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Introduction to MicrocalorimetryMalin SuurkuuskTA Instruments, Sweden

TA Instruments Design, Manufacture and Service Microcalorimeters Thermal analysis Instrumentation Dynamic Vapor Sorption Instrumentation Rheometers and Dynamic MechanicalAnalyzers Subsidiary of Waters Corp since 1995 ~500 employees worldwide Based in New Castle, DE Manufacturing/Support sites: Utah (US) - Microcalorimetry Pittsburgh (US) Hüllhorst (Germany)

Microcalorimetry Center~35 employeesFull Training centerFull Service centerApplications LabTA InstrumentsApplications support10 scientistsUS GermanyJapan SwedenChina IndiaMicrocalorimetryManufacturing / Service / Apps SupportLindon, Utah

MicrocalorimetryVirtually all chemical and physical processes result ineither heat production or heat absorption.Calorimetry quantifies the amount and rate of heat releasein terms of heat flow, heat and heat capacity.Calorimetry is a non-specific technique making it ideal forstudying almost all kind of physical and chemicalprocesses in life sciences, material sciences and withinthe pharmaceutical field.

How does calorimetry differ from otherphysical techniques?Calorimetry is nondestructive and noninvasiveAll kinds of processes; Chemical, Physical andBiologicalNot dependent on the physical shape of the sampleSolids, liquids and gases can be studiedNo chemical derivatization or immobilizationNo need for sample preparationNon-specificMicrocalorimetry continuously and directly measuresthe process under study - Real-time data

Microcalorimetric TechniquesIsothermal Titration Calorimetry – ITCFor characterization of binding reactions between proteins and ligands or othermacromoleculesFor enzyme kineticsDifferential Scanning Calorimetry – DSCFor the characterization of the thermal stability of proteinsIsothermal Microcalorimetry - TAMAmpoule calorimetryFor stability, compatibility, polymorhism & amorphicity assessmentRH perfusion calorimetryFor amorphicity & crystallinity assessment, hydration and swellingSolution calorimetryFor amorphicity & crystallinity assessmentTitration calorimetryEvaluation of drug effect on living cells

Scanning MicrocalorimetrySensitivemWµW nWMoreSensitiveDiscovery DSCMC DSCNano DSCDiffusion-bonded Sensor50-Sample AutosamplerSample Size: up to 20 mgScan Rate: 0.1 – 100°C/min3 – samples1 – referenceSample Flexibility≤ 1 mL sample Vol.Scan Rate: 0 - 2°C/min1 – sample1 – referenceIn-solution sample300 µL active cell Vol.Scan Rate: 0.001 - 2°C/minAutomated sample handling

Isothermal MicrocalorimetrySensitivemWµW nWMoreSensitiveTAM AirTAM III & TAM 48MC DSCNano ITC8 – samples8 – reference20 mL sample Vol.Temp Range: 5 to 90°C3 – samples1 – referenceSample Flexibility≤ 1 mL sample Vol.Temp Range: -40 to 150°C1-48 – samplesSample Flexibility1-20 mL sample Vol.Temp Range: 15 – 150°C1 – sampleMax Sensitivity1 mL or 190 µL cellsTemp Range: 2 – 80°C

Biopharmaceutical Characterization ToolsAnalytical techniques to accurately evaluate and monitor:StructureFunctionCompatibilityProduct MicroheterogeneityProduct Stability

Microcalorimetry in BiopharmaceuticalCharacterizationDifferential Scanning Calorimetry (DSC)• Domain Structure• Thermal stability• Pre-formulation evaluationIsothermal Titration Calorimetry (ITC)• Ligand – Target interactions / binding• Thermodynamic binding profile• Inhibitor Identification & Characterization• Isothermal Microcalorimetry (IMC)• Stability and compatibility

Thermodynamic Driving ForcesSpecific BindingIntermolecular – ligand bindingIntramolecular – protein foldingProteins - Complex BiomoleculesMacroscopic systems surrounded by solventStructure Energetics – complexFavorable and unfavorable forcesEnergetic Driving Forces∆H (Enthalpy) ∆G (Gibbs free energy)∆S (Entropy) ∆C p (Heat capacity)

