冻干显微镜下的冻干工艺-英国凯文沃德博士课件

FormulationDesignandCharacterisationforSuccessfulFreeze-DryingCycleDevelopmentDr.KevinWard,MRSC凯文.沃德博士

DirectorofR&D,BiopharmaTechnologyLtd.,WinchesterUK英国Biopharma

技术有限责任公司研发总监

ChairofPharmaceutical&HealthcareSciencesSocietyFreeze-DryingSpecialInterestGroup冻干显微镜下的冻干工艺

1SynopsisofPresentationSomegeneralrulesforformulationdevelopment…andafewverybasicrulesforcycledevelopmentMethodsofformulationcharacterisationFreeze-DryingMicroscopy(FDM)DTA/electricalimpedanceanalysis(Zsinφ)Residualmoistureanalysisinthelyophilisedproduct2TheIdealFormulation?Insomerespects,theactiveingredient/materialaloneisthebestformulation:Lowestsolutedensity(therefore,lowerresistancetovapourflow)Noexcipient(in)compatibilityissuesLowercostofmanufactureHowever,theactiveingredientmayneedtobestabilisedpriortoandduringfreeze-drying3FormulationIssues(1)Itshouldberememberedthat:FREEZINGinvolvesCONCENTRATION“Freezingisinitselfaformofdehydration”(FelixFranks)DryingmayrisktheremovalofwaterinvolvedinmaintainingthestructureoftheAPI(especiallyforproteins)4FormulationIssues(2)Lowvolumeofhigherconcentrationshouldfreeze-dryfasterthanlargervolumeofalowerconcentrationManyproteinsloseactivitywhenfreeze-driedfromlowconcentrationsHowever,somematerials(especiallyorganisms)aredifficultorimpossibletoconcentratethemwithoutdamage5FormulationIssues(3)Additionally,someproductsarenotstableinsolution,therebyrequiringpHbufferingand/orotherstabilisationevenbeforefreeze-dryingstartsHowever,rememberthatpHbuffersaredesignedtoworkinsolution–therearenoguaranteesforthefrozenordriedstate!6APIcharacteristicsCrystallineoramorphous?Teu/Tg’/TcBulkcharacteristicswhenfreeze-driedSolubilityConcentrationrequiredpriortoFDpH-stabilityplotIEP&aggregationissuesforproteins7WhatareweformulatingtopreventfortheAPI?DestabilisationinliquidstateDamagebythefreezingprocessLossofactivityduringdryingDegradationduringstorageTherefore,needexcipientsthatarechemicallycompatiblewiththeAPIduringalltheabovestages8Formulationforfreeze-dryinginvolvestheuseofexcipientsto…providemechanicalstrength(bulk)affordthermalstability

(ahighTcritical)duringlyophilisationandinthedriedproductprotecttheactiveingredient(s)fromdamagebefore,duringandafterprocessinggivecorrectpH,andtonicitywhererequired(sometimesachievedbyreconstitutingmediumratherthanstartingsolution)9CommonExcipients:pros&cons

