Assessment of formulation robustness for nano-crystalline suspensions using failure mode analysis or derisking approach

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Assessment of formulation robustness for

nano-crystalline suspensions using failure mode analysis

or derisking approach

Mostafa Nakach, Jean-Rene Authelin, Cecile Voignier, Tharwat Tadros,

Laurence Galet, Alain Chamayou

To cite this version:

Mostafa Nakach, Jean-Rene Authelin, Cecile Voignier, Tharwat Tadros, Laurence Galet, et al..

As-sessment of formulation robustness for nano-crystalline suspensions using failure mode analysis or

derisking approach. International Journal of Pharmaceutics, Elsevier, 2016, 506 (1-2), p. 320-331.

�10.1016/j.ijpharm.2016.04.043�. �hal-01593327�

Assessment

of

formulation

robustness

for

nano-crystalline

suspensions

using

failure

mode

analysis

or

derisking

approach

Mostafa

Nakach

a,

_*

_,

_Jean-René

_Authelin

a

_,

_Cecile

_Voignier

c

_,

_Tharwat

_Tadros

b

_,

Laurence

Galet

c

_,

_Alain

_Chamayou

c

a_{SanofiR&D,13,quaiJulesGuesde,94403VitrysurSeine,France} b_{89NashGroveLane,Wokingham,BerkshireRG404HE,UK}

c_{EcoledesMinesd’Albi,CampusJarlard,RoutedeTeillet83013AlbiFrance,France}

Keywords: Nano-crystallinesuspension Wettingagent Dispersingagent Flocculation Aggregation Shearrate ABSTRACT

Thesmallparticlesizeofnano-crystallinesuspensionscanberesponsiblefortheirphysicalinstability duringdrugproductpreparation(downstreamprocessing),storageandadministration.Forthatpurpose, thecommercialformulationneedstobesufficientlyrobusttovarioustriggeringconditions,suchasionic strength,shearrate,wetting/dispersingagentdesorptionbydilution,temperatureandpHvariation.In ourpreviousworkwedescribedasystematicapproachtoselectthesuitablewetting/dispersantagentfor thestabilizationofnano-crystallinesuspension.In thispaper,we describedtheassessmentofthe formulationrobustness(stabilizedusingamixtureofsodiumdodecylsulfate(SDS)and polyvinylpyr-rolidone(PVP)and)bymeasuringtherateofperikinetic(diffusion-controlled)andorthokinetic (shear-induced)aggregationasafunctionofionicstrength,temperature,pHanddilution.Theresultsshowed that,usingtheSDS/PVPsystem,thecriticalcoagulationconcentrationisaboutfivetimeshigherthanthat observedintheliteratureforsuspensioncolloidalystableathighconcentration.Thenano-suspension wasalsofoundtobeverystableatambienttemperatureandatdifferentpHconditions.Desorptiontest confirmed the high affinity between API and wetting/dispersing agent. However, the suspension undergoesaggregationathightemperatureduetothedesorptionofthewetting/dispersingagentand disaggregationofSDSmicelles.Furthermore,aggregationoccursatveryhighshearrate(orhokinetic aggregation)byovercomingtheenergybarrierresponsibleforcolloidalstabilityofthesystem.

1. Introduction

Nano-crystalline suspensions are used in pharmaceutical industry toenhance biopharmaceutical performances of highly water insoluble active pharmaceutical ingredient (API). Their colloidalparticlesizerangeofferstheadvantageoflargesurface areaperunitvolumethatprovidestherequiredpropertiesofthe finalproduct(ShegokarandMüller,2010).Forthepreparationof nano-crystalline suspensions, the particle size reduction is the mostcommonlyusedmethodduetothepossibilityofcontrolling particlesizebysuitableselectionofwetting/dispersingagent,as wellas bycontrolof millingprocess parameters(Peltonenand

Hirvonen, 2010). The ability of nano-crystalline suspension to

remaininitsoriginalstateduringdrugproductpreparationand administration(processingorinusehandling)iscriticalforsince anychangecouldnegativelyimpactitsperformances.Infact,all suspensions having particlesize less than 1

m

mare inherently thermodynamicallyunstableduetothenaturaltendencydecrease thelargespecificsurface areaand excess surfaceenergy (Patel, 2010). For this purpose, wetting/dispersing agents are used to stabilizethesuspensionagainstflocculation(Holthoffetal.,1996;

Lautenetal.,2001)andcrystalgrowth(Ostwaldripening(Ostwald,

1901)).

In a previous work (Nakach et al., 2014), we described a systematicapproachtoselectasuitablewetting/dispersingagent forthepreparationofnano-crystallinesuspensions.Theobjective ofthispaperistodescribethederiskingapproachimplementedfor therobustnessassessmentoftheselectedformulationwithregard totheidentifiedrisksthatarelistedbelow:

(i)Particlesagglomeration duringmilling: Duringhigh-energy milling,thesizeofparticlesdecreasestosomecriticalvalues.

* Correspondingauthor.

E-mailaddresses:mostafa.nakach@sanofi.com(M.Nakach),

Jean-Rene.Authelin@sanofi.com(J.-R.Authelin),cecile.voignier@mines-albi.fr (C.Voignier),tharwat@tadros.fsnet.co.uk(T.Tadros),laurence.Galet@mines-albi.fr (L.Galet),alain.chamayou@mines-albi.fr(A.Chamayou).

