A Design of Experiment approach to predict product and process parameters for a spray dried in fluenza vaccine
GauravKanojiaa,b,1,Geert-JanWillemsa,HenderikW. Frijlinkb,Gideon F.A.Kerstena,c, PeterC.Soemaa,*,2,Jean-PierreAmorija,2,3
aIntravacc(InstituteforTranslationalVaccinology),AntonievanLeeuwenhoeklaan9,3720ALBilthoven,TheNetherlands
bUniversityofGroningen,DepartmentofPharmaceuticalTechnologyandBiopharmacy,AntoniusDeusinglaan1,9713AVGroningen,TheNetherlands
cDivisionofDrugDeliveryTechnology,LeidenAcademicCentreforDrugResearch,LeidenUniversity,Einsteinweg55,2333CCLeiden,TheNetherlands
ARTICLE INFO
Articlehistory:
Received4May2016
Receivedinrevisedform28July2016 Accepted10August2016
Availableonline12August2016
Keywords:
Spraydrying Processoptimization Processparameters Qualitybydesign Nozzlegasflowrate
ABSTRACT
Spraydriedvaccineformulationsmightbeanalternativetotraditionallyophilizedvaccines.Comparedto lyophilization,spraydryingisafastand cheapprocessextensivelyused fordryingbiologicals. The currentstudyprovidesanapproachthatutilizesDesignofExperimentsforspraydryingprocessto stabilize whole inactivated influenza virus (WIV) vaccine. The approach included systematically screeningandoptimizingthespraydryingprocessvariables,determiningthedesiredprocessparameters andpredictingproductqualityparameters.Theprocessparametersinletairtemperature,nozzlegasflow rateandfeedflowrateandtheireffectonWIVvaccinepowdercharacteristicssuchasparticlesize, residualmoisturecontent(RMC)andpowderyieldwereinvestigated.Vaccinepowderswithabroad rangeofphysicalcharacteristics(RMC1.2–4.9%,particlesize2.4–8.5mmandpowderyield42–82%)were obtained. WIV showed no significant loss in antigenicity as revealed by hemagglutination test.
Furthermore,descriptivemodelsgeneratedbyDoEsoftwarecouldbeusedtodetermineandselect(set) spray dryingprocess parameter.This was used togeneratea driedWIV powderwith predefined (predicted)characteristics.Moreover,thespraydriedvaccinepowdersretainedtheirantigenicstability even after storage for 3 months at 60C. The approach used here enabled the generation of a thermostable, antigenic WIV vaccine powder with desired physical characteristics that could be potentiallyusedforpulmonaryadministration.
ã2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).
1.Introduction
Manyexistingvaccinesarecurrentlydistributedandadminis- teredinaliquidform.Liquidvaccinesneedtobestoredat2–8Cto remainstable. Thisdependency on a steady cold chain makes vaccinedistributioncomplexandexpensive,especiallyindevel- opingcountries(Wang,1999).Driedvaccinescanovercomethis requirementforacoldchain,astheypossessalongershelflifeat elevatedtemperatures(Geeraedtsetal.,2010;Smithetal.,2015).
Moreover,drypowdervaccineformulationshavethepotentialto be used for alternative vaccine delivery routes, such as the intranasal,pulmonaryororalroutes(Dickoetal.,2000;Giudice andCampbell,2006;Tonnisetal.,2012;Amorijetal.,2010).
An established method to produce dried biologics is spray drying. Spray drying has the advantageover traditional drying techniques(suchasfreeze-drying)thatitisrelativelyfastandhas loweroperating costs.Moreover, it results in a dispersible fine powder compared toa dry cake as obtainedby freeze-drying, which may enable further powder handling and usage for alternativedeliveryroutes.