*fulfilthebasicrequirementofremainingamorphousbutprotectiveabilitydependsonAPI

**PEGoftenprovidescryoprotectionbutnotnecessarilylyoprotectionasitcancrystalliseExcipientBulkThermalProtectionMannitol(whencrystallised)GoodGoodPoorDisaccharides:SucroseGoodOKGood*LactoseGoodOKGood*TrehaloseGoodOKGood*MaltoseGoodOKGood*GlucosePoorPoorGood*DextranGoodGood?*PVPGoodGood?*PEGGoodGood?**BSA/HSAGoodGood?*Aminoacids/dipeptidesVariableVariableSomegood*10“Lyo-friendly”buffersCitrateTrisGlycine/HistidinePhosphateoftenbestavoidedduetopHshiftsonfreezing,resultingfromdi-sodiumsaltcrystallisingoutOtherbufferssuchasacetate,HEPES,borate,phthalate,arelesswellstudiedforfreeze-dryingbutmaybesuitable11Otherissuesinformulatingforfreeze-dryingMixingamorphousand“crystallising”componentstogether:Phaseseparation(ice+glass+crystals)Unpredictable“criticaltemperature”Possiblemicrocollapse/micromeltingInhibitionofcrystallisationResultingmetastablecomponentscouldchangeovertimeindrystate12“Extrascientific”issuesaffectingexcipientselectionEthicalacceptabilityintargetmarketPreviousacceptancebyregulatorybodies(FDA,MHRAetc.)foreachmodeofuse(e.g.in-vitro,PO,SC,ID,IP,IM,IV)GradeofpurityavailableCostSupplychainreliability13Vialsoffreeze-driedproductGoodOKPoorPoorTheproductinthe“Poor”vialshasbecomesoftanddenseduringfreeze-drying,becauseithasbecomewarmerthanits“CriticalTemperature”!14“WhatistheCriticalTemperatureforourproduct?”The“CriticalTemperature”willbe:Theeutectictemperature(Teu)forcrystallinematerialsThecollapsetemperature(Tc)foramorphousmaterials(somewhereatorabovetheglasstransitiontemperature)Theloweroftheabovetemperaturesformixedsystems

(dependingonwhethermicro-collapseisacceptable)Wecananalysethecriticaltemperatureofaformulationbeforefreeze-dryingit,forexampleusing:Freeze-DryingMicroscopy(FDM)Impedance(Zsinφ)andThermalAnalysis15Freeze-dryingmicroscopy(FDM)FDMisthestudyoffreeze-dryingatthemicroscopiclevelFDMallowsdeterminationofcollapse,meltingand“qualitativephenomena”suchasskinformation16WhatisaFreeze-DryingMicroscope?Effectivelya‘microfreeze-dryer’wherethefreeze-dryingofasmallsamplemaybeobservedFirstdesignsinthemid-1960sNowmanufacturedcommercially17SamplePreparationforFDMSampleholderSideDoorBlockSampleloadingtakesabout60seconds.Routineanalysisusuallytakes30–90minutes18SampleFormatinLyostat2Temperature-ControlledBlockLightSource(frombelow)ApertureQuartzcoverslip(16mmdia.)Glasscoverslip(13mmdia.)2µlofsampleObjectiveLens(usually10x)MetalSpacer(70µmthick)19IdeallytherawformulationisusedSometimesnecessarytousesamplesthathavepreviouslybeenfrozenorlyophilisedAfterloadingthesample,theLyostat2issettocooltothedesiredtemperatureThesampleisallowedtocoolandfreeze(Note:foreutecticmaterials,therewillbemorethanonefreezingevent!)SampleLoadingandCooling20Whensamplereachestheholdingtemperatureandhasbeenobservedtofreeze,vacuumpumpisswitchedonanddryingbegins.Sublimationinterfacecanbeseenmovingthroughthefrozensample.FrozensampleDriedsampleSublimationfrontOn-lineplotTemp/timetableINITIALFDMIMAGE21Increasingordecreasingthetemperatureofthesampleallowsyoutoviewitsfreeze-dryingcharacteristics.Byexaminingthefreeze-driedstructurebehindtheinterface,thecollapsetemperatureofthematerialcanbedetermined.ThetemperaturemaybecycledinordertoevaluateTcmorecloselyFrozensampleSublimationfrontCollapsedmaterialINTERPRETATIONOFEVENTS22Samplestructurelostwhencollapsetemperaturewasexceeded.Structureregainedassamplewasre-cooledtobelowitscollapsetemperature.FrozensampleCollapsedsampleRegainedstructureSublimationfrontINTERPRETATIONOFEVENTS23100%structurehasbeenregainedbyloweringthesampletemperature.Sampletemperaturewasagainincreasedtoaboveitscollapsetemperature,causingthesampletocollapse.DriedsamplewithstructureCollapsingagainonreheatingFrozensampleSublimationfrontINTERPRETATIONOFEVENTS24‘Micro-collapse’(seee.g.Wang,2004)BelowTcofamorphousphaseAboveTcofamorphousphaseAsimilareffectmayalsobeobservedduetothemeltingofcrystallinecomponent(s)ontoarigidamorphousstructure