Furtherenergysupplytotheseparticlesoflimitingsizecauses furtherdeformationofparticles,energyaccumulationinthe volumeoratthesurfaceofparticles,andsubsequentlyleadsto aggregation. To evaluate such risk, a longmilling duration (13h)athighshearratewascarriedinordertoevaluateifthe suspensionundergoesnegativemillingphenomenonwhichis incloserelationwithaggregationandagglomeration(

Ander-sonandLekkerkerker,2002).

(ii)Aggregationduringstorageoradministrationduetothelack ofelectrostaticstabilization:Aggregationoccursiftheenergy barrier is small or negligible. According to Sato and Ruch

(1980), an energy barrier of 15kBT is sufficient to prevent

aggregation since thermal energy is 1kBT, where, kB is

Boltzmannconstant andTistheabsolutetemperature.The height of the energy barrier depends on the electrolyte concentrationwhichisdirectlyrelatedtothethicknessofthe doublelayer.Basically,whenasaltisaddedtothesuspension, theelectricaldoublelayerrepulsionisscreened,andvander Waalsattractionbecomesdominantandinducesfast aggre-gation of particles(Yuand von Gottberg, 2002).Atcritical electrolyte concentration (CCC) the energy maximum dis-appearsleadingtofastaggregationofparticles.Inourwork, theCCCwasdeterminedbyquantificationofaggregationrate asafunctionofelectrolyteaddition.

(iii)Shear-inducedaggregation(orthokineticaggregation): Aggre-gation processes are always carried out under conditions wheresuspensionissubjectedtosomeshearbystirringorby flowleadingtoincreaseofcollisionfrequency(Potanin,1991). Inourstudy,Orthokineticaggregationwasevaluatedundera fixedshearrate.

(iv)Aggregationbydesorptionofwetting/dispersingagentfrom particles surface: Robustness to dilution is important for nano-crystalline suspension to ensure that the particles formed have similar properties at different dilutions to achieveuniformdrugreleaseprofile andtoensurethatthe drugwillnotgetaggregationathigherdilutionsinvivowhich maysignificantlyimpactbiopharmaceuticalattributesofthe product.Wehadpreviously,demonstratedthattheadsorption of polymeric surfactants (PVP K30) led to high affinity isothermimplyingthatsuchprocessisirreversible(Nakach etal.,2014).Tochecktheirreversibilityofadsorbedsurfactant, desorptionof surfactantfromparticlessurfacewas investi-gatedbydilutingthesuspensioninwater.

(v)CrystalgrowthduetoOstwaldrepiningand/orflocculationby depletionathightemperature:Duringmillingorautoclaving (incaseofsuspensionuseinparenteraladministration),the suspensioncouldbesubmittedtohightemperature.In this case, large particlesgrow with time at theexpenseof the smallerones(Ostwald,1901)duetothewell-knownKelvin effect (Hiemenz and Rajagopalan,1997).Furthermore, high temperature can lead to desorption of polymer molecules which become more soluble (Tadros, 2012) or precipitate when the temperature is higher than polymercloud point

(Cortietal.,1984).Consequently,thenon-adsorbingpolymer

molecules can induce flocculation by depletion interaction betweencolloidalparticles(JenkinsandSnowden,1996).In this paper, we evaluated the sensitivity of suspension to Ostwald repining and/or flocculation by depletion at high temperature.

(vi)Flocculationdue topHvariation: According totherouteof administration,anAPI experiencesawide rangeof physio-logicalpH.Accordingly,nano-suspensionneedstoberobustly designed with regardstopHvariations which may signifi-cantly affect the effectiveness of electrostatic stabilization

(Kiratzisetal.,1999).Indeed,pHisanimportantparameterto

betakenintoaccountbecausetheparticlesurfaceisstrongly

modifiedbyacid-base equilibriums,andtheparticlecharge mayvaryfromnegativetozeroandtopositivevalues.Infact, byvaryingthepH,theisoelectricpoint(IEP)canbereached. TheIEPisthepHvalueatwhichthezetapotentialvalueis zero,implyingnoelectricchargeonthesurfaceofaparticle. For that purpose, the sensitivity of our formulation to pH variationswasinvestigated.

2. Experimental 2.1.Materialandmethods 2.1.1.Materials

AhydrophobichighlyinsolubleAPIpowderwas providedby SanofiR&D(Paris).Itwasmicronizedbyjetmillingbeforeuse.The physico-chemicalpropertiesoftheAPIaregiveninTable1.

Polyvinylpyrrolidone PVP (K30) (Molar mass: 30,000g) was purchasedfromBASF(France),sodiumdodecylsulfate(SDS)was purchased from Univar (France), Vitamin E TPGS1 _(d-alpha

tocopheryl polyethylene glycol 1000 succinate) was purchased from Eastman Chemical Company (Netherlands), Solutol1

HS15 was purchased from BASF (France) and Sodium chloride waspurchasedfromSigmaAldrich(France).

2.1.2.Methods

2.1.2.1.Suspensionpreparation

2.1.2.1.1.Preparationofmilledsuspensionforevaluationofperikinetic aggregation,orthokineticaggregation,sensitivitytotemperatureand pHvariation. ThemilledsuspensionswerepreparedusingAPIat concentration of 20% (w/w)and SDS/PVPas wetting/dispersing agent at concentration of 1.2% (w/w). An aliquot of 50ml suspension and 50ml of Cross-linked Polystyrene beads (500

m

m diameter supplied by Alkermes (USA)) were introduced in Nano-mill 011 _{(annular mill purchased from}

Alkermes, having a stator of 80mm diameter and rotor of 73mm).Themillwas operatedduring150minat10.8m/s.The milltemperaturewasmaintainedat10!_C.

