adsorption and function of recombinant factor viii at solid–water interfaces in the presence of...

BIOTECHNOLOGY

Adsorption and Function of Recombinant Factor VIII atSolid–Water Interfaces in the Presence of Tween-80

OMKAR JOSHI,1,2 JOSEPH MCGUIRE,1 D.Q. WANG2

1Department of Chemical Engineering, Oregon State University, Corvallis, Oregon 97331

2Bayer HealthCare LLC, Berkeley, California 94701

Received 3 August 2007; accepted 21 December 2007

Published online 13 March 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21333

Omkar JoshBerkeley, CA 94

Corresponden6306; Fax: 541-

Journal of Pharm

� 2008 Wiley-Liss

ABSTRACT: The adsorption, structural alteration and biological activity of a recombi-nant Factor VIII was investigated in the presence of the surfactant Tween-80, athydrophilic and hydrophobic solid–water interfaces. Hydrophilic and silanized, hydro-phobic silica surfaces were used as substrates for protein and surfactant adsorption,which was monitored in situ, with ellipsometry. At the hydrophobic surface, thepresence of Tween in the protein solution resulted in a reduction in amount of proteinadsorbed, while rFVIII adsorption at the hydrophilic surface was entirely unaffected bythe presence of Tween. These observations were attributed to high binding strengthbetween Tween and the hydrophobic surface, and low binding strength between Tweenand the hydrophilic surface. Colloidal particles bearing hydrophilic and hydrophobicsurfaces, and net positive or negative surface charge, were used as substrates for rFVIIIadsorption in evaluation of tertiary structure change and biological activity retention atinterfaces. Fluorescence emission spectroscopy showed that rFVIII tertiary structure waschanged upon exposure to hydrophobic nanoparticle surfaces. Similarly, the biologicalactivity of rFVIII (based on the activated partial thromboplastin time) was reduced athydrophobic surfaces. At high surfactant concentration, these properties were betterpreserved. This was attributed to Tween adsorption sterically inhibiting rFVIII adsorp-tion. While hydrophilic surfaces were associated with relatively high rFVIII adsorption,they did not induce large changes in structure or activity. This was attributed to theformation of a tightly packed, ordered adsorbed layer on these surfaces, governed byelectrostatic attraction and not mediated by the rFVIII active site. � 2008 Wiley-Liss, Inc.

and the American Pharmacists Association J Pharm Sci 97:4741–4755, 2008

Keywords: adsorption; desorption; fo
rmulation; nanoparticles; protein binding;protein structure; proteins; surfactants
INTRODUCTION

Factor VIII (FVIII) is a high-molecular weight(280 kDa) multidomain protein that is an essen-

i’s present address is Bayer HealthCare LLC,701.ce to: Joseph McGuire (Telephone: 541-737-737-4600; E-mail: [email protected])

aceutical Sciences, Vol. 97, 4741–4755 (2008)

, Inc. and the American Pharmacists Association

JOURNAL OF PHARM

tial blood coagulation factor. FVIII has a domainstructure of A1-A2-B-A3-C1-C2, in which theheavy chain is composed of the A1, A2, and Bdomains and the light chain is composed of the A3,C1, and C2 domains.1 FVIII exists as a hetero-dimer of the heavy and light chain and isassociated with 50-fold excess von WillebrandFactor (vWF) when present in plasma.2 In theblood coagulation cascade, FVIII serves as acofactor for Factor IXa in the activation of Factor

ACEUTICAL SCIENCES, VOL. 97, NO. 11, NOVEMBER 2008 4741

4742 JOSHI, MCGUIRE, AND WANG

X to Factor Xa.3 FVIII is first cleaved by thrombinto the active form, Factor VIIIa which is aheterotrimer consisting of the A1 and A2 domainsand the light chain.4 The thrombin proteolysis ofFVIII to FVIIIa thus results in the removal of theB domain.5 The A2 and A3 domains interactwith Factor IXa while the C-terminal region of theC2 domain interacts with vWF and, upon activa-tion, with phosphatidyl-L-serine-containing mem-branes. A functional deficiency in FVIII causeshemophilia A, a congenital bleeding disorder.

There are relatively few reports on the structureof the full-length FVIII molecule. The heavy andlight chains are held together by metal ion-dependent and hydrophobic interactions.6 Sudha-kar and Fay7 evaluated the hydrophobic sites onthe surface of FVIII and FVIIIa using intrinsic andextrinsic probe fluorescence. An identical bindingand emission pattern for fluorescent probe bisani-linonapthalsulfonic acid of FVIII and FVIIIa, whichlacks the B-domain, suggested that the B domaindoes not contain any hydrophobic sites on thesurface. They identified two hydrophobic sites eachon the isolated heavy and light chain. They furtherproposed that one hydrophobic site each on theheavy and light chains is retained on the surface ofreconstituted FVIII while the other two hydro-phobic sites participate in inter-subunit associa-tion. Fowler et al.8 used electron microscopy tostudy the domain structure of Factor V and FVIII.Each Factor V and FVIII molecule contained alarge globular domain 12–14 nm in diameter. Asingle tail up to 50 nm was also often resolved inpreparations containing a high-molecular weightheavy chain. Fowler et al. present a molecularmodel of FVIII in which the A and C domainsconstitute the globular head and the connecting Bdomain is represented by a two-stranded tail.Stoilova-McPhie et al.9 used the method of two-dimensional crystallization of B domain-lackingFVIII onto phospholipid monolayers followed byelectron microscopy and crystallography to solvethe structure of such FVIII. The domain arrange-ment displayed A3 domains in close associationwith the C domains near the membrane surface.Four C2 loops are embedded within the lipidmonolayer. The C1 domain is nearly perpendicularto C2, with the C1 long axis almost parallel to themembrane. Looking toward the membrane, A1appears to be fully covering C1, while C2 ispartially overlapped by the A2 domain of theadjacent molecule.