(dependingonwhichhasthelowercriticaltemperature)Macroscopicallysimilarbutisit:Wetter?Lessstable?Moredifficulttoreconstitute?25FDMimageofanaqueoussolutionof2%Mannitol+1%Glucose-41oC,aroundTcforglucose.Possibleevidenceofvisiblemicro-collapse(Dryingfront)FrozenmaterialRegionswithgooddriedstructure.Justmannitol?Regionsof(micro)collapse.Justglucose?26So,whatelsecanFDMtellus?Eutecticmeltingtemperature27NaClBelowEutecticTemperatureFrozenDry28NaClAboveEutecticTemperatureNotechangesinappearanceoffrozenstructureEutecticliquid29So,whatelsecanFDMtellus?EutecticmeltingtemperatureMaygivesomeindicationofskin(crust)formationpotentialofaformulation30LayerofconcentratedsoluteatedgeofsampleCrustformation(1)31CrustFormation(2)Dryingonlyoccursthroughbreaksinthecrust32So,whatelsecanFDMtellus?EutecticmeltingtemperatureMaygivesomeindicationofskin(crust)formationpotentialofaformulationWhetherheat-annealingmaybeofbenefitToincreaseicecrystalsize–andwhatconditionsarerequiredforthis(aboveTg’?)Toencouragesomecomponentstocrystallise33EffectofannealingonicecrystalsizeSamplecooledto-40°C,thenwarmedto-10°CSamesampleafterafurther15minutesat-10°CExperimentscanbecarriedouttocompareratesofchangeatdifferenttemperatures,inordertoestablishwhatannealingtemperaturemightbemostefficienttouseinthefreeze-dryer.34FDMsetupwithpolarisedlightPolariserAnalyserSampleCamera35Effectofannealingonsolutebehaviour:FDMwithpolarisedlightfunctionSamplequenchcooledbelow-40°CNosignofcrystals(nolightrotation)SameSamplenowdryingat-18°CPolarisershowspresenceofcrystals(whiteareas)36FurtherapplicationsofFDMItispossibletoexaminedifferencesinrelativedryingrates:FordifferentformulationsForaspecificformulationatdifferenttemperaturesRef:Zhai,S.,Taylor,R.,Sanches,R.andN.K.H.Slater(2003).MeasurementofLyophilisationprimarydryingratesbyfreeze-dryingmicroscopy.Chem.Eng.Sci.

58,2313-232337DTAandElectricalImpedanceanalysis(Zsinφ)ofFrozenFormulations38DifferentialThermalAnalysis(DTA)EffectiveyetsimpleandinexpensivemethodofanalysingfrozensolutionsGivesexothermicandendothermicevents,whichcanindicate:Glasstransitions(amorphous)Eutecticmelts(crystalline)Crystallisations(amorphoustocrystalline)AtBiopharma,weusethisincombinationwithelectricalimpedance(Zsinφ)analysistogiveamorecompletepicture39ElectricalImpedance(Zsinφ)AnalysisThisisamoresophisticatedversionofelectricalresistance(R)analysisImpedance(Z)isacombinationofResistance+Inductance+CapacitanceLookingatZ(ormorespecificallyZsinφ)cangivemoredetailedinformationaboutfrozensolutemobility(Rey,1999).40InvestigatingZsinφ–Methods(1)Wehavedevelopedadevice(Lyotherm2)incollaborationwithProf.LouisRey,whichiscapableofanalysingImpedanceatafrequencyof1000HzLyotherm2allowsbothDTAandImpedance(Zsinφ)analysistobecarriedoutonasampleinthefrozenstate–largesamplevolumetogivestrongersignal41InvestigatingZsinφ–Methods(2)Approximately100single-anddual-solutesampleswereanalysedinordertovalidatethedeviceagainstpublisheddataFormanyformulations,theonsetofmobility(atapointwedefinedas“TZonset”)wasobtained,evenwhereModuledTemperatureDifferentialScanningCalorimetrywasunabletoshowathermaleventAnapplied,in-depthstudywasthencarriedoutincollaborationwithDr.PaulMatejtschukofNIBSC(UK)inordertoestablishhowZsinφanalysismightberelevanttoafurther40multi-componentbiologicalproductformulationsandplacebos42InvestigatingZsinφ-PracticalZsin