2.1.2.1.2. Assessment of suspension behavior during long milling duration trial. In ourpreviouswork,thescreening of wetting/ dispersingagentwascarriedoutintwoparts(Nakachetal.,2014): (i)Part 1 focused on qualitative screening to select the lead candidate.Attheendofthisparttwoformulationsappeared clearlysuperiortotheothers:SDS/PVPandVitaminETPGS1_.

(ii)Part2focusedonquantitativescreeningaimedtooptimizethe selected lead. For this part, the SDS/PVP made from ionic surfactant (SDS) and polymer (PVP) considered as more relevantwasselectedfortheoptimization.

Inthepresentstudy,we decidedtocomparethesuspension madeofSDS/PVPtothatmadeofvitaminETPGS1_{(asreference)in}

Table1

Physico-chemicalpropertiesoftheAPIusedforthisstudy.

Averageparticlediameter 5mm Molecularweight(g/mol) 497.4 Watersolubility(mg/ml) 0.2

LogPa _6.9

Density(g/ml) 1.42

Meltingpoint(!_{C) 156.7}

termsoftheirresistancewhentheyaresubmittedtohighshear rateduringalongmillingduration(13h)

Themilledsuspensionswerepreparedasfollows:

" SuspensionAmadeofAPIatconcentrationof 20%(w/w)and SDS/PVPaswetting/dispersingagentatconcentrationof1.2%. " SuspensionBmadeof APIatconcentrationof20% (w/w)and

VitaminETPGS1_{aswetting/dispersingagentatconcentrationof}

3%.

50ml aliquot of each suspension and 50ml of Cross-linked Polystyrene beads(500

m

m) wereintroducedin Nano-mill011_.

Themillwasoperatedduring13hat11.6m/s.Themilltemperature wasmaintainedat10!_C.

Themeanshearrategeneratedduringmillingcanbecalculated accordingtheEq.(1)(Spiceretal.,1996).

_

g

¼ ffiffiffiffiffiffi Pv

h

s ð1Þ where,

h

isthesuspensionviscosityandPvisthepowerdensity determinedaccordingtheequation

Pv¼_VP ð2Þ

Pis thenetpowerdraw(58W)measuredduringmillingusing wattmeterandVisthesuspensionvolume(50ml)

Themainshearratewasfoundabout22000s&1_.

Theoverallstraingeneratedduringmillingcanbecalculated accordingtheEq.(3)

g

¼ _

g

't¼1:0109 ð3Þ wheretisthemillingduration

2.1.2.1.3. Preparation milled suspension for desorption evaluation. For desorption assessment, we compared the suspension made of SDS/PVP to that made of solutol1 _(as

reference) whichwasgatedoutfromthescreeningdoneinour previouswork(Nakachetal.,2014).Themilledsuspensionswere preparedasfollows:

" SuspensionAmadeofAPIatconcentrationof 20%(w/w)and SDS/PVPaswetting/dispersingagentatconcentrationof1.2%. " SuspensionCmadeof APIatconcentrationof20% (w/w)and

Solutol1_{aswetting/dispersingagentatconcentrationof3%.}

50ml aliquot of each suspension and 50ml of Cross-linked Polystyrene beads(500

m

m) wereintroducedin Nano-mill011_.

The mill was operated during 150min at 10.8m/s. The mill temperaturewasmaintainedat10!_C.

2.1.2.2.Perikineticevaluationofmilledsuspension. Therearetwo regionsofcoagulation:diffusion-controlled(fastcoagulation)and reactionlimitedcoagulation(slowcoagulation)(Elimelechetal., 1995). It shall be assumed that every collision is effective in forming an aggregate (collision efficiency=1), so that the aggregation rate constant is the same as the collision rate constant(PittandHounslow,2015).

Theaggregationprocesswasconsideredtoberepresentedbya second orderkinetic(describedby theEq.(4))assuggestedby Schmoluchowski(forthefastcoagulationrate)andbyFuchsfor theslowcoagulationrate(Fuchs,1936;Schmoluchowski,1917). dnt

dt ¼ka(n2t ¼numberofcollision ð4Þ

where nt, tand ka arethe total number of particles, time and

secondorderconstantrespectively.TheEq.(4)canbeintegrated withtheinitialconditionnt=n0(n0istheinitialconcentrationof

particles)togiveEq.(5). n0 nt¼ 1þ t

t

;

t

¼ 1 Kan0 " # ð5Þ Thecharacteristic time

t

is often referredto asthehalf-life of aggregation. At this time the total number of particles in the dispersionhas been reduced bya factor of 2. In the regionof reactionlimitedcoagulation,therateofcoagulationisreduceddue to the additional repulsive force from electrostatic or steric interaction.Inthisregion,noteverycollisionresultsincoagulation. Onlya fraction1/Wof collisionsis successful. Wis commonly definedasthestabilityratio,whichis theratioof thediffusion limited(fast)coagulationratetotheslowcoagulationrate.When W=1,thecoagulationisinthediffusion-limitedregion.