Powders with different physiochemical and morphological propertiescanbeobtainedbyspraydrying.Thepowderproperties dependontheappliedprocessparametersandcompositionofthe liquid feed(Croweet al.,1994; Jain and Roy,2008). The spray drying process consists of nebulization of a liquid product, generating aerosols, into a heated gaseous drying medium, resultingina drypowder(Fig.1).Thelargesurfaceareaofthe aerosolsresultsinarelativerapiddryingprocess.Dependingon Abbreviations: QbD, qualityby design; DoE,Design ofExperiments; QTTP,
qualitytargetproductprofile;WIV,wholeinactivatedinfluenzavirus;XRD,X-ray diffractometry;DSC,differentialscanningcalorimetry;RMC,residualmoisture content.
*Correspondingauthor.
E-mailaddress:peter.soema@intravacc.nl(P.C. Soema).
1Firstauthor.
2Sharedlastauthors.
3Currentaddress:VirtuvaxBV,TheNetherlands.
http://dx.doi.org/10.1016/j.ijpharm.2016.08.022
0378-5173/ã2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
ContentslistsavailableatScienceDirect
International Journal of Pharmaceutics
j o u r n al h o m ep a g e: w w w . el s e v i e r . c o m / l o c at e / i j p h a r m
thesizeofthespraydrierandairflowrate,thedryingmaytake between 0.2–30s (Anon., 1999). During drying, the protein in evaporating droplets may experience reversible or irreversible denaturation. This could be due to the loss or weakening of hydrogen bonds and simultaneous increase in hydrophobic interactionsduringevaporationofwater.However,theself-cooling effectofdropletsduetowaterevaporationpreventsthetempera- ture increase of the droplet surface above the wet bulb temperature (temperature of drying aerosols achievedthrough evaporationcooling)(Katja,2011).Thus,spraydryingmaybean appropriateprocedurefordrying thermolabilevaccinesand has beenusedtoproduceexperimentaldrypowdervaccinesagainst measles(Linetal.,2011;Ohtakeetal.,2010),influenza(Lovalenti etal.,2016;Salujaetal.,2010;Scherliessetal.,2014;Souetal., 2015),tuberculosis(Wongetal.,2007)andhepatitisB(Chenetal., 2010). Moreover, dry powdered measles vaccine has showed promisingresultsinphase1 clinicaltrials(MVDPauthor group etal.,2014).Therefore,spraydryingmightbeasuitablealternative methodtoobtaindrypowdersofavarietyofvaccines(Amorijetal., 2008).
Thespray-dryingprocessusedtoproducethesepowderswith desired product characteristics is usually optimized by a one- factor-at-a-time (OFAT) approach, where the effect of process parametersontheproductareassessedinalinearfashion,one-at- a-time.Thisconsumesalotof timeand resources.Moreover,it
requiresalargenumberofexperimentsandinteractionsbetween parametersarefrequentlymissed.ADesignofExperiments(DoE) approachcanbeusedinsteadinordertosystematicallyscreenand optimizeprocesses.DoEisastructuredapproachthatcanbeused toidentifycriticalandnon-criticalparameters,andtheirrespective interactions,ofaproductionprocess.Moreover,itcanbeusedto quantifytheimpactofrawmaterialsandprocessparameterson theproductcharacteristicsandquality(Cooketal.,2013).Several studieshaveemployedaDoEapproachtoinvestigateandoptimize thespraydryingprocessofproteins(Prinnetal.,2002;Maltesen et al., 2008) and liposomal adjuvants (Ingvarssonet al., 2013).
However,thepotentialofutilizingDoEforproducingspray-dried powdervaccineshasnotbeenexploredsofar.
Tomaintainthestructuralintegrityduringandafterthedrying process, biological products such as proteins or vaccines often requireexcipientsthatactasstabilizersintheirformulation.The sugar trehalose is an excipient commonly used for stabilizing vaccines, due to its good protein-stabilizing characteristics (Geeraedtsetal.,2010;Ogainetal.,2011).Includingtrehalosein the vaccine formulations for spray drying might therefore be essential to obtaina stable vaccineproduct after spray drying.
