Raman Spectroscopy for Chemical Development, Formulation Development, and Fermentation Applications Brochure

Raman Spectroscopy for Chemical Development, Formulation Development, and Fermentation Applications IFPAC, Cortona, ItalySeptember 19-22, 2010• What is Raman Spectroscopy?• Why use Raman Spectroscopy?• Raman Instrumentation• PAT Applications using Raman• SummaryWhat is Raman Spectroscopy?Raman spectroscopy is a spectroscopic technique used to study vibrational, rotational, and other low-frequency modes in a system. It relies on inelastic scattering, or Raman scattering of monochromatic light, usually from a laser. The laser light interacts with phonons or other excitations in the system, resulting in the energy of the laser photons being shifted up or down gives information about the phonon modes in the system. . I?=sLCII? = Raman intensitys = Raman cross sectionL = PathlengthC = ConcentrationI = Instrument parametersAnalytical Raman SpectroscopySample• What is Raman Spectroscopy?• Why use Raman Spectroscopy?• Raman Instrumentation• PAT Applications using Raman• SummaryWhy Raman?• Composition and Structural Information with Fiber Optic Sampling – the specificity of mid-IR, but with the ease of use of near-IR • Not an absorption process, spectral normalization avoids potential problems associated with differences due to sample shape, sample positioning, particle size, density, and humidity• No sample preparation required, non destructive measurement of most forms of sample - liquids, slurries, pastes, solids, powders, vapors, gases.• Real Time, In-situ, Remote Measurements• Compatible with complete Pharma Lifecycle, from discovery to final manufacturing Chemical Specificity of Mid-IRIdeal for low concentration high potency based formulations040008000185 685 1185 1685Raman Shift (cm-1)Relative Intensity90 mg TabletAvicel Powder (Excipient)Acetaminophen Powder (API) 0.000.200.400.601100 1500 1900 2300Wavelength (nm)IntensityNIR - Majority of signal from ExcipientRaman - Majority of signal from Crystalline API0.940.960.981.001.021.041.061.081 3 5 7 9 11 13 15 17 19 21 23 25 27 29Sample Number%RSD 1325cm-1 APAP Peak Area3mm Dynamic Sampling w/ Normalization3mm Dynamic Sampling w/o NormalizationWith Normalization%RSD = 0.09%Max = 100.2%Min = 99.8%Without Normalization%RSD = 2.89%Max = 105.3%Min = 98.5%Raman spectra of a caffeine sample (which was moved in and out of a sample chamber between spectral acquisitions) with and without normalization. Spectral normalization significantly reduces the effects of sampling (such as particle size, powder compression effects, physical shape of sample) that can be major problems for absorption spectroscopy techniques Instrument RepeatabilityAdvantage of Spectral Normalization• What is Raman Spectroscopy?• Why use Raman Spectroscopy?• Raman Instrumentation• PAT Applications using Raman• SummaryRamanRxn2 Hybrid AnalyzerIdeal PAT tool for Pharma and Biotech ApplicationsAvailable with 532nm, 785nm, and 1000nm lasers offering the best combination of sensitivity and fluorescence suppression for liquids, solids, and gases.• Integrated cart allows for easy sharing between laboratories without the need to IQ/OQ each time• Integrated Cal-Check™ and Auto-Cal™ ensures high quality spectra necessary for multivariate modeling and calibration transferiC Raman™ 4.1 SoftwareDesigned in partnership with METTLER TOLEDO and built on the iC framework, it incorporates Kaiser’s experience in spectroscopy by providing Raman-proven methods, pre-processing steps, and univariate analysis tools. iC Raman™ 4.1 provides instrument configuration, data acquisition, and reaction analysisRamanRxn3 Process AnalyzersManufacturing Ready• Available as 532nm and 785nm Hybrid and 4-Channel systems• Available as 1000nm single channel system• ATEX Certified for Hazardous installations• Compliant Process Software • Documentation Package (GAMP V Model)Window/lensprobeheadSealed adapterFiber cableLaboratory MR ProbeFlexible Sampling OpticsWorking DistanceDepthOf FieldLens