Historically, FVIII was derived from humanplasma. The risk of pathogen transmission and

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 11, NOVEMBER 20

the limited availability of plasma have led to thedevelopment of a recombinant FVIII (rFVIII).10

rFVIII is the largest molecule ever successfullycloned by genetic engineering techniques and isthe largest and most complex protein currentlymanufactured.11 The rFVIII molecule is sensitiveto both chemical and physical degradation. Thedegradation involves changes in higher orderstructure and may be caused by a number ofpathways including aggregation, precipitation oradsorption onto surfaces.12 Surface adsorption ofrFVIII is rapid.13 About 50% of rFVIII productmay be lost due to adsorption during sterilefiltration alone.14 In many studies involvingrFVIII, activity loss is the only indicator foradsorption. This paper addresses the rFVIIIadsorption issue with three indicators: adsorbedamount, higher order structure, and biologicalactivity. These indicators are complementary.Assimilation of data recorded from adsorptionand elution kinetic experiments, secondary andtertiary structure determination at interfaces,and biological activity determination at inter-faces, is expected to facilitate a comprehensiveunderstanding of rFVIII adsorption.

In the past, human serum albumin (HSA) wasadded to rFVIII solutions in order to preventadsorption during manufacturing and packaging.This practice was discontinued due to the risk ofinfectious agents originating from HSA. Thenonionic surfactant Tween-80 is now added as areplacement for HSA since surfactants are gen-erally effective in reducing protein adsorption. Forexample, Fatouros and Sjostrom15 have reportedthat the agitation-induced denaturation of rFVIIIis significantly reduced upon addition of 0.20 mg/mL Tween-80 or Tween-20. But the specificmechanisms underlying Tween action in mini-mizing adsorption loss and aggregation are notwell understood. In particular, even for formula-tions that are considered ‘‘optimized’’ for chemicaland physical stability of the protein, the effec-tiveness of the surfactant will depend verystrongly on the chemistries of the interfacespresent (whether gas–liquid, liquid–liquid, andsolid–liquid) in a given circumstance. Nonionicsurfactants, when introduced to an adsorbedprotein layer, do not generally affect the amountadsorbed on hydrophilic surfaces but do have aneffect on the amount adsorbed on hydrophobicsurfaces, presumably because of the difference insurfactant binding strength at the interface.16

Recently, we completed a comprehensive studyof rFVIII behavior at solid–water and air–water

08 DOI 10.1002/jps

RECOMBINANT FACTOR VIII AT INTERFACES 4743

interfaces, in the presence of Tween-80.17 Theconcentration of surfactant, as well as the methodof surfactant and protein introduction to thesurfaces (in sequence or combined) was varied inorder to identify the separate roles of protein,surfactant, and the protein-surfactant complex indetermining adsorption outcomes. In this article,we describe rFVIII adsorption, structural altera-tion, and activity at solid–water interfaces, in thepresence of Tween-80.

MATERIALS AND METHODS

Protein, Surfactant, and Buffers

The recombinant Factor VIII (rFVIII) used in thiswork was a gift from Bayer HealthCare (Berkeley,CA). The rFVIII used in ellipsometry experimentswas obtained in a frozen liquid formulation at aconcentration of 533 mg/mL. The protein wasformulated in 0.02 M 3-(N-morpholino)propane-sulfonic acid hemisodium salt (MOPS, Sigma),220 mM NaCl, 25 mM CaCl2, 1% sucrose and 80ppm Tween-80. The frozen rFVIII solution wasthawed and aliquoted into 2 mL Eppendorf vials.The Eppendorf vials were then placed in a �808Cfreezer and were thawed just prior to use. TheMOPS buffer used for incubation and rinsingduring ellipsometry experiments contained noTween. Tween-80 (J.T. Baker) was dissolved indistilled, deionized water to obtain concentratedstock solutions at 10000 and 50000 ppm. TheTween stock solutions were aliquoted into 2 mLEppendorf vials and frozen at �808C and thenthawed just prior to use.

The rFVIII used in the fluorescence and activityassays was formulated in the KG-2 bufferconsisting of 30 mM NaCl, 2.5 mM CaCl2, 22 g/L glycine, 3.1 g/L L-histidine and 10 g/L sucrose atpH 6.8. The buffer excipients were provided byBayer HealthCare. The protein solution containedabout 100 mg/mL rFVIII and 20 ppm Tween.

Preparation of Hydrophilicand Hydrophobic Silica

Silicon (Si) wafers (WaferNet, Inc., San Jose, CA,crystal grade, type N, boron doped, orientation1-0-0, thickness 525� 18 mm, resistivity 0.01–0.02 ohm-cm) were oxidized in air (1 atm, 10008C)for 18 min to obtain an oxide film thickness ofabout 300 A.18 Wafers were cut into 1� 3 cmplates using a tungsten pen, rinsed with acetone,then cleaned using a standard acid/base cleaning

DOI 10.1002/jps JOURNA

procedure. In brief, the plates were first immersedin a solution of NH4OH/H2O2/H2O (1:1:5 volumeratio), held at 808C for 10 min, and rinsed incopious amounts of distilled, deionized water(DDW). They were then transferred to a solutionof HCl/H2O2/H2O (1:1:5 volume ratio), held at808C for 10 min, rinsed with DDW again and thendried under a flow of nitrogen. At this stage thesilica plates exhibit hydrophilic surfaces, asevidenced by a water contact angle between 0and 108. Hydrophilic silica samples were stored inethanol until use.