Previously,spraydryingofinfluenzavaccineshasbeendescribed usingvarioussugars,Maa etal.(Maa etal.,2004)werefirstto describetheuseoftrehalose,Souetal.(2015)combinedtrehalose with leucine, whereas Scherliess et al. (2014) showed the Fig. 1.Spraydryingprocess:Aliquidinfluenzavaccine(wholeinactivatedinfluenzavirusvaccine)wasspraydriedwithtrehaloseasanexcipienttoproduceapowdervaccine.
Investigatedprocessparametersareindicatedinred.Aspiratorcapacitywaskeptfixedat22m3n/h(highestaspiratorsettingpossible).
superiorityoftrehaloseovermannitol.Audouyetal.(2011) and Salujaetal.(2010)describedtheuseofthespraydryingprocess withinulinasstabilizer.
The goal of this study was to investigate theuse of a DoE approachtosystematicallyscreenandoptimizethespraydrying processparameters(inletairtemperature,nozzlegasflow rate, andfeedflowrate)andpredictingtheprocesssettingsneededto achieve the targeted product quality parameters like outlet temperature,particle size, powder yield and residual moisture content.As a model antigen, whole-inactivated influenza virus (WIV)vaccinewasused,withtrehaloseasastabilizingexcipient.
Using DoE software, an experimental design was generated to assess the impact of various process parameters (inlet air temperature, nozzle gas flow rate, and feed flow rate) and trehalose concentrations on the final product characteristics (particle size, powder yield and residual moisture content).
Regressionmodelswerefittedonthemeasured outputparam- eters, and theprediction powerof the model was assessed by selecting three untested combinations of process parameters within the investigated design space. Finally, the antigenic recoveryandthermostabilityoftheobtainedspray-driedvaccines wasassessed.
2.Materialsandmethods 2.1.Influenzavaccine
Influenza A/PR8/34 WIV was obtained by inactivating egg- propagated influenza virus with b-propiolactone as described previously (Hendriks et al., 2011). The bulk vaccine was concentratedwithCentriprep centrifugalfilters(Millipore)with amolecularweightcut-off(MWCO)of10kDa,formulatedinHBS (20mMHEPES,125mMNaCl,9mMCaCl2,5mMMgCl2).Thefinal WIV stock contained 800mg/mL HA that was determined by
surface plasmon resonance as described previously (Hendriks etal.,2011).Thevaccinesolutiontobespraydriedwaspreparedby mixing the WIV stock with D-(+)-trehalose dihydrate (Sigma- Aldrich)solutioninPBS(resultinginweightratiohemagglutinin protein(HA)/trehalose:1/400).Theratioofproteintosugarwas chosenbasedonpreviousresearchexperiencewithspray-freeze dryingofinfluenzavaccines(Geeraedtsetal.,2010).Thetrehalose solutionwasfilteredusinga0.45mmMillex-HVfilter(Millipore) priortomixingwithWIV.
2.2.Spraydryingofinfluenzavaccineformulation
WIVpowderswereproducedusingaBüchiminispray-drierB- 290inconjunctionwithahighperformancecycloneandaB-296 dehumidifier(bothfromBüchiLabortechnikAG).Alltheexperi- ments were performed in a closed loop configuration using nitrogenasdryingmedium.
Nitrogen being inert in nature was preferred to avert any unwantedreactionthatmightoccurinthepresenceofairasdrying medium.Atwo-waynozzlewithorificediameterof0.7mmwas usedin aco-current modewithnitrogenasatomizinggas.The nitrogen pressure was set constant at 5bar. The spray drying parameters were varied in accordance with the experimental design matrix (Table 2). The feed flow rate is displayed in percentage(%)ontheequipmentandfeedflowrateof5,10or15%
corresponding toexperimentally determined flow rates ranged from1.0,3.4and4.5mL/min,respectively.Anatomizingairflowof 7.317.5L/mincorrespondstoasettingfrom30to50mm(normal liter[Ln]isthevolumeat0Cand1atm).Theaspiratorrateswere setat22m3n/hinallexperiments;thiscorrespondedtoinstrument settingof100%.