W¼

_t

t

ð_slowfastÞ

ð Þ ð6Þ

Thecharacteristictime

t

canbecalculatedfromtheslopeofline representedbyn0/ntversustime(t).Thetotalnumberofparticles

at each time can be calculated from the weight-weight % of suspensionandthemassofasingleparticlethatcanbecalculated fromitsvolumeanddensityaccordingtothefollowingequations, mtotal¼ masseofsampleÞ₁₀₀ (ð%ofsolidÞ

" # ð7Þ mSP¼

r

(V¼

r

(4_{3 (}

p

(r3 ð8Þ r¼d₂ ð9Þ nt¼m_mtotal sp ð10Þ

where,mtotal,msp,

r

,V,randdarethetotalmassoftheparticles,

themassofsingleparticle,thedensityofparticle,thevolumeof singleparticle,particleradiusandparticlediameter,respectively. Theaggregationratemeasurementwascarriedoutbyfollowing the particle size as a function of time using dynamic light scattering(DLS).Differentsolutionsofvaryingionicstrengthwere preparedbydilutingtheappropriate5.0Msodiumchloride(NaCl) standardsolutionwithpurifiedwater.Themilledsuspensionwas dilutedwithpurifiedwaterfrom20%w/wto0.1%w/w.10

m

lofthis dilutedsuspensionwereaddedto1mlNaClsolutioncoveringa wide range of concentrationand theresulting suspension was gentlymixedbyhandintheDLScuvetteandthenplacedintothe measuringcelloftheDLSinstrument.

2.1.2.3. Orthokinetic evaluation. In addition to the Brownian motion, particles movements and their collision rates can be modified by applying an orthokinetic force (shear-influenced aggregationinducebyfluidtransport).Therateofchangeinthe total concentration of particles with time due to Orthokinetic aggregationisexpressedasfollow(LeBerreetal.,1998;Tolpekin

etal.,2004).

dnt

dt ¼&

2'

a

' _

g

'd3'n2t

where,ntisthetotalconcentrationofparticlesinsuspensionat

timet,

a

isthecollisionefficiencyfactor, _

g

isthemeanshearrate anddistheparticlediameter.

Theortho-kineticevaluationwasassessedonsuspensionof20% API using a kinetic experiment by measuringthe particle size (Laserdiffraction). Theevaluationwas performedusingannular millwithoutbeadsat3880rpm(15m/s),during7h.Thetestwas performedatnativeionicstrength(withoutsaltaddition)andat 0.17 molar of Nacl (corresponding to 0.24(CCC according to

Sommer (2007)).The applied shearrate and shearstrain were

foundequalto26,000s&1_and6.5(108_{,respectively.}

2.1.2.4.Measurementofdesorption. Thedesorptionofthewetting/ dispersingagentwas carriedout usinga kineticexperimentby followingtheparticlesizeasafunctionoftime(upto45min)using laserdiffraction(MalvernMastersizer2000)inrecirculationmode at1800rpm.Thesuspensionwasdilutedbyfactorof1000. 2.1.2.5.EvaluationofpH-inducedaggregation. Theaggregationrate measurementwascarriedoutbymonitoringtheparticlesizeasa function of time using dynamic light scattering. Solutions of different pH were prepared by diluting the appropriate 1.0M solutionofhydrochloricacid(HCl)and1.0MsolutionofSodium Hydroxidesolutionwithpurifiedwater.Themilledsuspensionwas dilutedwithpurifiedwaterfrom20%w/wto0.1%w/w.10

m

lofthis dilutedsuspensionwereaddedto1mlofacidicorbasicsolution coveringa wide range of pHand theresulting suspensionwas gently mixed in the DLS cuvette and then placed into the measuringcelloftheDLSinstrument.

2.1.2.6.Evaluationofsensitivityofformulationtotemperature. In orderto evaluate thesensitivityof the formulation toOstwald ripening and flocculation, solubility of API was measured as functionofconcentrationofwetting/dispersingagent(SDS/PVP)as wellasfunctionoftemperatureinwater.Then,thecrystalgrowth wasevaluatedusingtemperaturestresstest.

2.1.2.6.1. Assessment of API solubility in water and in SDS/PVP solution. To assess API solubility a reverse phase HPLC-UV

method was developed. The concentration of API was determinedusing a HPLC system composed ofVarian Prostar1

230pump(suppliedbyVarianFrance),injectorWaters1_717plus

(supplied by Waters1 _{France) and UV absorbance detector}

(suppliedbyThermo-Fisher1_{France)setat232nm.Themobile}

phasewasacetonitrile/PhosphatebufferpH3.510mM(60/40,v/v). AXTerraRP1850(2.1mm,3.5

m

mcolumn(suppliedbyWaters1

France) was used withthe flow rate setat 1.0ml/min and the temperaturesetat45!_C.

Forthesamplespreparation100mgoftheAPIwereintroduced invialcontainingtheadequatesolution.Then,thesuspensionwas agitatedusingmagneticstirreratfixedtemperatureduring4hand let settle for 1h. The obtained supernatant was then filtered through0.22

m

mPVDF1

filter(suppliedbyMillipore1_{)and the}

filtrate was diluted using ethanol. The injected volume of the filtrate and the analysis time were fixed at 5

m

l and 2min respectively.ThechromatogramswereanalyzedusingEmpowerTM

chromatographysoftware(suppliedbyWaters1_France).