Thermodynamic Driving Forces Definition∆GGibbs Free Energy of formationspontaneous reactions have a negative G valueK aBinding constantn∆H∆S∆C pStoichiometry# of binding sites per molecule of targetEnthalpyindicates specific hydrogen bond formation, van derWaals forces and ionic interactionsEntropyindicates hydrophobic or non-specific bond formationHeat capacityindicates conformational changes occurring upon binding

Thermodynamic Driving Forces Relationships∆G= ∆H−T∆S∆G= −RTlnK B∆C p=∆HT22− ∆H−T11

ITC & DSC: Complimentary Tools∆G, ∆H, ∆S and ∆Cp are properties ofany associating systemThey are composed to varying degreesof contributions from:Binding reactionConformational changes during a reactionChanges in a molecule’s solvation statusProtonation status

DSCDifferential ScanningCalorimetryMain calorimetric technique used in thecharacterization of the thermal stability ofproteins

110100908070605040302010DSCT mArea = ∆H cal∆C p50 60 70 80 90 100Temperature (°C)Partial Molar Heat Capacity (kJ K -1 mol -1 )

Why use Nano DSC?DSC is the only technique that allows thedirect measure of T m , ∆C p and ∆H. DSC allows for calculation of entropy (∆S) andfree energy (∆G).Equally useful for macromoleculesCompatible with essentially any buffer oradditiveRequires very small sample concentrationsand volume

Nano DSC SensitivityHow much protein is required for a DSC experiment?200HEW Lysozyme in 0.20 M Glycine Buffer, pH 4.0 (2 °C/minute)180Molar Heat Capacity (kJ K -1 mol -1 )1602 µg1401205 µg10010 µg806025 µg4050 µg100 µg20400 µg055 60 65 70 75 80 85 90 95Temperature (°C)

Nano DSC Design

Benefit of Capillary Sample CellData obtained with a DSC with “Coinand Cylindrical ” shaped sample cellData obtained with a Nano DSC withcontinuous Capillary sample cell4020070.788.95040Stable baselineafter unfoldingheat rate / µJ s -1-20-40-60-80Precipitating proteinafter unfoldingheat rate / µJ s -1302010-100-1200-140-1040 50 60 70 80 90 100 11040 50 60 70 80 90 100 110temperature / °Ctemperature / °CPurified human IgG 1 monoclonal antibody in physiological buffer; 0.5 mg/mL

Nano DSC ApplicationsBiopolymer – Conformation &SolvationAbsolute Heat Capacities, isrequired to correct temperaturedependency of ITC dataBiopolymer StabilityProtein / DNA / RNA DenaturationBiopolymer StructureDomain and Subunit OrganizationBio-EngineeringMutant ProteinsLigand InteractionsDrug Binding to Proteins orNucleic AcidsMembrane StructureLipid Bilayers, MembraneproteinsPressure PerturbationLipid/biopolymer structureand solvation (G, H, C p , C v ,α [thermal coefficient ofexpansion], β[compressibility])

Types of Experiments Addressed by Nano DSCDetermine the T m Temperature at which half the molecules are unfolded. Indication of the stability of the molecule.Analyze a single macromolecule, or macromolecularcomplex Stability Measure binding affinity (high affinity)Measure the heat capacity change (∆C p ) of amoleculeMeasure the enthalpy (∆H) of unfolding ofmacromolecules or macrolmolecular complexes (Tm).Experimental approaches are applicable to allmacromolecules

DSC – Domain Structure252015Data: SAMPLE5DSC_cpModel: M2StateChi^2 = 3361644.85449Tm1 60.00 ±0.1541H1 1.14E5 ±2.44E3Tm2 69.83 ±0.0890H2 1.47E5 ±2.21E3kcal/mole105020 30 40 50 60 70 80 90Temperature ( o C)

Power of DSCPlasminogenStructure of plasminogen(MW=83,000 Da) is organized in 7cooperative domains which meltindependently over differenttemperature rangesDSC Strength:Deconvolution analysis of themelting profilesEnthalpy and temperature arethermodynamic parametersspecifying the system and theirfunctional dependenceInformation on the states of thesystem populated over theconsidered temperature range* Peter L. Privalov and Anatoly I. Dragan, 2006

IgGT max(˚C)∆H cal(kJ/mol)∆S cal(kJ/molK)mutA 57.1 570 1.7mutB 57.6 669 2.0WT 64.9 529 1.6IgG control (red)Mutated #A (green)Mutated #B (blue)Wen, J.; Jiang, Y. American Pharm. Rev., 2008, 11, 98.