Afterspraydryingthespraydriedproductwascollectedand, aliquoted(100mg)invials(3mLvial,NuovaOmpi)inaglovebox underarelativehumidityof<3%(TerraUniversalInc,Series100) andsealed.Theyieldwasdefinedastheratiobetweentheamount ofpowderobtainedandtheamountofsubstanceintroducedinthe liquidfeed(Eq.(1))(Maltesenetal.,2008).Theweightofbuffer saltswasnotincludedinyieldcalculationastheywereconstantfor allformulations.
PowderYield½%¼Collectedpowderweight½g
Trehaloseinthefeed½g 100 ð1Þ
2.3.Targetproductprofile
The quality target product profile (QTTP) was defined, and processparametersthatmayhaveanimpactonresidualmoisture content(RMC),particlesize,powderrecoveryandoutlettemper- ature were determined from previous studies on spray drying (Prinnetal.,2002;Ingvarssonetal.,2013;Ogainetal.,2011).The QTPPdescribesthecharacteristicsofthefinalproduct(Table1).
Theparametersinvestigatedwereinlettemperature,atomization airflow rate(nozzlepressure),feedflowrateand feedexcipient concentration. Additional factors such as raw materials and operatorwerekeptconstant.Theinvestigatedspacewouldresult inadesignspacewhereoneormoreofthetargetedproductprofile couldbeachieved.
2.4.DesignofExperiments(DoE)
TheDoEmodelwaspreparedandevaluatedusingMODDE10.0 (UmetricsAB).Modelswerefittedwithmultiplelinearregression (MLR) and adjusted by removing non-significant model terms.
Priortoourstudy,screeningexperimentswereperformedusinga fullfactorialdesigntodeterminethemostrelevantinputprocess parameters that affected the output process and product parameters.SincetheweightratioofHA/trehalosewas1/400,it was decided to find the appropriate process input parameter rangeswithoutusingtheantigen.Thereby,assumingthatsuchlow
Table1
Qualitytargetedproductprofile.DescribesthedesiredqualitytargetprofileforadrypowderWholeinactivatedinfluenzavirusvaccine.
Desired response
Target Rationale
Particlesize 1–5mm Particlesizethatissuitableforinhalationalandparenteraladministrationafterreconstitution.
Residual moisture content
3%orless Withintheprescribedlimitsfordriedbiologicals(Mayetal.,1992;TheFoodandDrugAdministration, 1990)
PowderYield 70%ormore Powderyieldshouldbesufficienttomaketheprocesseconomicallyfeasible.
Stability Lessthan10%lossinHAtitersduring storagefor12monthsat2–8C
Powdervaccineshouldbeatleastasstableasthedescribedstabilityforwholeinactivatedliquid influenzavaccines(Farnsworthetal.,2011;Kumruetal.,2014).Thehumandose15mgofHAcanbe calculatedtheoreticallybasedonpowderyieldofthevaccine.
antigenweightratiowouldnotsignificantlyinfluencetheoutput parameters. The model was further optimized using a Central Composite Family (CCF) design, including WIV as the model antigen. A reduced CCF design was used for optimization, consisting of in total 23 experimental runs. The choice of a reduced CCFdesign was made dueto fewerexperimental runs giving the same amount of information as the CCF. To reduce systematicerrors,alltheexperimentswerecompletelyrandom- ized.
2.5.Antigencharacterization
2.5.1.WIVhemagglutinationtiter
TodeterminethehemagglutinationtiteroftheWIVindried vaccine powder, a hemagglutination assay was performed as describedpreviouslybySoemaetal.(2014).Spray-driedvaccines werereconstitutedaccordingly inpurifiedwater(MilliQ)corre- sponding toa HA concentrationof 0.1mg/mL. Finally, PBS was
added to the reconstituted vaccine to obtain a 1:10 dilution.