Fig.2.MilledsuspensionofVitaminETPGS1_{showingthatthesuspensionisgel}

like.

Fig.1.Longmillingdurationtrial(n=1):particlesizedistributionofSDS/PVPorvitaminETPGS1_{atinitialtimeandafter13hofmilling.ThefigurereflectsthatPVP/SDS}

Thelimitofquantificationofmethodusedwasfoundat0.1

m

g/ ml.

Eachmeasurementwasdonetwice.Arepeatabilitystudywas performedon6samples.Astandarddeviationofabout1.8%was determined.

2.1.2.6.2. Assessment of suspension stability as a function of temperature. The suspension stability was monitored by using kineticexperimentwhereparticlesizewasmeasured(usinglaser diffraction)asfunctionofstoragetimeatdifferenttemperature: 20!_,40!_,50!_and60!_C.

2.1.2.7.Particlesizemeasurement

2.1.2.7.1.Laserdiffraction. Theparticlesizedistributionofmilled suspension was measured using Laser diffraction (Malvern Mastersizer 2000). This method is based on measurement of angleoflightdiffractedbyparticles,whichdependsontheparticle radius using Fraunhofer diffraction theory. This method can measure particle sizes down to 1

m

m. For smaller particles, forward light scattering is measured with application of Mie Theoryoflightscattering.Bycombiningresultsobtainedwithlight diffractionandforwardlightscattering,onecanobtainparticlesize distributionsintherange0.02–10

m

m(Swithenbanketal.,1976). Thesymmetryofdistributioncanbeevaluatedusing polydis-persityindex(PI)thatisdescribedbythefollowingequation: PI¼ln d10'd90

d2 50

!

ð12Þ where,d10,d50,d90arethecharacteristicdiametersofparticles distributionreferringrespectivelytodiameterof10%,50%and90% ofparticlespopulation

Three situations may be encountered. They are described below:

(i)PI=0forlog-normaldistribution

(ii)PI<0fordissymmetricdistributiontowardssmalldiameters d50/d10>d90/d50

(iii) PI>0fordissymmetricdistributiontowardslargediameters

TheMastersizeris equippedwithlenshavingfocallengthof 550mmand cellmeasurementhavingthicknessof2.4mm.The samplewasdilutedin100mlofpurifiedwaterandintroducedin MS1 sampler. The suspension was stirred at 1500r.p.m and recirculatedthroughthemeasurementcell.Thedilutionfactorwas adjustedinordertoensureanobscurationintherangeof2.5–4.5. Themeasurementswerecarriedoutatroomtemperature.Each measurementwas performedduring20sand repeated3times. TherefractiveindexoftheAPIanddispersingwerefixedat1.61and at1.33,respectively.

A repeatability study was performed on 10 samples. The standarddeviationofabout1%wasdetermined.

2.1.2.7.2.Dynamic lightscattering. The dynamic lightscattering (DLS)wasusedfortheaggregationratemeasurementsasfunction ofpHandionicstrengthusingMalvernZetasizerinstrument.The methodreferredtoasphotoncorrelationspectroscopy(PCS).The methodisbasedinmeasuringtheintensityfluctuationofscattered light as the particles undergo Brownian diffusion. From the intensityfluctuationonecancalculatethediffusioncoefficientD from which the particle diameter d is estimated using the Stoeckes-Einsteinequation (see Eq. (12))(Pecora,1985)where, Disthediffusioncoefficient,kBisBoltzmannconstant,Tis the

absolutetemperature,

h

istheviscosityofthemediumanddhis

Table2

Rateofaggregationandstabilityratioasfunctionofsodiumchlorideconcentration. TheTableshowsthathigheristhesodiumchloride,higheristheaggregationrate andloweristhestabilityratio.

NaClconcentration(M) Rateofaggregation;t(s&1_{) Stabilityratio;W}

3.03 3.0010&11 _1.00 2.51 1.0010&11 _3.00 2.00 1.0010&11 _3.00 1.50 5.0010&12 _6.00 1.01 3.0010&12 _10.00 0.75 2.0010&12 _15.00 0.50 9.0010&13 _33.00 0.25 1.0010&13 _300.00

Fig.3. n0/nt(wheren0istheinitialnumberofparticles,ntisthenumberofparticlesaftertime(t))asfunctionoftimefordifferentelectrolyteconcentrations(n=1).Thefigure outlinesthatthehigheristheconcentrationofelectrolytesthehigheristheaggregationrate.

thehydrodynamicdiameteroftheparticles. D¼₃

_ph

kBT_d

h ð13Þ

Themeasurementswerecarriedoutusingascatteringangleof 90!_.

Eachmeasurementwasrepeated3times.Arepeatabilitystudy was performed and 10 samples. The standard deviation was determinedas0.8%.

3. Resultsanddiscussion 3.1.Longmillingdurationtrial

The results reveal that the SDS/PVP system leads to a suspension with particle size in the nanometric range (see

Fig.1)havingmono-modaldistributionandpolydispersityindex of0.003indicatingthattheparticlesizedistributioniscloseto Ln-normal distribution. In contrast, thevitamin E TPGS1 _{led toa}

Fig.4.Stabilityratio(W)asfunctionofelectrolyteconcentration(n=1).Thefigure givesaroughestimateoftheCCCofNaCl(*0.7molar)forthedesigned nano-suspension.