WindowRaman back to collection opticsExcitation Laser inLong Focus best for clear liquids Short Focus best for crystallizations or slurriesProcess WetHead ProbesScale up/Pilot Plant ApplicationsWetHeadTM-MaxWetHeadTM-MiniProcess PhAT ProbesPowders, Solids and Slurry ApplicationsAirHeadTMProbeGas and Vapor ApplicationsÎ Direct Insertion / Multi-pass Probe designÎ Sealed Optical DesignÎ 100°C temp / 650 psiÎ Fiber lengths up to 30 metersÎ Constructed of SS 316Sample FlowSapphire WindowFocusing OpticsReflectorProcess Pilot-E ProbesATEX compatible probe designHermetically sealed Pilot-E process immersion probeDesign & Construction Process Certified by Notified Body (TUV)Designed, and Verified to meet the Pressure Equipment Directive (PED)Special window design to meet Impact Tests Special Fiber Optic Connectors to ensure explosive atmosphere compatibility• What is Raman Spectroscopy?• Why use Raman Spectroscopy?• Raman Instrumentation• PAT Applications using Raman• SummaryPAT Applications using Raman SpectroscopyChemical Development• Polymorph Screening• Co Crystal Screening• Reaction Optimization• Reaction Safety Analysis• Polymorphic Monitoring• Lyophilization Monitoring• API dryingFormulation Development• Wet Granulation• Blending• Continuous Blending• Hot Melt Extrusion• Tablet Content Analysis• Tablet Coating AnalysisBiotech Development• Cell CulturePolymorphic ScreeningPolymorphic ScreeningForm IdentificationH‚ X?@?@‚ P‚ O?@?@‚ P‚ P?@?@‚ P‚ Q‚ X?@?@‚ P‚ O?@?@‚ P‚ P?@?@‚ P‚ QPolymorphic ScreeningForm IdentificationForm IdentificationPolymorphs may have different properties - solubility, dissolution rate, stability, or bioavailability.Different crystal forms provide intensity and frequency shifts in the Raman spectrum.1600 1200 800 400 200 Raman Shift, cm–11000 600 1400 Reaction OptimizationCatalytic Hydrogenation ReactionArne Arne ZillianZillian, , SolviasSolvias (Novartis)(Novartis)Catalytic Hydrogenation ReactionIntermediate (hydroxylamine) is a potential thermal safety hazardPreferred pathway excludes the intermediate speciesReactantIntermediateProductCatalytic Hydrogenation ReactionReactantIntermediateProductCatalytic Hydrogenation ReactionRaman provides a clear understanding of nitro-compound hydrogenation to primary amino-compounds.Raman spectroscopy was used to examine the mechanistic and kinetic properties of the reaction.In-situ measurements were possible even in the presence of heterogeneous catalyst.Solvent subtraction was unnecessary using RamanReaction Safety AnalysisReal Time Grignard Monitoring Dave am Dave am EndeEnde & Tim Houck, Pfizer& Tim Houck, PfizerGrignard Reagent FormationR-X +Mg R-Mg-XX= Cl, BrGrignard Reagent FormationSummary:Raman was used to follow the direct formation of a Grignard Reagent.Reaction initiation was followed in real time, eliminating uncertainty in a highly exothermic reaction.The results of the Raman experiment were confirmed by conventional calorimetric analysis.Raman is an ideal tool to monitor hazardous reaction environments.Polymorphic Monitoring“An Investigation of Solvent-Mediated Polymorphic Transformation of Progesterone Using In Situ Raman Spectroscopy,”Wang, F., Wachter, J.A., Antosz, F.J., and Berglund, K.A., Organic Process Research & Development, Vol. 4, No. 5, 2000, 391–395.Polymorphic MonitoringPolymorphs may have Different Properties- i.e. Solubility, Dissolution Rate, Stability, or Bioavailability.Raman is able to Discriminate between Polymorphs because Different Crystal Forms Provide Intensity and Frequency Changes in the Raman Spectrum.The Raman Technique can be Applied without Sample Preparation and Allows for Non-Destructive and In-SituMeasurements.Raman Spectra of ProgesteroneCrystal Forms I and IIRaman Shift (cm-1)1600 1650 1700 0 1000 2000 3000Form IIForm I? 5 cm-1¾ For this Study the C=O Stretching Vibration was used to Quantitate Form I and Form II Polymorphs. Form I @ 1662 cm-1. Form II @ 1667 cm-1.Progesterone Solvent-MediatedPolymorphic Transformation at 45° CRaman Shift (cm –1)16801660Form I @1662Form II @1667167015355575950 20 40 60 80Transformation time (min)Conc. of FormForm IForm II(45° C)¾ Crystallizations were monitored over the temperature range from 5 to 45° C .