Liquid Formulation Stability Increases in T m correlate with improved stability of IL-1R DSC data can reliably predict the rank order of stability for various additivesExcepient Mole Ratio T m ( o C)Control - 48.1SugarsManitolGlucose2037203746.749.6Polymers /PolyolsPEG (300)EthanolEthanol7779761749.448.743.8SaltsNaClCaCl 271771753.141.1SurfactantsPluronic F68Tween 804546.645.8Glucose/NaCl 2037 / 717 52.2Remmele, et. al., Pharmaceutical Res. 15, 200-208 (1998)

Liquid Formulation Stability Evaluation of Preservatives and IL-1RT m %AggregationControl 50.8 1.5Phenol 50.3 3.0m-Creosol 48.4 5.1Benzyl alcohol 45.2 16.5Control in 100 mM NaCl in 20 mM NaCitrate% Aggregation determined by SECRemmele, et.al.,Pharmaceutical Res. 15, 200-208 (1998)

Membranes/membrane protein interactions Membrane proteins are mostlyhydrophobic, DSC can provide indication of a proteininserted into a specific membrane Specific changes in lipid thermogram indicateshow protein interacts with membrane Most techniques are affected bycloudiness, light scattering. DSC is notaffected DPPC – green, 2-3 molecules ofmembrane protein added to DPPC (othercurves). Shift in T m identifies that proteinembeds into DPPC bi-layer.

Importance of van’t Hoff enthalpyIs N ↔ U ?K eq = [U]/[N];∆G = -RTlnK eq ;∆G = ∆H - T∆S Fit DSC data to two-state model to determine K eq at temperatures intransition, evaluate ∆H vH . Curve is the van’t Hoff plot per mol of N ↔ U‘cooperative unit’. Fit enthalpy under curve is ∆H vH . ∆H cal is directly measured energy per mole protein. ∆H vH is calculated energy per mole cooperative unit. If ∆H vH = ∆H cal , then co-operative unit = protein, and N ↔ U

Importance of van’t Hoff enthalpyComparison of measured (∆H cal ) and calculated (∆H vH )enthalpy can provide information into the biopolymerstructure.If ∆H vH = ∆H cal , then N ↔ UMW Co-operative unit = MW protein (two-state model valid)If ∆H vH < ∆H cal , then MW Co-operative unit < Mw proteinIntermediate unfolded states are likely present, N ↔ I ↔ U(two state model invalid)If ∆H vH > ∆H cal , then MW Cooperative unit > MW proteinProtein forms oligomers N n ↔ nU (two-state model valid)

Importance of heat capacityThe heat capacity of a folded protein isdue to its specific sequence (majorfactor), hydration (minor) and noncovalentinteractions (2 o and 3 o structure)(minimal).Change in heat capacity during unfoldingprimarily reflects changes in hydrationKnowing molecular weight andconcentration of the protein allows thepartial molar heat capacity of the proteinthroughout the scan (e.g. 37 o C) to bedetermined.Practical implications: protein with lowerheat capacity is more rigid, moreselective when binding a ligand. Proteinwith higher heat capacity is more flexible,less selective.

OligomerizationEffect of sample concentration dependence of T m is a test foroligomerization.

How to determine if unfolding is kineticallycontrolled? Scan rate dependence of T m indicates that N and U are not inequilibrium. Their concentrations change at a rate equal to the sum of theunfolding and refolding reactions. Increasing temperature faster thansystem responds distorts T m and shape.

Membrane protein structureMicrograms of a membrane proteincan represent weeks of work andrequire detergents for solubilizationNano DSC obtained high-quality scansusing 20 µg (0.3 picomoles) of a70,000 Da complex (non-palmitoylated andpartially palmitoylated SNAP-25) consisting of3 membrane proteins Thermogram very well fit by 3transitionsPartial chemical derivatization andstabililization of complex easily verifiedby DSC