Hydrophobic silica plates were made by immer-sion of hydrophilic silica plates in a solutioncontaining 1% dichlorodimethylsilane in xylenefor 1 h. The plates were then rinsed sequentiallywith xylene, acetone, and ethanol. The silaniza-tion procedure rendered the silica plates hydro-phobic, as evidenced by a water contact anglebetween 90 and 1008. The hydrophobic plates werestored in ethanol until use.

Evaluation of the Surface Chargeof Silica Adsorption Substrates

Silica wafers were cut into 3.8� 6.35 cm plates,then cleaned and silanized as described above.Surface charge properties were quantified usingan ElectroKinetic Analyzer (EKA, Anton Paar)which measures the streaming potential. TheMOPS buffer, with no Tween and diluted 20-foldwith DDW, was used as the electrolyte solution.The calculated zeta potentials for the hydrophilicand hydrophobic silica plates were �37.95 and�29.47 mV, respectively. The sign of the zetapotential corresponds to sign of the surface chargeand the magnitude correlates with charge density.

Nanoparticles Conforming to DifferentSurface Characteristics

Nanoparticles provide a large surface area forinterfacial processes to take place while remain-ing dispersed in colloidal suspension. Thus,nanoparticles enable the application of solutionphase assays to study of phenomena occurring ona solid substrate. Nanoparticles conforming tofour distinct surface characteristics were obtainedand used as supplied. Table 1 describes theproperties of the various nanoparticles used.The sizes of the nanoparticles were selected soas not to introduce particle curvature effects onadsorption behavior.

L OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 11, NOVEMBER 2008

Table 1. Properties of the Colloidal Particles Used in Evaluation of Structure and Activity

Description

SurfaceCharacteristics

MeanDiameter (nm) SupplierWettability Charge

Sulfated white polystyrene latex Hydrophobic Negative 75 Interfacial Dynamics Corp., Eugene, ORAmidine white polystyrene latex Hydrophobic Positive 78 Interfacial Dynamics CorpNyacol1 9950 silica Hydrophilic Negative 100 EKA Chemicals, Moses Lake, WABindzil1 CAT 80 alumina-coatedsilica

Hydrophilic Positive 40 EKA Chemicals


Evaluation of rFVIII Secondary Structure

Circular dichroism (CD) spectra of rFVIII (0.533 mg/mL, formulated in MOPS buffer) with Tween-80concentrations of 80, 160, and 280 ppm wereperformed to evaluate the effect of Tween-80 onprotein structure in solution (it was not possible tostudy rFVIII at a Tween concentration lower than80 ppm without simultaneously diluting theprotein). The CD spectra were obtained using aJ-720 UV Spectrum spectropolarimeter (JASCO).All experiments were carried out at 258C. Acylindrical cuvette with a 100 mm pathlength wasused. The CD spectra were recorded with every1 nm increment in wavelength, starting at 300 nmand ending at 194 nm. In order to increase thesignal-to-noise ratio, six scans were recorded foreach sample and then averaged. The CD spectra ofprotein-free, Tween-containing buffer were sub-tracted from the rFVIII CD spectra in every case.

Evaluation of rFVIII Adsorption Kinetics

rFVIII adsorption kinetics were recorded in situ,by ellipsometry.17 An automatic ellipsometer(L-104SA, Gaertner Scientific Corp., Chicago,IL) with a 1 mW He-Ne light source was used.The angle of reflection was set equal to the angle ofincidence at 708. Each silica plate (hydrophobic orhydrophilic) was suspended in a trapezoidal, fusedquartz cuvette (Hellma, Plainview, NY) whichwas equipped with a magnetic stir-bar, and filter-ed MOPS buffer was added. After stable opticalproperties of the bare substrate were measuredfor 30 min (at 15 s intervals), a rFVIII, Tween,combined rFVIIIþTween, or protein-free, surfac-tant-free buffer solution was introduced to thecuvette as described in the following subsections.

The adsorbed layer changes the optical proper-ties of the reflected laser beam which can then berelated to the adsorbed mass.19 A one-film-modelellipsometry program20 was used for the calcula-


tion of adsorbed mass. The program uses thevalues of the ratio between molar mass and molarreflectivity (M/A) and partial specific volume (V) ofthe adsorbing species. It is difficult to assign thesevalues for rFVIII, since rFVIII is a highlyglycosylated protein. In the absence of accuratevalues specifically for rFVIII, the values for amodel globular protein, lysozyme, were usedinstead.17 The M/A and V used in this case were3.841 g/mL and 0.761 mL/g, respectively. Protein-specific values of V and M/A were used todetermine the adsorbed mass in both the presenceand absence of surfactant, as it is not possible toassign a correct value to these parametersfor mixed, protein/surfactant films.21,22 Thisapproach does not influence any of the trendsobserved in these experiments.

The experimental scheme involved the intro-duction of Tween together with rFVIII (coadsorp-tion), after introduction of rFVIII (sequentialadsorption), and before the introduction of rFVIII(‘‘pre-coat’’). For adsorption on hydrophobic silica,different Tween concentrations (8, 28, and 88 ppm)were used in each case. For adsorption on hydro-philic silica, experiments were performed in thepresence of Tween at low (8 ppm) or high (88 ppm)concentrations. It must be noted that rFVIIIadsorption kinetics with no Tween could not berecorded since the protein was obtained in a liquidformulation containing 80 ppm Tween and wasused without any further modification. Thus thelowest Tween concentration studied was 8 ppm,obtained by the 10-fold dilution of the proteinsample during the ellipsometry procedure.

