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)