Measuring Molecular Interactions with DSCDSC Binding Data Increasing amounts ofinhibitor shows T m shiftas more binding occurs Binding constant can beestimated Tight binders up to 10 20M -1 can be runExcess Cp / kJ mol -1 K -180706050403020100Rnase A – 2’CMP0.3 mM0.15 mM0.075 mM0.05 mM0 mM0.75 mM1 mM1.25 mM1.5 mM DSC is a quick way ofscreening whether twomolecules interact-1035 45 55 65 75 85Temperature / °C

Nano DSC Automation SystemNano DSC Autosampler System: True program-and-walk-away functionality Most reliable, sensitive Nano DSCavailable Reliable liquid handling autosampler fromSpark Holland Easy conversion of Nano DSC tosingle sample (or manual) configuration Utilizes minimum bench space Superior signal-noise at lowsample concentration Both Nano DSC and Autosamplerprogrammed from same software Temperature controlled sample storage(4°C – Ambient) Two 96-well sample plates Programming for up to 96 samples withmatching buffers

Nano DSC AutosamplerWash Buffer forSample HandlingSyringeSample HandlingSyringeSample DeliveryLine to Nano DSCTemperatureControlledSample Storage AreaSample PlateBuffer Plate

Nano DSC Autosampler PerformanceRaw Heat Rate / µJ/s302520151050-5Raw Heat RateNano DSC AnalysisAutosampler System2 mg/mL1 mg/mL0.5 mg/mL0.2 mg/mL0.1 mg/mLGlycine Buffer30 35 40 45 50 55 60 65 70 75 80Temperature / °CExperiment Parameters:Instrument: Nano DSCSample: LysozymeScan Range: 10 – 80ºCScan Rate: 1ºC / minSample Buffer: GlycineSample Vol: 1mLA/S & DSC Protocol:1. Initial cell wash2. Cell conditioning scan3. Cell wash4. Buffer scan5. Cell wash6. Sample scan7. Cell wash8. Buffer scan9. Final cell wash2 SamplesEachmg/mL

Nano DSC Autosampler PerformanceMolar Heat Capacity / kJ/mol·K4035302520151050-5-10-15Nano DSC AnalysisAutosampler SystemLowest concentration 0.1 mg/mLMolar Heat CapacityExperiment Parameters: Instrument: Auto Nano DSC Sample: Lysozyme Scan Range: 10 – 80ºC Scan Rate: 1ºC / min Sample Buffer: Glycine Sample Volume inautosampler tray: 1mL Small shift in magnitude oflowest concentration sample,which could indicate errors inactual concentration used toconvert to Molar HeatCapacity units.40 45 50 55 60 65 70Temperature / °C

Nano DSC Autosampler Performance ResultsLysozyme SampleConcentrationT m(ºC)∆H(kJ / mol)2 mg/mL 55.28 ±0.02 399.0 ±2.81 mg/mL 55.35 ±0.07 398.5 ±2.10.5 mg/mL 55.48 ±0.07 397.0 ±1.40.2 mg/ml 55.70 ±0.01 395.0 ±2.80.1 mg/mL 56.00 ±0.01 393.0 ±1.4Published Values @ pH 2.41-10 mg/mL55.25 – 58.75 377 – 439

Summary DSC is the only technique for directly determining the enthalpy of theunfolding of a biological polymer. DSC is a direct measurement of the thermal stability and molecularstructure of biopharmaceuticals Comparison of ∆H cal to ∆H vH provides unique information about theunfolding pathway (oligomerization, intermediates, aggregation). Sample concentration dependence of T m is a sensitive test of higherorderassociation. Scan rate dependence of T m is the key test for equilibrium unfolding.

ITCIsothermal Titration CalorimetryMain calorimetric technique used incharacterization of binding reactionsbetween proteins and ligands or othermacromolecules

ITC∆G=∆H-RTlnK a=∆H-T∆SK an

Why use Nano ITC? Completely general technique ITC measures the production and absorption of heat Direct measure of Enthalpy Provides information on thermodynamics of thereaction Technique of choice for affinity measurements Natural, unmodified ligands and substrates can beused Requires neither immobilization or labeling Equally useful for macromolecules and smallmolecules Compatible with essentially any buffer oradditive Reactions conducted isothermally at anytemperature between 2 to 80 o C

