of interaction is thehydrophobic effect.6 The importance of this mech-anism was illustrated with hGH when it was foundthat despite its weak binding to Tween, the opti-mal level of protection against agitation-inducedaggregation was rendered when the protein wasformulated with sufficient surfactant to saturateall the binding sites on the protein’s surface.

CONCLUSIONS

We have evaluated the utility of nonionic surfac-tants Tween 20 and Tween 80 as excipients forprevention of surface-induced denaturation andaggregation of recombinant fusion protein, Al-butropin. The results of this work show thatTween 20 and Tween 80 both confer significant

Table 2. Thermodynamic Parameters Obtained from Guanidine HydrochlorideUnfolding Studies

Surfactant UsedThermodynamic

Parameters(GdnHcl)D0.5 M

DG of UnfoldingKcal/mol

DDGKcal/mol

With Tween 20 Transition 1 2.1� 0.2 5.3� 0.7 1.3� 0.5Transition 2 4.4� 0.3 5.8� 0.8 N/S

No Tween 20 Transition 1 1.8� 0.1 4.0� 0.2 —Transition 2 4.5� 0.2 5.9� 0.6 —

With Tween 80 Transition 1 2.0� 0.1 4.8� 0.4 0.6� 0.2Transition 2 4.4� 0.3 5.9� 0.5 N/S

No Tween 80 Transition 1 1.9� 0.2 4.3� 0.3 —Transition 2 4.4� 0.2 5.9� 0.5 —

Data presented are the means and standard deviations obtained from triplicate measurements.N/S, not significant.



protection against agitation-induced damage toAlbutropin, even at concentrations below theCMC, provided the stoichiometric molar ratioidentified by the binding studies is used. Tweenprotects Albutropin against aggregation byincreasing thermodynamic stability of the pro-tein, thus decreasing its propensity for denatura-tion and subsequent aggregation. Despite therelatively weak interaction, binding of Tween20 and Tween 80 by Albutropin provides 0.6–1.3 kcal/mol of stabilization to the protein. Sa-turation of the hydrophobic surface areas ofAlbutropin by Tween may also provide sterichindrance by decreasing intermolecular contactsthat precede the onset of aggregation. Finally, thecurrent study provides strong evidence that arational approach to surfactant formulation ofprotein pharmaceuticals could minimize the risksassociated with a given excipient, yet providesufficient benefit on protein stability.

ACKNOWLEDGMENTS

We greatly appreciate the generous gift of Al-butropin from Human Genome Sciences, Inc. Thiswork was partly supported by HGSI and NIHLeadership in Biotechnology Fellowship given toDKC.

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Effects of Tween 20 and Tween 80 on the Stability