¾ Slurry: 2 grams Progesterone (25ml Organic Sol.) added to 500ml H2O .¾ Temperature control and stirring were provided by a LabMaxautomated lab reactor.¾ Polymorph concentration was determined from the C=O stretch band center position.¾ Raman measurements were made in-situConclusions¾ Raman can Distinguish Form I and Form II Progesterone Crystals.¾ It was shown to accurately follow the Polymorphic Transformation(Form II to Form I) In-Situ.¾ Transformation Rates were found to Increase with Increasing Temperature.¾ The In-Situ Monitoring of this System Permits the Rate of Polymorphic Transformation to be Predicted over a Wide range of Process Temperatures.¾ The use of Fiber-Optic Sampling Simplifies the Transfer of In-SituRaman Monitoring to Pilot and Production Plants.Wet Granulation"Comparison of Techniques for In-line Monitoring using Raman Spectroscopy."H Wikstrom, l.R. Lewis and L.S. Taylor, Appl. Spectrosc., 59, 934-941 (2005) - Purdue University Wet GranulationWet Granulation of Nitrofurantoiny = x - 2·10-6R2 = 0.99920204060801000 20 40 60 80 100PredictedObservedy = x - 4·10-6R2 = 0.97320204060801000 20 40 60 80 100PredictedObservedSmall Spot- Immersion SamplingPhAT Technology- Wide AreaWet Granulation of Wet Granulation of NitrofurantoinNitrofurantoin anhydrousmonohydrateRaman: Clearly identifiable bands are observed for both the monohydrate and anhydrous forms. NIR: Spectrum dominated by free water. The free water prevented quantification of the forms during the transformation.Solids Blending“A Novel Approach to Measuring Unit Operations using Raman Spectroscopy”F. LaPlant & S.Romero, Pfizer MI Labs, MPPCC Meeting 10/05Solids BlendingSolids BlendingPhAT SystemprobeheadSapphire window sweeps by laser as bin rotatesSolids BlendingSolids Blending“A Novel Approach to Measuring Unit Operations using Raman Spectroscopy”, F. LaPlant & S.Romero, Pfizer MI Labs, MPPCC Meeting 10/05API Scores vs. Blender Revolutions-8.00E-02-4.00E-020.00E+004.00E-028.00E-021.20E-011.60E-010 50 100 150 200 250 300Blender Revolutions Score Value on PC110Hz, API loaded first785 nm0.1 sec acquisitionNo blender modificationHot Melt ExtrusionOff-line and On-line Measurements of Drug-loaded Hot-Melt Extruded Films Using Raman SpectroscopyVenkat S. Tumuluri a, Mark S. Kemper b, Ian R. Lewis b, Suneela Prodduturi c, Soumyajit Majumdar a, Bonnie A. Avery a, and Michael A. Repka a*a Department of Pharmaceutics, The University of Mississippi, University, MSb Kaiser Optical Systems, Ann Arbor, MIc Food and Drug Administration, St. Louis, MOHot Melt ExtrusionHopperHigh T ScrewExtrudate-2-1012341581.47 1603.28 1625.08 1646.89 1668.69 1690.49 1712.3 1734.1 1755.91 Amorphous I - 1 Amorphous II - 1 Amorphous II_I - 110_1 - 1 110_2 - 1 110_3 - 1 110_4 - 1 110_5 - 1 120_1 - 1 120_2 - 1 120_3 - 1 120_4 - 1 120_5VariablesCrystallinity PeakIncreasing Temperaturez Optimize processing conditions to enhance propertieszModify delivered drug’s solubility usinga polymer matrixzMeasure properties without processing equipment modificationExtrudate and FilmsReplace Traditional Raman measurements forpolymer films, transdermal patches, topical dental patches etc 1%2%4%5%7.5%10%15%Pure ketoprofen-300-200-10001001640 1650 1660 1670 1680 2ndDerivativeRaman shift (cm-1)Note: Spectra have been normalizedCrystallinityMeasurementSuspension Content AnalysisReliable and fast quantitative analysis of active ingredient in pharmaceutical suspension using Raman spectroscopySeok Chan Park a, Minjung Kim a, Jaegeun Noh a, Hoeil Chung a,*,Youngah Woo b, Jonghwa Lee b, Mark S. Kemper ca Department of Chemistry, Hanyang University, Seoul 133-791, South Koreab Korea Institute of Toxicology, Daejon 305-343, South Koreac Kaiser Optical Systems, 371 Parkland Plaza, Ann Arbor, MI 48103, United StatesSuspension Content AnalysisConcentration of the active pharmaceutical