Fig.5.Ortho-kineticevaluation(n=1):monitoringoftheparticlesizeasfunctionoftime(n=1)at2ionicstrengths(nativewithoutaddedsaltand0.17molarofNaCl).The figureshowsthatwithoutsaltaddition(A)theshearinducedaggregationrateismuchhigherthanwhenthesaltisaddedat0.17molar(B).

suspensionwithparticlesizeinthemicronrangeexhibiting bi-modal distribution (see Fig. 1) with polydispersity index of 1.874indicatingthattheparticlesizedistributionisdissymmetric towardslargediameters.Furthermore,asillustratedinFig.2,the milled suspensionmadefromvitamin E TPGS1 _{exhibiteda gel}

aspectaftermilling.Whereas,thesuspensionsmadeofSDS/PVP remainedfluid.It isnoteworthythatduringourpreviouswork, after1hofmilling,thesuspensionmadeofvitaminETPGS1_hada

mono-modal distribution innanometric range.We proposethe following interpretations to explain the result obtained with vitaminETPGS1_:

" Theaggregationcanbeduetotheappliedstressduringalong periodandabsenceofelectrostaticstabilization(Andersonand

Lekkerkerker,2002).Infactinourpreviouswork,wefoundthat

SDS/PVP system had a zeta potential of &54mV and its stabilizingmechanismiselectro-stericrepulsion,while,vitamin E TPGS1 _{has a zeta potential of}

&22mV and its stabilizing mechanismisstericrepulsion.

" Anothermechanismthatcanexplaintheobservedresultisthe gelationofpolymerundershearflow(Omarietal.,2003).We assumethatfree andadsorbedvitaminETPGS1_on

thenano-particles bridge together under shear stress and form gel networkthatbindsthenanoparticlestogether.

3.2.Evaluationofperikineticaggregation:measurementofcritical coagulationconcentration

Themilledsuspensionusedforthisevaluationhadaparticle sizedistributionsimilartothatshowninFig.1(milledsuspension usingSDS/PVPsystem)

Fig. 3 shows the variation of n0/nt as function of time.

Noteworthy, n0/nt starts to increase with time when NaCl

concentration was larger than 0.25M. The plots gave straight linesindicatingthattheprocessofaggregationfollowsasecond orderkinetic.Thecharacteristictimes

t

werecalculatedfromthe slopeofthefittedlines.

ThestabilityratioswerecalculatedaccordingtoEq.(6)where thefastaggregationcorrespondstoionicstrength3MinFig.3. Indeed,furtherincreaseinionicstrengthdoesnotincreasetherate ofaggregation.Consequently,thislimitwasconsideredasthefast aggregationrate(

t

(fast)).

Theresultsfor

t

andWaresummarizedinTable2.

Thestabilityratio,W,asfunctionofelectrolyteconcentrationis plotted in Fig.4.One canestimatethat ata critical electrolyte concentration(*0.7MofNaCl),alltherepulsiveforceshavebeen effectivelyscreenedandcoagulationprocesswaspurelycontrolled bydiffusion.

ThevalueofmeasuredCCCindicatesahighcolloidalstabilityof designed system.In fact,it isaboutfivetimeshigherthan that observedintheliteratureforsuspensioncolloidalystableathigh concentration(Heetal.,2007;Serraetal.,2016).

3.3.Evaluationoforthokineticaggregation

The resultsshowed that, in the absence of added salt (see

Fig. 5A), aggregation and increase of polydispersity index (dissymmetricdistributiontowardslargediameters)(Fig.6)are observedovertime.Incontrast,inthepresenceof0.17MofNaCl (Fig.B) lessaggregationandlessincreaseofpolydispersityindex (Fig.6)wereobservedovertime.Theseresultsmayseem counter-intuitive.Indeed,underhighshearrate,ahighcolloidalstability awsobservedationicstrengthof0.24(CCCthanatverylowionic strength(withoutsaltaddition).Thisobservationisin contradic-tion with results observed during perikinetic evaluation. We

propose the following interpretations to explain the obtained results.

Atlowionicstrength,thePVPmoleculesarehighlysolublein water. Under highshear rate, thepolymers chainsare may be extractedfromnanoparticles.Hence,the stericrepulsion and a majorpartofelectrostaticrepulsionareeliminated.Furthermore, thehighshearratecanovercometheelectrostaticbarrierresulting inorthokineticaggregation.Incontrast,inthepresenceof0.17M NaCl,thelowsolubilityofPVPmoleculesinthemediumleadstoa strongadsorptionontotheparticles.Inthiscase,theshearinduced aggregationisprevented.Itisnoteworthythatonecanestimate whichmechanismprevailsbymeansofthePecletnumberofthe particles defined as the ratio of the time scale of convective transportduetoshearoverthetimescaleofdiffusivetransport (seeEq.(14))(Ehrletal.,2009).InourcasethePeclet’snumberwas foundabout77which ismuchhigherthan1outliningthatthe aggregationtakesplaceinorthokineticregimeandtheenergyused toapproachparticlesbetweenthemis likelymuch higherthan DLVOpotentialbarrier.

