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
• Summary
What 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.
.
Iλ=σLCI
Iλ = Raman intensityσ = Raman cross sectionL = PathlengthC = ConcentrationI = Instrument parameters
Analytical Raman Spectroscopy
Sample
• What is Raman Spectroscopy?
• Why use Raman Spectroscopy?
• Raman Instrumentation
• PAT Applications using Raman
• Summary
Why 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 formulations
0
4000
8000
185 685 1185 1685
Raman Shift (cm-1)
Rel
ativ
e In
tens
ity
90 mg TabletAvicel Powder (Excipient)Acetaminophen Powder (API) 0.00
0.20
0.40
0.60
1100 1500 1900 2300
Wavelength (nm)
Inte
nsity
NIR - Majority of signal from Excipient
Raman - Majority of signal from Crystalline API
0.94
0.96
0.98
1.00
1.02
1.04
1.06
1.08
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29Sample Number
%R
SD 1
325c
m-1
APA
P Pe
ak A
rea
3mm Dynamic Sampling w/ Normalization3mm Dynamic Sampling w/o Normalization
With 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
• Summary
RamanRxn2 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 transfer
iC Raman™ 4.1 Software
Designed 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 analysis
RamanRxn3 Process Analyzers
Manufacturing 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/lens
probehead
Sealed adapter
Fiber cable
Laboratory MR ProbeFlexible Sampling Optics
Working Distance
DepthOf Field
Lens Window
Raman back to collection optics
Excitation Laser in
Long Focus best for clear liquids Short Focus best for crystallizations or slurries
Process WetHead ProbesScale up/Pilot Plant Applications
WetHeadTM-Max
WetHeadTM-Mini
Process PhAT ProbesPowders, Solids and Slurry Applications
AirHeadTM ProbeGas and Vapor Applications
Direct Insertion / Multi-pass Probe designSealed Optical Design100°C temp / 650 psiFiber lengths up to 30 metersConstructed of SS 316
Sample Flow
Sapphire Window
Focusing Optics
Reflector
Process Pilot-E ProbesATEX compatible probe design
Hermetically sealed Pilot-E process immersion probe
Design & 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
• Summary
PAT Applications using Raman Spectroscopy
Chemical Development• Polymorph Screening
• Co Crystal Screening
• Reaction Optimization
• Reaction Safety Analysis
• Polymorphic Monitoring
• Lyophilization Monitoring
• API drying
Formulation Development• Wet Granulation
• Blending
• Continuous Blending
• Hot Melt Extrusion
• Tablet Content Analysis
• Tablet Coating Analysis
Biotech Development• Cell Culture
Polymorphic Screening
Polymorphic ScreeningForm Identification
H
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Polymorphic ScreeningForm IdentificationForm Identification
Polymorphs 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–1
1000 600 1400
Reaction Optimization
Catalytic Hydrogenation Reaction
Arne Arne ZillianZillian, , SolviasSolvias (Novartis)(Novartis)
Catalytic Hydrogenation Reaction
Intermediate (hydroxylamine) is a potential thermal safety hazardPreferred pathway excludes the intermediate species
ReactantIntermediateProduct
Catalytic Hydrogenation Reaction
ReactantIntermediateProduct
Catalytic Hydrogenation Reaction
Raman 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 Raman
Reaction Safety Analysis
Real Time Grignard Monitoring
Dave am Dave am EndeEnde & Tim Houck, Pfizer& Tim Houck, Pfizer
Grignard Reagent Formation
R-X +Mg R-Mg-XX= Cl, Br
Grignard Reagent Formation
Summary:
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 Monitoring
Polymorphs 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 II
Raman Shift (cm-1)
1600 1650 1700
0 1000 2000 3000
Form II
Form I
∆ 5 cm-1
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° C
Raman Shift (cm –1)16801660
Form I @1662
Form II @1667
1670
15
35
55
75
95
0 20 40 60 80Transformation time (min)
Con
c. o
f For
m
Form I
Form II(45° C)
Crystallizations were monitored over the temperature range from 5 to 45° C .Slurry: 2 grams Progesterone (25ml Organic Sol.) added to 500ml H2O .Temperature control and stirring were provided by a LabMaxautomated lab reactor.Polymorph concentration was determined from the C=O stretch band center position.Raman measurements were made in-situ
Conclusions
Raman can Distinguish Form I and Form II Progesterone Crystals.
It was shown to accurately follow the Polymorphic Transformation(Form II to Form I) In-Situ.
Transformation Rates were found to Increase with Increasing Temperature.
The In-Situ Monitoring of this System Permits the Rate of Polymorphic Transformation to be Predicted over a Wide range of Process Temperatures.