ingredient in both clear and turbid suspensions can be accurately measured with the use of the wide area illumination (WAI) scheme. By using a laser that illuminates a relatively large sample area, spectra could be obtained that were more representative and morereproducible compared to the conventional small-spot scheme. Tablet Content Uniformity – In TransmissionAPI Concentration & ConfirmationTransmission Accessory~4 mmRaman CollectionLaser Excitation~7 mmAnalyzed Volume of tablet by transmissionTablet Content Uniformity – In TransmissionBuffered Aspirin with a TiOBuffered Aspirin with a TiO22Coating Coating –– pure componentspure components-0.010.190.390.590.790.991.191.391.59200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400Raman Shift (cm-1)Arbitrary IntensityAcetylsalicylic AcidCalcium CarbonateTitanium DioixdeTablet Content UniformityTablet Content UniformityComparisons of Raman GeometriesBuffered Aspirin with a TiO2 CoatingRaman Shift, cmRaman Shift, cm--11Traditional Microscope 2Traditional Microscope 2--8 8 µµm spot , Backscatterm spot , Backscatter6 mm, 6 mm, PPhhATAT, Backscatter, spectra from all components , Backscatter, spectra from all components 6 mm, 6 mm, PPhhATAT, Transmission reduces spectral component from surface coatings, Transmission reduces spectral component from surface coatings400 600 800 1000 1200 Tablet Coating AnalysisValidation of Raman spectroscopic procedures in agreement with ICH guideline Q2 with considering the transfer to real time monitoring of an active coating processJoshua Müller, Klaus Knop, Markus Wirges, Peter Kleinebudde*Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstr 1, 40225 Düsseldorf, GermanyArticle history:Received 4 March 2010Received in revised form 15 June 2010Accepted 19 June 2010Available online 25 June 2010Tablet Coating AnalysisTablet Coating Analysis00 1010 2020 3030 4040 5050 6060 7070001122334455Time (Min.)Time (Min.)Relative Relative IntensityIntensityCoating stoppedRaman Spectroscopy for CHO Cell Culture ApplicationsDr. H Lamb, N Seabrook, A Williams, S Rucker, and J Kelly of the NC State University (Department of Chemical and the Biomolecular Engineering) and BTEC, the BiomanufacturingTraining and Education CenterFermentation StudiesIntroduction• Investigating in-line process monitoring, controland optimization of therapeutic protein production viafed-batch culture of Chinese hamster ovary (CHO)cells.• In this work, multivariate partial least squares (PLS)calibration models were developed from lab standards and real-time process Raman data for a specific CHO cell culture process.• Ultimate goal is closed-loop control of a fed-batch cell culture process based on bioprocess feedback.2L CC BioreactorKey Features¾Top-mount agitator¾Pitched blade impeller¾Sintered metal sparger¾Rotameters for air, O2, CO2, and N2to sparger¾Rotameter for air to overlay¾Heating blanket (not jacketed)¾3 addition pumps (acid, base and anti-foam) ¾PID control of T, pH, and DOCell Culture Bioreactor:Critical Control Parameters• Closed Loop Control• Temperature (37 ± 0.5ºC)• pH (± 0.1 unit)• Dissolved oxygen (± 5%)• Anti-Foam*• Level*• Monitor/Trend• Cell density• pCO2• Osmolality• Glucose• Ammonium• LactateGlucose TrendsAdded NutrientsStart of Day 4End of Day 11Lactate TrendsStart of Day 4End of Day 11Glucose vs LactateResultsGood quantitative analysis and preliminary method transfer results for Glucose, Lactate, Glutamine, Glutamate, VCC, TCC, ViabilityFurther CHO cell culture research is currently beingconducted at several industrial sites. All results to date show capability to quantify the above constituents in situ, and in real-time.Summary• Raman spectroscopy is routinely used as a PAT tool for the development of drug substance. (reaction analysis and polymorph identification) • Raman is now being deployed for API manufacturing having met the ATEX and compliant software requirements • Raman spectroscopy is being accepted as a PAT tool for formulation applications (Tablet content uniformity and coating quality)