Nano ITC DesignChoice of Sample Cell Volume190 µL1.0 mL

Binding interactions Measure the affinity of bindingbetween two or more molecules(complex)Protein-Protein, Protein-DNA, Protein-RNA,Protein-LipidProtein-Carbohydrate, Protein-Metal ionDNA-DNA, DNA-RNA, DNA-Metal ionProtein-Nanomaterial, DNA-Nanomaterial,Antibody-NanomaterialEtc. – Any interacting moleculesProtein-ProteinInteractionResearchers identify the affinity of an interaction, so they can then try andblock/strengthen the interaction to influence a therapeutic-cellular response.Drug discovery,Enthalpy and stoichiometry provide additional informationType of binding (hydophobic/H-bonding)

ITC ApplicationsMolecular Binding StudiesQuick and accurate affinitiesBinding stoichiometriesInhibition of protein protein interactionInvestigate structure-function relationshipsAffinity and mechanism of action screeningSpecific vs. non-specific bindingQuality and process controlProtein engineering assessmentValidate virtual modelsReaction KineticsK M, V max, k catEnzyme Inhibition

Enthalpy/entropy compensation in drug design1 2 310kcal/mole50-5-10∆G∆H-T∆S-15-20Entropy Hydrophobic interactions Solvation entropy due to releaseof water (favorable)Enthalpy Directly associated to number andstrength of H-bonds broken or formed Choice of solvent important

ITC: Biopharmaceutical Characterization

Proton LinkageM + L + BM + LB + H +ML + BH + ; ∆H reactionML + H + ; ∆H bindingBH + ; ∆H protonP/ nW2000-200-400-600-800∆H reaction-1.0000 50 100 150 200Time/min∆H protonIGF-I to the soluble extracellularIGF-I receptorData from Hallén, D., Thermometric Application Note 22024 (1997)

Quality Control-250% active0Protein Quality Anti-quinidine antibodybatches comparedkcal/mole of injectant-4-6-8-10Fully active Activity of antibodiesimmobilized on metalbeads quantitativelymeasured0.0 0.5 1.0 1.5 2.0Molar Ratio

Why run Both ITC and DSC?The complete thermodynamic profile of anassociating system is composed of :Binding reactionSpecific interactionsNonspecific interactionsConformational changes during a reactionUnfolding and refoldingStability changesChanges in a molecule’s solvation statusWater accessible polar and nonpolar surfacesProtonation statusSolvent ionic strength

Power of DSC and ITC TogetherITC provides global thermodynamic profile of energeticsof molecular binding reactions.DSC provides details on energetics of molecularconformation changes, subunit folding, complex stability,etc.Together ITC and DSC enables the understanding ofcomplex contributions of molecular and sub-moleculardriving forces, binding mechanisms and subunit andcomplex stability.

Studying enzyme kinetics by ITCEssentially all enzyme reactions produce heat. Studyingkinetics by ITC is fast, easy, with little methodsdevelopment required: simply titrate a known amount ofsubstrate into the enzyme solution, and measure the totalheat produced. When heat production stops, the reactionis finishedRate of an enzyme reaction is calculated by:Rate of heat production per unit substrate is known, so:

Enzyme application 1: Simple kinetics, single injection Single injection method: versatile, fast, canbe used to study fast and slow kineticsInject [S] > K M , [E]Penicillin: 15 µL, 10.2 mM injectionPenicillinase: 950 µL 7.7 x 10 -11 Mheat rate / µW1614121086420AA: raw data (instrument response)B: rate of hydrolysis (normalized for [E])-20 500 1000 1500 2000 2500 3000 3500time / s1800Replot B as Lineweaver-Burk plot(1/velocity vs. 1/[S]); V max = y-intercept,K M = x-intercept, k cat = V max /[E total ]rate / s -11600140012001000800B600K M = 30.7 µMV max = 1.4 x 10 -10 mol s -1k cat = 1950 s -140020000 20 40 60 80 100 120 140 160[S] / µMEssentially no methods development

Enzyme application 2: Enzyme inhibition, single injection2.52ABlue: 10 µL 5.1 x 10 -7 M trypsin injectedinto 950 µL 1.44 x 10 -4 M BAEERed: plus 1.36 x 10 -4 M benzamidineTotal heat identical (∆H = -6.33 kcal/moleBAEE)(-) inhibitor: K M = 4.17 µM; V max = 0.091µMol/s, k cat = 17.8 s -1(+) inhibitor: K M = 35.1 µM; V max = 5.9 x10 -4 µMol/s, k cat = 0.11 s -1 , K i = 18.4 µMheat rate / µW1.510.500 500 1000 1500 2000 2500 3000 3500 4000time / s20rate / s -118B16141210864200 20 40 60 80 100 120 140[S] / µM