Tween R rFVIII Coadsorption

Frozen rFVIII vials were thawed by holding ina378C water bath. rFVIII solutions (0.5 mL, 0.533mg/mL protein, 80 ppm Tween) were used as is ormixed with 20 mL of concentrated Tween solution

08 DOI 10.1002/jps


to obtain 280 or 880 ppm Tween-containingprotein samples. These samples were vortexedbriefly to ensure good mixing. The Tweenþ rFVIIImixture was then added to the cuvette (containing4.5 mL filtered formulation buffer) to obtain afinal protein concentration of 0.053 mg/mL and aTween concentration of 8, 28, or 88 ppm. Adsorp-tion was monitored for 30 min. The sample wasthen rinsed by flowing buffer through the cuvetteat a flow rate of 30 mL/min for 5 min. Adsorbedmass was monitored for an additional 25 min.

rFVIII—Tween Sequential Adsorption

A rFVIII solution containing 8 ppm Tween wasadded to the cuvette to give a final concentrationof 0.053 mg/mL, and adsorption kinetic data wererecorded for 30 min. The sample was then rinsedby flowing buffer through the cuvette as above,with adsorbed mass monitored for an additional25 min. A Tween solution (0.5 mL) was thenadded such that the final Tween concentrationwas 8, 20, or 80 ppm. Tween was allowed tocontact the surface for 15 min, after which thesample was rinsed with buffer. Adsorbed masswas monitored for an additional 25 min.

Tween R rFVIII Coadsorption at aSurface Precoated with Tween

In this case, 4.4 mL filtered phosphate buffer wasadded to the trapezoidal cuvette. One-tenth (0.1)mL Tween solution was then added such that thefinal Tween concentration in the cuvette after theaddition of protein would be 8, 20, or 80 ppm.Tween adsorption was monitored for 45 min, afterwhich 0.5 mL rFVIII solution was added tothe cuvette to obtain a final protein concentrationof 0.053 mg/mL. Adsorption from the TweenþrFVIII mixture (i.e., the rFVIII which was addedand the Tween that was already present in thecuvette) was monitored for 30 min. The surfacewas then rinsed with buffer for 5 min andadsorbed mass monitored for an additional25 min.

rFVIII Adsorption at a SurfacePrecoated with Tween

This procedure was similar to that described inthe preceding paragraph up to the time of proteinaddition. Here, after 45 min contact with Tween,


the sample was rinsed with surfactant-free,protein-free buffer for 5 min and adsorbed massmonitored for an additional 25 min. Then 0.5 mLrFVIII solution was added to the cuvette to obtaina final protein concentration of 0.053 mg/ml.Adsorption was monitored for 30 min. The samplewas then rinsed with phosphate buffer for 5 minand adsorbed mass monitored for an additional25 min.

Evaluation of rFVIII Tertiary Structure

The tertiary structure of rFVIII, in the presenceand absence of colloidal particles, was studiedusing fluorescence emission spectroscopy.23,24

Tryptophan is a well documented intrinsic fluor-ophore,25 and rFVIII has 37 tryptophan resi-dues.26 Tryptophan fluorescence depends on thelocal environment of the amino acid residueswithin the protein molecule, and is related toprotein tertiary structure. Analysis of fluores-cence emission spectra often involves the calcula-tion of the wavelength at which the fluorescenceintensity is at a maximum (lmax). If the lmax oftryptophan spectra within the protein dissolved inan aqueous medium is shifted to shorter wave-length in relation to the lmax of free tryptophan inwater, the tryptophan is considered internal andin a nonpolar environment.27

The rFVIII stock samples in these testscontained about 100 mg/mL protein and 20 ppmTween-80, formulated in the KG-2 buffer. Thesewere diluted with KG-2 buffer (containing noTween) so that the final Tween concentration was8 ppm. A small volume of concentrated Tweenstock solution was then added to 2 mL rFVIIIsamples to obtain 20 and 80 ppm Tween-contain-ing solutions. The volume of each nanoparticlesuspension added was determined based on thespecific surface area of each suspension and themolecular dimensions of rFVIII. The longest axisof the rFVIII molecule is reported to be 14 nm.8

One rFVIII molecule was assumed to occupya 14� 14 nm area on the surface. The arearequired for the adsorption of all molecules wascalculated by taking the product of the surfacearea occupied per molecule, and the number ofrFVIII molecules in the 2-mL sample volume. A50% excess area was then provided in each case toaccount for molecular proximity effects and toensure that rFVIII molecules had sufficient sur-face area for adsorption. The volume of nanopar-ticle suspension can then be easily estimated by



dividing the area required by the specific surfacearea of the suspension.

The sulfated and amidine polystyrene latticeswere obtained as 8% and 4% (g/100 mL) suspen-sions, respectively and were used as supplied. Thesilica and alumina-coated silica nanoparticles,obtained as 50% and 42% suspensions, werediluted 10-fold using KG-2 buffer just prior touse. The volume of suspension added corre-sponded to the same surface area provided ineach case. The samples were then equilibratedovernight by placing on a tube rotator. Protein-free buffer samples were prepared in an analogousmanner for controlled comparison. After over-night equilibration, samples were transferred to aquartz cuvette and tested for internal fluores-cence. Emission spectra of rFVIII solutions andrFVIII-nanoparticle suspensions was obtainedusing a PTI QuantaMaster fluorometer (PhotonTechnology International). The excitation wave-length was set at 295 nm to selectively excite thetryptophan residues within rFVIII. Emissionspectra were recorded at 1 nm increments from305 to 405 nm. The excitation and emission slitwidths were set at 0.25 and 2.0 mm, respectively.Three scans were recorded and averaged in eachcase in order to increase the signal-to-noise ratio.The data were corrected for protein-free back-ground, and each experiment was performed intriplicate. In order to estimate the wavelength atwhich maximum fluorescence emission (lmax)occurred, the background-corrected data weredifferentiated using the FeliX32 software sup-plied by PTI. The wavelength at which thedifferentiated curve intersected the x-axis wasrecorded as lmax.