Dilutedvaccinesolutionweretransferredtoa96-wellsV-bottom plate(Greiner)andseriallydilutedtwo-foldwithPBS.Allthewells consistedof 50mLafterdilution.Next,anequalvolumeofa1%
suspensionofturkeyerythrocytes(Harlanlaboratories)wasadded to the wells and the plates were incubated for 1h at room temperature.The titerwas determinedbyvisualobservationof agglutinationofRBCinthewellsandsubsequentlyexpressedas the reciprocal of the highest dilution that yielded complete hemagglutination.The titers weredetermined onreconstituted powdersjustafterspraydrying(t=0)andwererepeatedonstored vaccinepowder over a period of 3 monthsat intervals of one month.HAtiters(activity)ofthesamplesateachconditionwere calculated relative to the starting liquid mixture of WIV with excipientat4C.TheLog2HAtiterswereexpressedinpercentage forcomparisonbetweendifferentsamples.
2.5.2.Dynamiclightscattering(DLS)
The size of WIV after reconstitution was measured using a Zetasizer Nano-ZS system (Malvern Instruments). DLS
measurements were done in triplicate with 0.2mL of the reconstitutedWIVsamples.Sampleswerepreparedwithvaccine powderreconstitutedbygentleshakinginpurifiedwater(MiliQ) (HA0.1mg/mL)atanoperatingtemperatureof25C.Homogeneity ofthesizedistributionwasreflectedinthepolydispersityindex (PdI).
2.6.Residualmoisturecontent(RMC)
The RMC of spray dried influenza vaccine samples was determined using a C30 Compact Karl Fischer Coulometer (Mettler-Toledo). Samples of approximately 100mg of dried powder vaccinein vialswerereconstituted in1mLHYDRANAL Coulomat A (Sigma-Aldrich)andsubsequently injectedintothe titration vessel. Each sample was measured in triplicate. The relativeandabsolutemoisturecontentwerecalculatedfromthe standardplot,basedontheweightofthedriedproductinthevial, volumeofthereconstitutedreagent,volumeofextractedsample injectedintothetitrationvesselandtheblanktitration.
2.7.Physicalcharacterizationofthevaccinepowders
2.7.1.Geometricparticlesize
Thegeometricparticlesize(X50definedasthemedianparticle size) of spray dried powder product was analyzed by laser diffractionwithaHelos system(Sympatec GmbH). Thepowder wasdispersedintotheHelossystemusinganaspirosdispersing system operatedatadispersing pressureof1.0bar.Thevaccine powderwasmeasuredwithlenshavingameasuringrangeof0.1/
0.18–35mm. Furthermore,tocheckforanyaggregatesthedried vaccine powder was measured again with a lens having a measuringrangeof4.5–875mm.Moreover,increasingthedisper-
sionpressureto5bardidnotresultin changeof themeasured particlesizedistribution,whichindicatesthatthesizedistribution oftheprimaryparticleswasobtainedat1bar.Resultsarethemean ofthreemeasurements.Thespanof theproducedpowderswas calculatedusingtheequation:span=(X90X10)/X50.Thespanisa Table2
Experimentaldesignmatrix.Thedesignmatrixshowstheinputparameterssetforspraydrying(inlettemperature,nozzlegasflowrate,feedflowrate,trehalose concentration)andtheoutputparametersthatwereexperimentallydetermined[Particlesize(X50medianparticlesize)andspan,outlettemperature,RMCandpowder yield].Aspiratorwassetatmaximuminstrumentsetting.
Exp No.