Pe¼6(

p

(

h

(r

3_{( _}

_g

KB(T ð14Þ

where,

h

is thedynamic viscosity, _

g

is theshear rate, r isthe characteristiclength-scale(particleradius),KBis theBoltzmann

constant,andTistheabsolutetemperature. 3.4.Desorptionevaluation

Fig.7Arevealsthattheparticlesizedistributionofsuspension madeofSDS/PVPsystem isunchangedovertimeindicatingthe highcolloidalstability of thenano-suspension. In contrast, the resultsobtainedwithSolutol1_{systemshowedanotablechangeof}

particlesizedistributionasfunctionoftime(seeFig.7B)indicating the aggregation of the nano-suspension. These results are in agreementwiththoseobservedinourpreviouswork.Infact,the SDS/PVP system was found to exhibit high affinity adsorption isothermwhichisknownasirreversibleprocess.Incontrast,the Solutol1_{adsorptionisweakandreversible.}

Fig.6. Ortho-kineticevaluation:polydispersityindexasfunctionoftime(n=1)at 2ionicstrengths(nativewithoutaddedsaltand0.17molarofNaCl).Thefigure showsthatwithoutsaltadditionthepolydispersityindexismuchhigherthanwhen thesaltisaddedat0.17molar.

3.5.Sensitivitytoostwaldripeningandflocculation

3.5.1.APIsolubilityasfunctionofwetting/dispersingagent(SDS/PVP) concentrationinwater

Fig.8representstheAPIsolubilityasfunctionofPVP,SDSand SDS-PVP (atratio of 40–60%(w/w)) concentration.It hasbeen observedthat:

"The API solubility does not change with increase of PVP concentration,

"TheAPIsolubilityincreaseswithSDSconcentration

"TheAPIsolubilityincreasessharplywithSDS-PVP(atratioof40– 60%w/w)concentration.

TheimpactofSDSconcentrationonAPIsolubilityis straight-forward.Itmeans,abovethecriticalmicellarconcentration(CMC), theformedmicellescansolubilize‘N’numberofAPImoleculesper micelle. The dissolved amount of API by micelle is therefore

proportionaltoformedmicellesasdescribedbytheEq.(15)

S¼S0þNAPI(MAPI _MC&CMC SDS(Nag

" #

ð15Þ where,S,S0,NAPI,MAPI,C,CMC,MSDSandNagaretheAPIsolubility

in SDS/PVP solution, API solubility in water, number of API moleculespermicelle,APImolecularweight,SDS/PVP concentra-tion,criticalmicellarconcentrationofSDS,SDSmolecularweight andnumberofmicellesaggregation,respectively.

AsPVPalonedoesnotsolubilizetheAPI,theincreaseofAPI solubilitywhenPVPisaddedtoSDSisdifficulttounderstand.This phenomenoninfactcanbeexplainedbythesubtlemechanism: AccordingtoShirahamaetal.(1974)andCabane(1977),atcritical aggregationconcentration(wellbelowtheCMC),polymerchains interactwithsurfactanttoformsmallmicellesofsurfactantinside thepolymerchain(seeFig.9).Therefore,inpresenceofpolymer muchhighernumberofmicellesareformed.Thehighernumberof micellesexplainsthehighsolubility.

Fig.7. Assessmentofdesorptionofthewetting/dispersingagent(n=1):monitoringoftheparticlesizeasfunctionoftimeforSDS/PVPsystem(A)andforSolutol1_(B).The

figureemphasizedthattheSDS/PVPsystemexhibitsirreversibleadsorptionasnoaggregationwashighlightedduringdilution.Incontrast,highaggregationwasobserved withSolutol1_.

3.5.2.APIsolubilityasfunctionoftemperaturein(SDS/PVP)solutionat concentrationof1.2%

The obtained results show that the API solubility does not changewithincreaseoftemperaturewhenwaterisusedasvehicle. However,whensolutioncontaining1.2%ofSDS/PVPatratioof40– 60%(w/w)isused,theAPIsolubilitydecreasessurprisinglywith increase of temperature (see Fig. 10A). This result can be interpreted by the disaggregation effect of temperature (above roomtemperature)onSDSmicelles.Infact,increaseof tempera-ture causes disruptionofthe structuredwatersurrounding the hydrophobicgroupswhichdisfavorsmicellization(SakhawatShah

andEjaz-Ur-Rehman, 1987).Furthermore,thisphenomenoncanbe

heightenedbysolubilisationofPVPmoleculesathightemperature. BoththeobservationsindicatethattheheatinducedOstwald ripening hypothesis can be ruled out. Indeed, the diffusion of

dissolvedAPItosolidparticlescannottakeplacefromthemicellar systemasitisattachedtothesolidparticles.

3.6.Temperaturestresstest

Duringmilling,thesuspensionundergoeswideamplitudeof temperature. The impact of temperature on particle size of suspension(20%w/wofAPI)wasevaluatedintherangeof20– 60!_{C.Theresultsshowthatat20}!_{C,thedispersionremainsstable}

formorethan600h,showingnoincreaseinparticlesizewithtime. As thetemperature increasesto 40!_{, 50}! _{and 60}!_{C, significant}

increase of particle size with time is observed (see Fig.11A). Moreover, as can be seen in Fig. 11B, when the temperature increases,thepolydispersityindexincreasesovertimeindicatinga dissymmetric distributionof particles towards largediameters.

Fig.8. APIsolubilityasfunctionofconcentrationinSDS/PVPsolution(atratioof40–60%w/w),SDSsolutionandPVPsolutionat20!_{C(n=2).ThefigurehighlightsthattheAPI}

solubilityismuchhigherwhentheSDS/PVPsystemisusedasmediumthanwhenSDSorPVPareusedalone.ThisoutlinesthattheAPIsolubilityisdrivenbythesynergyof SDSandPVPmolecules.