Enzyme application 3: Enzyme catalysis, multiple injections Enzyme (between 5 x 10 -11 to 2 x 10 -8 M) insample cell, substrate (between 2 x 10 -5 to5 x 10 -1 M) in syringe Determine rate by measuring dQ/dt when 2-10 µL of substrate injected Spike due to heat of dilution of substrate Signal establishes new steady state,reaction occurs at steady state, negligiblesubstrate converted to product in this time Second substrate injection: substrateconcentration increased, thermal output ofreaction increased (dQ 2 /dt). Rate of heatgenerated proportional to rate of reaction. Typically 10-30 injections, 2-5 min apart Example: penicillinase (1 mL, 7.7 x 10 -11 M,30 o C. Penicillin (30.6 mM), 20 x 5 µL, 5 minintervals. K M = 101 mM.

ITC and enzyme kinetics summaryEssentially all enzyme reactions produce heat, and socan be studied by calorimetry.No spectroscopic probes or colorimetric substrates arerequired. The natural substrate can be used, makingcalorimetry a versatile and universal assay method.The same approach can be used for all enzymes,requiring little or no methods development.

Summary ITC is a completely general technique, since all reactions produce orabsorb heat ITC is the technique of choice for determining K a , n and ∆H ofbinding reactions Only ITC provides a complete thermodynamic analysis of the bindingreaction. This is advantageous since determination of ∆G alone provides littleinformation on the specificity of the binding reaction. The concentrations of reactants required for ITC should satisfy:10 < K a [M] T < 1000 ITC is also a powerful and straightforward approach for determiningkinetics constants

Isothermal microcalorimetry Isothermal Calorimetry is a technique for a direct measurement ofheat production or consumption of a sample The heat production rate is directly related to the rate of reaction Total heat produced is related to the extent of the reactionP H ⋅k⋅ f= ∆(c)Heat flow (µW/g)1800016000140001200010000800060004000200000 1 2 3 4Time (d)Energy (J/g)1800016000140001200010000800060004000200000 1 2 3 4Time (d)

Benefits Universal – if anything is happening in the sample it will likely bedetected Non-specific – everything going on in the sample will be detected Sample preparation is not necessary Not dependent on physical state of the sample Non-destructive Direct Continuous Highly sensitive – measurements can be performed at or close toambient. No need for accelerated conditions

Isothermal Microcalorimetry– A universal techniqueChemistryPharmaceuticalSciencesLifeScienceMaterialScience

TAM III –a flexible multichannel microcalorimetric system 1 – 48 individual & independent calorimeters nW - mW sensitivity depending oncalorimeter

Calorimeters for TAM IIINanocalorimeter

The Flexibility of TAM IIISample sizeAbsolute Sensitivity

Sample handling system for TAMAll sample handling systems areavailable in 1, 4 and 20 ml sizesMacrocalorimeter ampoules areavailable in 120 mlGas or LiqPerfusionRHPerfusionTitrationVacuumPressure

Static measurementsStatic measurementsStabilityCompatibilityReaction kineticsAmorphicityPolymorphismCuringSafety assementMicroorganism growthEtc...

Irreversible denaturation of proteinsStability study of a monoclonal antibody:Abbott X in phosphate bufferDSC traceNLumry-Eyring:k1,k 2←⎯⎯→U1rate = ∑ ∆ Hk3⎯⎯→i⋅ PiAIsothermal calorimetry trace ofa at 4 different pHZhu et al (2010) Thermochim. Acta 499

Titration setup– Possibilities for adding and mixing Evaluate binding constants andthermodynamics Complex formation Cmc determinations Enzyme kinetics Mixing enthalpies Dissolution kinetics Absorption Reaction kinetics Swelling Drug effect on livingcells Etc...P,µW4.53.01.50.0kJ/moldQ/dAmount402000.01 2 3 4Time,hourK 1 =2.541 . 10 5 M -1∆H=55.79694 kJ/mol0.6 1.2 1.8[L]/[M],

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