Table 2. The Effect of Nanoparticles on the rFVIIIActivity Assay

SampleClot Time (s)(Mean�SD)

Buffer 36.73� 0.70Negative, hydrophobic nanoparticles 38.00� 0.36Positive, hydrophobic nanoparticles 38.60� 0.89Negative, hydrophilic nanoparticles 39.00� 0.56Positive, hydrophilic nanoparticles 38.80� 0.36

Evaluation of rFVIII Biological Activity

The biological activity of rFVIII, in the presenceand absence of colloidal particles, was evaluatedusing a one-stage clotting assay based on theactivated partial thromboplastin time (aPTT).28

In this assay, test samples are incubated at 378Cwith a mixture of FVIII-deficient plasma andaPTT reagent. Calcium chloride is then added tothe incubated mixture to initiate clotting. Aninverse relationship exists between the timerequired for a clot to form and the logarithm ofrFVIII activity. Activity levels for unknownsamples were interpolated by comparing theclotting times of various dilutions of test sampleswith a curve constructed from a series of dilutions


of standard samples of known activity and werereported in International Units per mL (IU/mL).The rFVIII samples used for activity testing wereprepared as indicated for the fluorescence experi-ments. However, the samples were not equili-brated overnight but tested within 6 h ofpreparation. Samples were kept at room tempera-ture prior to actual testing. Samples were testedat least twice.

In order to evaluate the effect of nanoparticleson the assay, control experiments were performedin which a nanoparticle suspension (30 mL) orbuffer was added to plasma containing 1.0 IUrFVIII. Clotting was initiated and the timerequired for clot formation was recorded in eachcase. Tests were performed in triplicate. As shownin Table 2, clot times were similar in the presenceand absence of nanoparticles, indicating they hadno effect on the assay.

RESULTS AND DISCUSSION

rFVIII Structure as a Function of TweenConcentration

The CD spectra of rFVIII at Tween concentrationsof 80, 160, and 280 ppm are shown in Figure 1. TheCD spectra are similar, indicating that Tween hadno effect on rFVIII secondary structure. While CDspectra were not recorded for Tween concentra-tions below 80 ppm, the CD spectra of Figure 1correspond closely to results published by Grilloet al.,29 for rFVIII at low Tween concentration.The CD data were deconvoluted using the ‘‘cdsstr’’program.30 Results are presented in Table 3.rFVIII appeared to have a low helical content anda more pronounced b structure, comparing wellwith results recorded by Grillo. And, the composi-tion of structural elements was independent ofTween concentration.

Figure 2a shows representative results obtain-ed for rFVIII fluorescence in the presence of

08 DOI 10.1002/jps

Figure 1. Circular dichroism spectra of rFVIII in thepresence of Tween-80.

Figure 2. The effect of Tween-80 concentration on (a)fluorescence spectra of rFVIII, and (b) estimates of lmax.


Tween, and indicates spectra were nearly iden-tical at Tween concentrations of 8, 20, and 80 ppm.The calculated lmax for different Tween concen-trations were also nearly identical (Fig. 2b) andstatistical analysis revealed no significant differ-ence between groups (p> 0.05). In summary thesedata suggest Tween-80 did not alter the structureof the native rFVIII molecule in any substantialway.

Tween R rFVIII Coadsorption on Hydrophobic Silica

The adsorption kinetics of rFVIII in the presenceof 8, 28, and 88 ppm Tween on hydrophobic silicaare shown in Figure 3. A reduction in adsorptionwith an increase in Tween concentration wasobserved, with reduced amounts remaining at theinterface after rinse. These observations wouldbe consistent with Tween being able to locate atthe interface more rapidly than rFVIII (owing to

Table 3. Calculated Secondary Structure of rFVIII inthe Presence of 80, 160, and 280 ppm Tween-80

Secondary StructureComponent

Tween Concentration

80 ppm 160 ppm 280 ppm

a-helix 6 6 83/10 helix 4 5 4Extended b-strand 26 28 26b-turns 14 13 13Polyproline-like 3/1 helix 9 8 9Others 41 41 41


its smaller size and higher diffusivity), in this wayinhibiting rFVIII adsorption, and/or Tween asso-ciating with rFVIII in solution forming complexesof reduced surface affinity. Alternatively Tweenmay simply have a higher adsorption affinitythan rFVIII for the hydrophobic surface. The

Figure 3. Adsorption kinetics of rFVIII on hydropho-bic silica in the presence of 8, 28, and 88 ppm Tween.


Figure 4. Adsorption kinetics of rFVIII on hydropho-bic silica followed by buffer elution and the introductionof Tween at concentrations of 8, 20, 80, and 200 ppm.


adsorption kinetics for the rFVIII-Tween solu-tions at 28 and 88 ppm Tween were comparable tothat for pure Tween adsorption, suggesting thatTween may be dominating the adsorption at highconcentrations. (Tween adsorption in the absenceof protein appears in Fig. 5.)

A reduction in adsorbed amounts at thehydrophobic surface with an increase in Tweenconcentration was also observed for lysozyme-Tween coadsorption.17 The molecular weight ofrFVIII is about 20 times greater than that oflysozyme, and it is possible that faster diffusionof Tween may assume greater importance in theobserved reduction in rFVIII adsorption ascompared with lysozyme. A completely unambig-uous interpretation of coadsorption results iscomplicated by the lack of data for surfactant-free rFVIII adsorption. It is likely that rFVIII byitself would adsorb in a greater amount thanobserved upon coadsorption with 8 ppm Tween.In fact, we have recently recorded adsorption ofrFVIII (0.079 mg/mL) in KG-2 buffer including2.2 ppm Tween to be substantially higher thanthat shown in Figure 3 (between 0.4 and 0.5 mg/cm2 after rinse), on a hydrophobic surfaceprepared by silanization with hexamethyldisila-zane (HMDS).