Fig.9. PVPmoleculesandSDSmicellesarrangement.Theconformationreflects thatTheSDSmicellesaretrappedbythePVPchainswhichmayprovideahigh electrostericbarrier.

Fig.10.APIsolubilityinSDS/PVP(atratioof40–60%w/w)solutionatconcentration of1.2%w/wandinwateratdifferenttemperatures(n=2).Thefigureshowsthatthe increaseoftemperaturedoesnotimpacttheAPIsolubilitywhenwaterisusedas medium.Incontrast,theAPIsolubilitydecreaseswhenthetemperatureisincreased forSDS/PVPsolution.

Hypothetically, this increase could be due to either Ostwald ripeningorflocculation.Ostwaldripeningcanberuledoutsincea plotofcrystalgrowth(r3

(t)&r3(0))versustimedidnotgivealinear

relationship(seeFig.12)(LifshitzandSlyozov,1961;Wagner,1961). Inaddition,monitoringofparticlesizedistributionshowedthat theappearanceofcoarseparticlesisnotduetodisappearanceof fineparticles(seeFig.13).Thus,theincreaseinparticlesizewith time must be due to flocculation. These results from either desorptionofthedispersingagent(PVPmolecules)whichbecomes moresolubleathightemperature(Tadros,2012)ordegradationof SDSathightemperature.Indeed,prolongedheatingofSDSat40!_C

orgreatercausesdecompositionofalkylsulfatesintofattyalcohols andsodiumsulfate(Anon.,1970;Specification,2012).

3.7.SensitivitytopHvariations

TheimpactofpHonparticlesizeofsuspension(20%ofAPI)was evaluatedintherangeof2.0–9.5tomimicgastricpH(1.50–5.00),

intestinalpH(7.40–7.80)orplasmaticpH(7.35–7.45).Theresults show(seeFig.14)thatparticlesizedidnotchangeovertimewhen the pH was higher than 2 indicating high robustness of the designed formulation. At pH 2, a strong destabilization of suspension was observed. In fact, a spontaneous increase of particlesizewasobservedsoonafterintroductionofthesample withintheacidicsolutionfollowedbyacontinuouslinearincrease oftheparticlesize.Thismaybeduetothecontributionoftwo mechanisms:

(i)pKaofSDSisclosetothesecondacidityofsulfuricacid(1.9).At pHabovethepKa,theSDS losesitsprotonH+_{and become}

negativelychargedtoensureelectrostaticstabilization.AtpH belowpKa,theSDSrecoversitsprotonandlosesitsnegative chargesandthusitsfunctionaselectrostaticstabilizer. (ii)Degradation ofSDS.According tothehandbookof

pharma-ceutical excipients SDS (Rowe et al., 2012) under extreme conditions i.e., pH 2.5 or below, it undergoeshydrolysis to

Fig.11. Temperaturestresstest:Meandiameter(A)andPolydispersityindex(B)asfunctionoftimeatdifferenttemperatures(n=1).Thefiguredoesnotreflectanychangein particlesizeandpolydispersityindexat20!_{C.However,increaseofparticlesizeandpolydispersityindexisobservedwhenthetemperatureisincreasedto40}!_,50!_and60!_C.

Fig. 13.Crystalgrowthasfunctionoftemperature(n=1).Thefigureoutlinesthattheostwaldripeningcanbeexcludedashypothesisasthegraphsdonotgiveastraightlines. Fig.12.Temperaturestresstest:monitoringoftheparticlesizeasfunctionoftimeat20!_(A),at40!_(B),at50!_(C)andat60!_{C(D).Thefigureoutlinesthattheappearanceof}

lauryl alcohol and sodium bisulfate leading to a lack of electrostaticrepulsionbetweenparticles.

4. Conclusion

Developmentofarobustformulationstablealongtheoverall valuechain(frommanufacturingprocessuntiladministrationto patient)isvital.Theexperimentalresearchmethodologydescribed inthispaperrepresentsanefficientapproachforevaluatingthe formulation robustness of nano-crystalline suspension. The assessmentofsuspensiondestabilizationundervariousconditions suchas,ionicstrength,shearrate,temperature,pHanddilution allowedidentificationofcriticalparameterswhoselevelsmustbe tightlycontrolled tomaintainproductstability andthusin-vivo performances.Acarefulattentionneedstobepaidduring down-processingofsuspensionwithregardtotheappliedshearrateand high temperature mainly during heat based process such as autoclavingwhereflocculationoraggregationmayoccur.Itwould be interesting to determine the limits of shear rate in which variations in the levels have minimal or no effect onproduct stabilityorin-vivoperformances.

Acknowledgments

TheauthorsgratefullyacknowledgeJ.L. Laly(Global Headof PharmaceuticalSciencesOperationsatSanofiR&D)forhissupport. TheAuthorsacknowledgealsoNait-BoudaLahlouforhissupport forsolubilitymeasurementsBernardCabane(ESPCI,France)and Harivardhan-ReddyLakkiredy(Headofdrugdeliveryat Pharma-ceuticalSciences Operations(SanofiR&D-Paris)for their appre-ciatedcontributionstopre-reviewthispaper.

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