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 Granulation
Wet Granulation of Nitrofurantoin
y = x - 2·10-6
R2 = 0.9992
0
20
40
60
80
100
0 20 40 60 80 100Predicted
Obs
erve
d
y = x - 4·10-6
R2 = 0.9732
0
20
40
60
80
100
0 20 40 60 80 100Predicted
Obs
erve
d
Small Spot- Immersion Sampling
PhAT Technology- Wide Area
Wet Granulation of Wet Granulation of NitrofurantoinNitrofurantoin
anhydrous
monohydrate
Raman: 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 10/05
Solids BlendingSolids Blending
PhAT Systemprobehead
Sapphire window sweeps by laser as bin rotates
Solids BlendingSolids Blending
“A Novel Approach to Measuring Unit Operations using Raman Spectroscopy”, F. LaPlant & S.Romero, Pfizer MI Labs, MPPCC Meeting 10/05
API Scores vs. Blender Revolutions
-8.00E-02
-4.00E-02
0.00E+00
4.00E-02
8.00E-02
1.20E-01
1.60E-01
0 50 100 150 200 250 300
Blender Revolutions
Scor
e V
alue
on
PC1 10Hz, API loaded first
785 nm
0.1 sec acquisition
No blender modification
Hot Melt Extrusion
Off-line and On-line Measurements of Drug-loaded Hot-Melt Extruded Films Using Raman Spectroscopy
Venkat 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, MO
Hot Melt Extrusion
Hopper
High T Screw
Extrudate
-2
-1
0
1
2
3
4
1581.47 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_5
Variables
Crystallinity Peak
Increasing Temperature
Optimize processing conditions to enhance
properties
Modify delivered drug’s solubility using
a polymer matrix
Measure properties without processing
equipment modification
Extrudate and Films
Replace Traditional Raman measurements forpolymer films, transdermal patches, topical dental patches etc
1%2%4%5%7.5%10%15%
Pure ketoprofen-300
-200
-100
0
100
1640 1650 1660 1670 1680
2nd
Der
ivat
ive
Raman shift (cm-1)
Note: Spectra have been normalized
CrystallinityMeasurement
Suspension Content Analysis
Reliable and fast quantitative analysis of active ingredient in pharmaceutical suspension using Raman spectroscopy
Seok Chan Park a, Minjung Kim a, Jaegeun Noh a, Hoeil Chung a,∗,Youngah Woo b, Jonghwa Lee b, Mark S. Kemper c
a 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 States
Suspension Content Analysis
Concentration 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 & Confirmation
Transmission Accessory
~4 mm
Raman Collection
Laser Excitation
~7 mmAnalyzed Volume of tablet by transmission
Tablet Content Uniformity – In Transmission
Buffered Aspirin with a TiOBuffered Aspirin with a TiO22Coating Coating –– pure componentspure components
-0.01
0.19
0.39
0.59
0.79
0.99
1.19
1.39
1.59
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400
Raman Shift (cm-1)
Arb
itrar
y In
tens
ity
Acetylsalicylic AcidCalcium CarbonateTitanium Dioixde
Tablet Content UniformityTablet Content UniformityComparisons of Raman Geometries
Buffered Aspirin with a TiO2 Coating
Raman Shift, cmRaman Shift, cm--11
Traditional Microscope 2Traditional Microscope 2--8 8 µµm spot , Backscatterm spot , Backscatter
6 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 coatings
400 600 800 1000 1200
Tablet Coating Analysis
Validation of Raman spectroscopic procedures in agreement with ICH guideline Q2 with considering the transfer to real time monitoring of an active coating process
Joshua Müller, Klaus Knop, Markus Wirges, Peter Kleinebudde∗
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstr 1, 40225 Düsseldorf, Germany
Article history:Received 4 March 2010
Received in revised form 15 June 2010Accepted 19 June 2010
Available online 25 June 2010
Tablet Coating Analysis
Tablet Coating Analysis
00 1010 2020 3030 4040 5050 6060 707000
11
22
33
44
55
Time (Min.)Time (Min.)
Rel
ativ
e R
elat
ive
Inte
nsity
Inte
nsity
Coating stopped
Raman Spectroscopy for
CHO Cell Culture Applications
Dr. 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 Center
Fermentation Studies
Introduction
• 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 Bioreactor
Key FeaturesTop-mount agitator
Pitched blade impeller
Sintered metal sparger
Rotameters for air, O2, CO2, and N2to sparger
Rotameter for air to overlay
Heating blanket (not jacketed)
3 addition pumps (acid, base and anti-foam)
PID control of T, pH, and DO
Cell 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• Lactate
Glucose Trends
Added Nutrients
Start of Day 4
End of Day 11
Lactate Trends
Start of Day 4
End of Day 11
Glucose vs Lactate
Results
Good quantitative analysis and preliminary method transfer results for Glucose, Lactate, Glutamine, Glutamate, VCC, TCC, Viability
Further 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)