impedanceprobeSymmetry-balancedheatingbuiltintosampleholdingblock,togivesmoothbaselineDTAassemblySampleformatforLyotherm2analysisSampleswerecooledtobelow-100ºCandanalysedonrewarming43ExampleofZsinφ+DTAgraph44Exampleswhereonsetofmobilityincrease(atTZonset)observedwellbelowcollapsetemperatureFormulation(%valuesinw/v)TZonset(ºC)(Lyotherm2Zsinφ)OnsetTc(ºC)(Lyostat2FDM)NaCl(0.9%)+HSA(0.5%)-46-18.4NaCl(0.9%)+HSA(1.0%)-64-20.4NaCl(0.9%)+HSA(5.0%)-60-23.0Eggallantoicfluid(undiluted)-60-50PotassiumphosphatebufferedHSA(0.2%)+trehalose(0.1%)-58-50HSA(0.1%)+casein(0.3%)inPBS-59-5045OngoingstudiesfromthesedataThereal-lifepracticalimplicationsofthesedataarecurrentlybeingdeterminedbyfreeze-dryingsomeofthe140sampleswehaveanalysed:BelowTZonset(goodstructure)BetweenTZonsetandTcollapse

(micro-collapse?)AboveTcollapse

(macroscopiccollapse)…andexaminingtheirappearance,structure,watercontent,crystallinity,stability,reconstitutiontime.Earlyindicationsarethatsuchacorrelationdoesexist,andtheextenttowhicheachaspectofthefinalproductisaffecteddependsonthenatureoftheproduct46ConclusionsforImpedanceAnalysisImpedance(Zsinφ)analysishasprovidedavalueofTZonsetforanumberofsamples,indicatingmobilityevenwhennothermaleventwasseenDataindicatethatacorrelationexistsbetweenproducttemperatureduringlyophilisation(inrelationtobothTZonsetandTc)anddefinedaspectsofthelyophilisedproductImpedanceanalysismaythereforerepresentausefuladditiontothecurrentrangeofanalyticalmethodsappliedpriortofreeze-drying47CycleDevelopmentBasics(1)Cooltobelowthecriticaltemperatureoftheformulation(monitorproducttemperatureatbase)Assesscoolingrateeffects(butnotbyfreeze-thawing,asthawingcanbedamaging!)Temperaturesafetymargintoallowforvariability,measuringdeviceeffects,scale-up…Moresafetymarginrequiredforeutectics?48CycleDevelopmentBasics(2)PartialpressureofvapourshouldbelowerthanvapourpressureoficeatdryingfrontNumerousPAT/EndpointdeterminationmethodsavailableSecondarydryingmayneedtobeginbelow“Tgatseveral%water”FinalwatercontentshouldgivesufficientlyhighTgfordesiredstoragetemperature&time49CaseStudyProductCycleDevelopmentTheywerediscardingahighpercentageofeachbatchduetovisibledefectsoccurringintheproductduringfreeze-dryingAclientapproachedBTLwithaproductthatwasbeingfreeze-driedusingacyclethatwasoriginallydevelopedforanotherproduct50ProductCycleDevelopmentStep1–Informationwasobtainedonthecriticaltemperaturesand thermalbehaviouroftheproductusingtheLyostat2andLyotherm2instrumentsStep2–Thisdataconfirmedthelackofsuitabilityoftheexistingfreeze-dryingcycleStep3–Criticaltemperatureinformationwasusedtocreatea“firstapproximation”cycletailoredtotheneedsoftheproductStep4–Datafromthiscyclewasusedtodesignamoreoptimised cycleuntilasafeandefficientcyclewasachieved, minimisingcycletimewithoutjeopardisingproductqualityCaseStudy51Lyostat2Freeze-DryingMicroscopyAnalysisSampledrieswellat-50.0°C,butcollapsestartsasthetemperatureisincreasedto-45.7°C.ThiscanbeidentifiedbydefectsappearinginthedriedmaterialAsthetemperatureincreasesto-39.6°CthestructurecontinuestoweakenandcollapsebecomesmoreevidentCaseStudy52Lyostat2Freeze-DryingMicroscopyAnalysisThesampleisrepeatedbutthistimewithanannealingstep–frozenandcooledto-50.0°C,warmedto-15.0°Candre-cooledto-50.0°Cbeforedrying.Thesampledrieswithgoodstructureuntilthetemperaturereaches-31.4°CanddefectsappearAt-30.8°CthesampleistooweaktomaintainanystructureastheiceisremovedCaseStudy53Lyotherm2DTAandImpedanceAnalysis1234Seefulllabels1–4onnextslideCaseStudy54ExotherminDTAandincreaseinImpedanceindicatingastabilisation/rearrangementofthefrozenstructureIncreaseindownwardgradientofImpedancecurveindicatingasofteningofthefrozenmaterialOnsetofasharpendothermconsistentwiththemeltingoftheiceMinimumImpedanceindicatingcompletemobilitywithinthesolutestructureLyotherm2DTAandImpedanceAnalysisCaseStudy55InterpretationofAnalysisResultsTheinclusionofanannealingstepresultedinanincreaseinthecollapsetemperatureoftheformulationfrom-45.7°Cto-31.4°C,aswellasincreasingicecrystalsizeandnetworkingTherefore,themaximumallowableproducttemperatureduringsublimation(toavoidcollapse)wasraisedby14.3°Cbytheuseofannealing,therebyallowingdryingtobecarriedoutathighertemperatures,foramoreefficientcycle.Thehighertheproducttemperatureduringdrying,thefasterthedryingrate.Fromtheresultsoftheseanalyses,wemadethefollowingdeductions:CaseStudy56Client’sExistingCycle–70hoursTc=-45.7°C+20°C-15°C-50°C-40°CShelfTemperatureProductTemperatureChamberPressure1231–Freezing2–PrimaryDrying