Inlettemp (C)
Nozzlegasflowrate (L/min)
Feedflowrate (mL/min)
Trehaloseconc (mg/mL)
Particlesize
[X50](mm) Size(X90(span)X10)/X50
Outlet temp(C)
Moisture content(%)
PowderYield (%)
1 110 7.3 1.0 100 6.1 1.7 67 4.2 63
2 160 7.3 1.0 100 6.9 2.0 91 1.4 72
3 110 17.5 1.0 100 2.4 2.1 58 2.4 82
4 160 7.3 4.5 100 7.3 2.0 78 3.8 53
5 110 17.5 4.5 100 5.7 2.0 48 7.7 64
6 160 17.5 4.5 100 3.2 1.5 63 3.1 72
7 110 7.3 1.0 150 6.8 1.3 63 4.1 61
8 110 17.5 1.0 150 2.9 1.6 61 2.6 75
9 160 17.5 1.0 150 2.9 1.2 83 1.9 69
10 110 7.3 4.5 150 8.5 1.8 53 2.9 42
11 160 7.3 4.5 150 7.9 1.8 76 3.6 52
12 160 17.5 4.5 150 3.1 1.5 73 2.3 69
13 110 12.4 3.4 125 4.5 1.8 55 3.7 69
14 160 12.4 3.4 125 4.3 2.0 78 2.4 70
15 135 7.3 3.4 125 7.5 1.9 68 4.9 57
16 135 17.5 3.4 125 3.3 2.1 65 3.1 76
17 135 12.4 1.0 125 3.7 1.8 74 2.4 77
18 135 12.4 4.5 125 4.2 1.6 61 3.3 69
19 135 12.4 3.4 100 3.8 2.1 67 2.9 78
20 135 12.4 3.4 150 4.2 2.1 62 2.8 62
21 135 12.4 3.4 125 4.1 1.6 63 3.1 67
22 135 12.4 3.4 125 4.5 2.0 66 3.2 72
23 135 12.4 3.4 125 4.4 2.1 66 3.5 73
measureforthehomogeneity/polydispersityof theparticlesize distribution.
2.7.2.Differentialscanningcalorimetry
Modulated differential scanning calorimetry (mDSC) was conducted using a TA DSC Q100 (TA instruments). Samples weighing between 10.0–15.0mg were crimped in hermetically sealedpans for measurement.The glasstransition temperature (Tg)wasdeterminedbymodulatedDSC(MDSC);thesampleswere cooledto10Candthenheatedto180Catarateof2.0C/min.The modulation amplitude was set at 0.318C every 60s. The midpointindeflectioninthereverseheatflowwastakenastheTg.
2.7.3.Scanningelectronmicroscopy(SEM)
The morphologyof theparticles was visualizedusinga JSM 6301Fscanningelectronmicroscope(JEOL).Samples(Experiment 1,7,8,11and12)werepreparedbyplacingthepowdersondouble- sided sticky carbon tape on a metal disk. Subsequently, the particleswerecoatedwitha goldlayerofapproximately10nm usingaBalzers120Bsputteringdevice(BalzerUNION).Thevoltage usedforanalysiswas10kVwithaspotsizeof7–8.Imageswere takenatamagnificationof1000and5000.
2.7.4.PowderX-raydiffractometry(XRD)
Vaccinepowdersamples(Experiment1,7,8,11and12)were analyzedbyanD2PhaserdesktopX-raydiffractometerequipped
with a LynxEye Si strip one-dimensional detector (both from BrukerAXS).ThesampleswereexposedtoCuKa(X-rays)atan
angularrangingwasfrom5to602&z.Theta;withastepsizeof 0.01andadwelltimeof0.5s.Thecrystallinestatusofthepowder was assessed qualitatively by examination of the resulting diffractionpatterns.Theupperlimitofdetectionofcrystallinity is2%ofthetotalsamplevolume.
3.Results
A DoE approach was used to systematically investigate the effectsoffeedflowrate,inlettemperature,nozzlegasflowrate (nozzle pressure) and trehalose concentration (which were establishedtobetheimportantinputprocessparametersinprior screeningstudies)ontheoutputparameters(outlettemperature) andvaccinepowdercharacteristics.Togain moreinsight inthe relationbetweeninputparametersontheresponses,areduced CCF design was adopted for further optimization of process parameters (Table 2). After spray drying and analyzing the formulationsoftheCCFdesign,MLRmodelswerefittedforeach output parameter. Valid models were obtained for outlet temperature,particlesize,residualmoisturecontentandpowder yield, described in model fit (R2), prediction power (Q2), reproducibilityandavalidmodel.
Fig.2.PowdermorphologyofspraydriedWIVpowders.Scanningelectronmicrographsofrepresentativespraydriedtrehalose-stabilizedWIVvaccines[Top:Run12(X50: 3.1mm),Bottom:Run8(X50:2.9mm)].Magnificationimagesleft(1000)andright(5000).