The Tween concentrations selected in these testsare believed to include values below and above theCMC. In earlier experiments,17 we used interfacialtensiometry to identify the CMC of Tween-80 inrFVIII-Tween mixtures, using a rFVIII concentra-tion of 24.5 mg/mL (about one-half the concentra-tion used in these ellipsometry experiments). Theobjective in this kind of approach is to determinethe surfactant concentration at which steady stateinterfacial behavior is governed entirely by surfac-tant. While we were able to estimate a CMCbetween 55 and 60 ppm by this method, compar-ison of the steady state surface tension datarecorded for rFVIII-Tween mixtures with similardata recorded for Tween in the absence of rFVIIIsuggested that steady state interfacial behavior isgoverned entirely by surfactant at Tween concen-trations well below 55 ppm. In particular, werecorded no appreciable difference in the steadystate value of interfacial tension demonstrated byrFVIII-Tween mixtures and by Tween alone, oncethe Tween concentration reached about 18 ppm. Insummary, the CMC of Tween-80 in these rFVIIIformulations was not unambiguously determined,but the lowest and highest Tween concentrationsused here are believed to be below and above theCMC, respectively.


rFVIII—Tween Sequential Adsorptionon Hydrophobic Silica

The kinetics of rFVIII adsorption (in the presenceof 8 ppm Tween) followed by buffer elution andthe introduction of Tween at concentrations of 8,20, and 80 ppm are shown in Figure 4. Theadsorption and elution kinetics recorded duringthe first 60 min in each case were similar, as theyrepresent identical experimental conditions. Atthe end of 60 min, the adsorbed layer was likelycomposed of stably bound rFVIII and Tweenmolecules that resisted buffer elution. WhenTween solutions were introduced, adsorbed masswas observed to increase, consistent with Tweenadsorption at the empty sites on the surface andon the adsorbed protein layer. After rinsing,adsorption values decreased to values near thoserecorded prior to Tween addition suggesting thatthe Tween was loosely held and had no effect onthe amount of protein adsorbed. This is contraryto the results obtained with the lysozyme-Tweensystem,17 where a net removal of lysozyme wasobserved after introduction of 80 ppm Tween.rFVIII is a much larger molecule than lysozyme,allowing the formation of more noncovalentcontacts with the surface and tighter bindingthan observed for lysozyme at a similar surface. Infact, no reduction in adsorbed amount of rFVIIIwas recorded even when Tween was introduced at200 ppm (data not shown).

It is also instructive to compare the amountsadsorbed when 8 ppm Tween was introducedtogether with rFVIII (30-min value in Fig. 4) andsequentially (75-min value in Fig. 4). The 75-min

08 DOI 10.1002/jps


value was greater, suggesting history-dependentmolecular rearrangement enabling a more effi-cient interfacial packing of Tween molecules.31–33

Tween R rFVIII Coadsorption on HydrophobicSilica Precoated with Tween

Tween adsorption at the hydrophobic surface,followed by the addition of rFVIIIþ 8 ppm Tweenand elution with buffer are shown in Figure 5 (thesolution present in the cuvette at the time ofrFVIII introduction contained the Tween intro-duced to form the precoat). Data recorded forrFVIIIþ 8 ppm Tween adsorption with no precoatare superimposed on Figure 5 beginning at 45 min,for comparison with protein addition in the precoatexperiments. Tween adsorbed in comparableamounts when added at 8, 20, and 80 ppmconcentrations. For the 8 ppm precoat, the amountadsorbed after protein addition was similar to thatwith no precoat. The data for the 20 ppm precoatshowed a delayed increase in adsorbed amount,eventually reaching the value attained with noprecoat. The 80 ppm precoat showed nearly nofurther increase in adsorbed amount after additionof protein. If the Tween interfacial layers in eachcase were in similar states of orientation and‘‘packing’’, the difference in rFVIII adsorption tothe Tween-coated interface may then be attributedto the difference in Tween concentration insolution. Alternatively, Tween may experience aconcentration-driven ‘‘packing optimization’’ at theinterface which serves to minimize further rFVIIIadsorption.

Figure 5. rFVIIIþTween coadsorption on hydropho-bic silica precoated with Tween.


rFVIII Adsorption on HydrophobicSilica Precoated with Tween

In these experiments a rinsing step was intro-duced after the formation of a Tween precoat andprior to the addition of rFVIII. This step removedTween present in solution as well as any Tweenloosely held at the interface. Figure 6 shows thata large fraction of Tween molecules remainedadsorbed even after the hydrophobic surface wasrinsed with buffer. However, the interfacialTween was unable to prevent rFVIII adsorption.When rFVIII together with 8 ppm Tween wasadded at 75 min, a nearly instantaneous increasein adsorbed amount was observed. The adsorptionplateaus reached after rFVIII addition weresimilar, irrespective of the Tween concentrationused to generate the precoat.

The importance of direct interaction betweensurfactant and solid surface, relative to surfac-tant-protein association in solution, in the mod-ulation of rFVIII adsorption by Tween-80 isdifficult to quantify with certainty in experimentswith hydrophobic surfaces, as adsorption affi-nities are high for Tween as well as for rFVIII.Therefore, in order to more clearly isolate thecontribution of interfacial Tween to the observedrFVIII adsorption behavior experiments wereperformed with hydrophilic silica, for whichTween-80 has little affinity.

Tween and rFVIII Adsorption onHydrophilic Silica

Figure 7 shows adsorption kinetics for rFVIII inthe presence of 8 and 88 ppm Tween on hydro-philic silica, plotted with similar experimentsperformed with hydrophobic silica (replotted fromFig. 3). The amounts adsorbed on hydrophilicsilica were much greater than those on hydro-phobic silica. It is generally observed that proteinsadsorb in greater amounts on hydrophobic than onhydrophilic surfaces. In this case, the greaternegative charge density on the hydrophilic silicasupports an adsorption driving force of electro-static attraction rather than hydrophobic inter-action. The overall isoelectric point of rFVIII is6.8, but McGuire et al.34 have demonstratedthe importance of charge location and mobility(as opposed to overall charge) in governing theprotein adsorption process. It is probable that amobile, positively charged and solvent accessibledomain on the rFVIII molecule is able to bind tothe negatively charged surface with high affinity.