3–SecondaryDryingAA–ProductatriskofcollapseCaseStudy57ModifiedCycleCreatedByBTL–42hours+20°C-15°C-50°C-35°CShelfTemperatureProductTemperatureChamberPressure1234Tc=-31.4°C1–Freezing2–Annealing3–PrimaryDrying4–SecondaryDryingCaseStudy58Thisgraphshowsanenlargedsectionofthepreviousgraph+20°C-15°C-50°C-35°CShelfTemperatureProductTemperatureChamberPressureTc=-31.4°C1–Freezing2–Annealing3–PrimaryDrying4–SecondaryDrying3TheSublimationCoolingEffectTheloweringofproducttemperaturecausedbythesublimationoficeCaseStudy59TheNextStepsFromthepreviousrunwenowknow:Theextentofsublimationcooling,allowingustoincreasetheshelftemperature/chamberpressureashighaspossiblewhilstsublimationcoolingkeepstheproducttemperaturebelowTcWhensublimationwascompleteintemperature-probedsamples(whenproducttemperature=shelftemperature)ThephysicalappearanceofthecakesproducedbythecycleResidualmoisturewasmeasuredinthefinalproduct,inordertoestablishwhethertheextentofsecondarydryingwassufficientCaseStudy60ProblemSolved!FDMandDTA/Impedanceanalysisprovidesyouwithessentialinformationonthecriticaltemperaturesofyourformulation,enablingyoutogetthebestresultsfromyourproductDeterminationofcriticaltemperatures‘TrialandError’CycleresultinginlossofproductorcostlyinefficientcycleNewcyclebasedonactualparameters61ChangesintheproductFormanyAPIs,freeze-dryingwillgivea(partially)amorphousproductNeedtobeawareof:Thermodynamic/KineticstabilityChangesinpoly(a)morphsand/orhydratesthatmayaffectthe‘location’ofwaterovertimeFMSvs.KFforlookingatwater…62FrequencyModulationSpectroscopy(FMS)FMSisamethodthatdetectsmoleculesintheheadspaceofasealedcontainer(suchasavial)Beingnon-destructive,itallowsthesamevialtobeanalysedatdifferenttimepoints,thusreducinginherenttimepointvariabilityTheLighthouseInstrumentsFM