Figure 6. rFVIIIþTween coadsorption on hydropho-bic silica precoated with Tween (no Tween present inthe rinse buffer).


In fact, the B-domain in rFVIII is calculated tohave a pI of 9.5 and therefore a highly positivecharge, and may be mediating the adsorption tohydrophilic silica. The charge calculations arebased on the rFVIII primary sequence and do notconsider the effect of glycosylation.

There was no reduction in adsorbed amount onthe hydrophilic silica when Tween concentrationwas increased from 8 to 88 ppm. In addition, therewas no reduction in adsorbed amount on thehydrophilic silica upon rinsing, even when Tween-80 was dissolved in the rinse buffer at 80 ppm(data not shown). The dynamics of the protein-surfactant interaction in solution, as well asdiffusion of each component, should remain

Figure 7. rFVIIIþTween coadsorption on hydrophi-lic silica (^: 8 ppm Tweenþ rFVIII on hydrophilicsilica; &: 88 ppm Tweenþ rFVIII on hydrophilicsilica; D: 8 ppm Tweenþ rFVIII on hydrophobic silica;*: 88 ppm Tweenþ rFVIII on hydrophobic silica).


independent of surface hydrophobic–hydrophilicbalance. So the differences in adsorption behaviorat hydrophobic and hydrophilic surfaces is attrib-uted to the direct interaction between thesurfactant and surface. In summary, the Tween-hydrophilic surface association is weak in com-parison to the Tween-hydrophobic surface asso-ciation. Even if Tween is adsorbed at thehydrophilic surface, rFVIII is apparently able toreplace it owing to the weak Tween-surfaceinteraction in that case.

In order to verify the role of electrostaticattraction in the adsorption process, the concen-tration of NaCl was reduced from 220 to 22 mMNaCl during rFVIII adsorption, while all othercomponents remained unchanged. As shown inFigure 8, rFVIII adsorption increased when theionic strength was reduced. The presence of salt athigher concentrations more effectively shields thecharges on the surface and on the protein. Anincrease in protein adsorption upon decreasingsalt concentration is a well-documented outcomeof electrostatic forces playing a dominant role inadsorption.

Figure 9 shows that when Tweenþ rFVIIIcoadsorption takes place on hydrophilic silicaprecoated by the adsorption of Tween (80 ppm for45 min), rFVIII apparently replaced Tween at thehydrophilic surface, adsorbing to an extentsimilar to that recorded on bare hydrophilic silica(Fig. 8). Tween adsorbed very weakly to thehydrophilic silica, as evidenced by the low surfaceconcentration (<0.05 mg/cm2) and the unstablenature of the adsorption kinetics.17 The data ofFigure 9 indicate that rFVIII rapidly replacesTween at the hydrophilic surface, while very littlerFVIII adsorption is detected on the Tween layerat the hydrophobic surface. This underlines theimportance of direct interactions between adsorb-ing species and the surface, relative to surfactant-protein association in solution, in modulatingrFVIII interfacial phenomena.

rFVIII Structure in the Presenceof Nanoparticles

rFVIII fluorescence emission was evaluated in thepresence of nanoparticles possessing differentsurface chemistries. Figure 10 shows representa-tive fluorescence spectra and Figure 11 shows thecalculated values of lmax in the presence ofnanoparticles at different Tween concentrations.

The lmax shifted to longer wavelengths(338.5� 1.18 nm) as compared to native, when

08 DOI 10.1002/jps

Figure 8. Effect of salt concentration on the adsorp-tion of rFVIII (with 8 ppm Tween) at hydrophilic silica.


rFVIII was adsorbed on negative, hydrophobicnanoparticles at low Tween concentration. Thissuggests that the tertiary structure of rFVIII wassignificantly altered in a manner that exposestryptophan residues to the aqueous environment.It is possible that Tween binding to such exposed,hydrophobic regions attenuated the fluorescencesignal to some extent. As more Tween was added,the lmax upon adsorption approached the nativevalue, suggesting that the addition of Tweenprevented unfolding. This is consistent with theellipsometry results just discussed, where Tweenwas observed to adsorb strongly to (negativelycharged) hydrophobic silica and reduce rFVIIIadsorption through steric repulsion. When rFVIIIwas adsorbed on positive, hydrophobic nanopar-

Figure 9. Comparison of rFVIIIþTween coadsorp-tion on hydrophobic and hydrophilic silica, each pre-coated with Tween.


ticles in the presence of low Tween, the lmax

shifted to shorter wavelengths (331.6� 0.44 nm)as compared to native. Thus, there was someindication of a structure change upon adsorption.But in this case, the tryptophan residues appear tobe on average more buried and less exposed to theaqueous environment than the native molecule.The reason for the difference in the direction of thelmax shift on positively and negatively chargedhydrophobic surfaces is not clear but may possiblybe attributed to different orientations adopted bythe rFVIII in response to the surface charge. AsTween concentration was increased, the lmax uponadsorption approached the native value, consis-tent with the trend observed with negative,hydrophobic nanoparticles.

Fluorescence spectra can be sensitive to subtlechanges in tertiary structure that lead to aggre-gation of the protein, and aggregation phenomenacannot be summarily dismissed in these experi-ments. But protein adsorption at solid surfaces isan inherently irreversible process, in the sensethat spontaneous desorption by buffer dilutionis not generally observed. In this regard, smallchanges observed in the presence of nanoparticlesare interpreted here to be a result of postadsorp-tive structure change consistent not with aggre-gation (which would be associated with weakenedprotein-surface association) but with tighterbinding to the nanoparticle surface.

When rFVIII was adsorbed on negative, hydro-philic nanoparticles in the presence of low Tween,there was no significant shift in lmax as comparedto native. Ellipsometry results on negativelycharged silica indicated that rFVIII adsorbed inrelatively large amounts, as a result of electro-static attraction. rFVIII adsorption to the nega-tive, hydrophilic nanoparticles is likely governedby the same mechanism, but Figure 11c suggeststhis adsorption did not induce any unfolding. Thiswould be consistent with formation of a tightlypacked and highly ordered layer of rFVIIImolecules on the surface. In this case, withpositively charged domains mediating adsorption,unfolding would not be energetically favorable if itreduced the electrostatic attractive force. And, ata crowded interface unfolding would also beinhibited by steric constraints. As the Tweenconcentration was increased, the lmax appeared toshift to a shorter wavelength. The reason for thisremains unclear. The lmax in the presence ofpositive, hydrophilic nanoparticles displayed onlysmall deviations from the native sample at allTween concentrations studied.


Figure 10. rFVIII fluorescence spectra in the presence of (a) negative, hydrophobicnanoparticles; (b) positive, hydrophobic nanoparticles; (c) negative, hydrophilic nano-particles; and (d) positive, hydrophilic nanoparticles.


rFVIII Activity in the Presence of Nanoparticles

The biological activity of rFVIII samples contain-ing nanoparticles bearing various surface char-acteristics was evaluated with the one-stageclotting assay. Control samples with no nanopar-ticles were evaluated along with every experi-mental run. Results are presented in Figure 12.

A large decrease in activity was observed whennegative, hydrophobic nanoparticles were addedto the rFVIII sample at low Tween concentration.This decrease in activity is consistent with thesignificant loss in rFVIII tertiary structureobserved under similar conditions (Fig. 11a).Biological activity was recovered as Tween con-centration was increased, and this can be attrib-uted to reduced adsorption of rFVIII resultingfrom strong association between Tween moleculesand nanoparticle surfaces. The positive hydro-phobic surface also caused an activity loss at lowTween, and improved recovery with increasing


Tween. The hydrophilic, negative surface causedthe least loss in rFVIII activity. This suggeststhat while the protein molecules were adsorbed,their active sites remained solvent accessible anddid not mediate the adsorption. An unusuallylarge decrease in activity was noted in thepresence of hydrophilic, positive nanoparticles,and this may be related to the alumina coatingthey possess. Aluminum ions, even when presentat low concentration (e.g., 10 mM), have beenimplicated in the aggregation and inactivation ofrFVIII.35 It is likely that aluminum ions presentat the interface as well as any leached out insolution, caused the large decrease in rFVIIIactivity.

As the B-domain possesses a high positivecharge and is not required for rFVIII biologicalactivity,36 we would hypothesize that rFVIIIadsorbs on a negatively charged, hydrophilicsurface in an orientation that aligns the B-domaintoward the surface. Since a particular orientation

08 DOI 10.1002/jps

Figure 11. lmax in the presence of (a) negative, hydrophobic nanoparticles; (b) posi-tive, hydrophobic nanoparticles; (c) negative, hydrophilic nanoparticles; and (d) positive,hydrophilic nanoparticles.

Figure 12. rFVIII biological activity in the presence ofnanoparticles. Control samples contained no nanoparticles.



would be preferred in this instance, such anadsorbed layer is likely to be ordered and packed.This is consistent with our observations of highadsorbed amounts of rFVIII, and good preserva-tion of structure and activity, on negativelycharged, hydrophilic surfaces.

Adsorption on a hydrophobic surface is likely tobe more random due to the potential for bothhydrophobic and electrostatic associations. Uponadsorption, rFVIII active sites may not remainavailable to participate in the clotting cascade.This, in addition to entropically driven structuralalteration is consistent with our observations ofstructure change and activity loss on hydrophobicsurfaces at low surfactant concentration. Preser-vation of structure and activity at hydrophobicsurfaces in the presence of sufficient Tween-80 is a



result of Tween adsorption at the surface, at oncesterically inhibiting rFVIII adsorption, and limit-ing the space available for surface-inducedunfolding of any rFVIII molecules that happento be adsorbed.

CONCLUSIONS

The presence of Tween-80 in rFVIII solutionsdecreased rFVIII adsorption on hydrophobicsilica, and this reduction in adsorbed proteinincreased with Tween concentration in solution.In addition, if Tween is introduced to thehydrophobic surface at sufficiently high concen-tration prior to rFVIII addition, rFVIII adsorptioncan be reduced or even prevented with sufficientTween present in solution. The tertiary structureof rFVIII was altered and biological activity wasdecreased in the presence of hydrophobic surfacesat low Tween concentrations. At high Tweenconcentrations, structure and activity were betterpreserved. On the other hand, Tween was noteffective in modulating rFVIII adsorption onhydrophilic silica. These data indicate that strongTween-surface association is necessary to inhibitprotein adsorption, while the Tween-proteinassociation in solution is of relatively smallconsequence. Accordingly, the rapid diffusion ofTween (relative to protein) to the interface islikely to contribute to a reduction in proteinadsorption, structure change and activity loss,only if Tween-surface affinity is sufficiently high.While hydrophilic surfaces were associated withrelatively high rFVIII adsorption, they did notinduce large changes in structure or activity. Thiswas attributed to the formation of a tightlypacked, highly ordered adsorbed layer on thesesurfaces, not mediated by the rFVIII active site.

ACKNOWLEDGMENTS

This work was supported by Bayer HealthCareLLC, Berkeley, CA.

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