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©2007 Waters Corporation
John ShinodaJohn Shinoda
Senior Support SpecialistSenior Support Specialist
Waters CorporationWaters Corporation
November 5, 2009November 5, 2009
IMPURITY PROFILINGIMPURITY PROFILING
©2007 Waters Corporation 2
OverviewOverview
� Business Issues
� Analytical Changes
—Chromatography
— Detector considerations
— Software considerations
� Test Case Study
—Budesonide method development
— Budesonide batch analysis
� Leachables
— Polymer Additives
— Detector Considerations
©2007 Waters Corporation 3
Impurity ProfilingImpurity Profiling
Importance to OperationsImportance to Operations
� Accept or reject raw material for API or drug product production
— Impurity profiles define material acceptance or rejection
— Out of specification results can adversely impact production schedules, ship dates, and supplier relationships
o Rapid investigation and determination of OOS cause is imperative
— Long impurity profiling analyses impact raw and work-in-process inventory costs, especially in lean manufacturing environments
� Support process development and scale up
—Characterize impurities and intermediates
— Necessary when new or different reagents and solvents are proposed to support process optimization and change
o Typically changed with the intent of improving economics of process or yield of synthetic route
©2007 Waters Corporation 4
Impurity ProfilingImpurity Profiling
Importance to OperationsImportance to Operations
� Sign off of finished API or drug product for release
— Ensures quality of the product for customer confidence
o API Certificate of Analysis
— Impacts time to market and customer satisfaction
� Adhere to CGMPs and CMC regulatory requirements
— Preparation of regulatory filings is dependent on time to
generate, retrieve, and compile results for necessary studies
— Results must be accessible and confidently defendable to FDA
audits and queries
©2007 Waters Corporation 5
NDA and ANDA Filing ConsiderationsNDA and ANDA Filing Considerations
Reporting, Control, and Reporting, Control, and
Specification of ImpuritiesSpecification of Impurities
Guidance for Industry, Q3A Impurities in New Drug Substances, February 2003; \\CDS029\CDERGUID\4164fnl.docDraft Guidance for Industry, ANDAs: Impurities in Drug Substances, January 2005; J:!GUIDANC\6422dft.doc
Summarize actual and potential impurities
Provide analytical results for all batches of drug substance used for clinical, safety, stability testing; also for batches representative of proposed commercial process
Provide documented evidence that analytical procedures are
validated and suitablevalidated and suitable for the detection and quantificationdetection and quantificationof impurities
List impurities for specification based on impurities found in batch(es) manufactured by the proposed commercial process
Drug SubstanceDrug Substance
©2007 Waters Corporation 6
Impurity ThresholdsImpurity Thresholds
Drug SubstancesDrug Substances
Maximum Daily Dose
Threshold
Reporting Identification Qualification
≤≤≤≤ 2 g/day 0.05%
0.10% or
1.0 mg/day intake (lower)
0.15%or
1.0 mg/day intake (lower)
> 2 g/day 0.03% 0.05% 0.05%
The quantitation limit for the analytical procedure The quantitation limit for the analytical procedure
should not be more than the reporting threshold should not be more than the reporting threshold Guidance for Industry, Q3A Impurities in New Drug Substances, February 2003; \\CDS029\CDERGUID\4164fnl.docDraft Guidance for Industry, ANDAs: Impurities in Drug Substances, January 2005; J:!GUIDANC\6422dft.doc
©2007 Waters Corporation 7
Analytical ChallengesAnalytical Challenges
� Accurate and precise quantification of low-level impurities
in the presence of high amount of drug substance
� Developing impurity profile methods that support potential
future process or supplier variability
� Accessing current and historical results when needed for
FDA audit or regulatory filing
� Expedient response and investigation into OOS events
©2007 Waters Corporation 8
Chromatographic ConsiderationsChromatographic Considerations
� High resolution separations
—Maximize chromatographic performance using sub-2 µm particles
—Optimize resolution and selectivity with a variety of column
chemistries and dimensions
—Use temperature as a tool
� Smooth chromatographic baselines
—Provide efficient solvent mixing
� Consistent peak detection and integration
—Increase sensitivity and improve limits
of quantitation with low volume, narrow
chromatographic peaks
©2007 Waters Corporation 9
1.1
84
1.3
09
1.4
19
1.7
58
2.8
55
3.2
46
3.4
43
3.7
00
4.6
09
4.7
13
5.1
87
5.3
72
6.7
86
AU
0.000
0.002
0.004
0.006
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
Simvastatin Impurity Simvastatin Impurity
UPLC SeparationUPLC Separation
SimvastatinLovastatin
ACQUITY UPLC BEH C182.1 x 100 mm, 1.7 µmFlow Rate: 0.65 mL/minMobile Phase A: 15 mM ammonium bicarbonate, pH 9.0Mobile Phase B: AcetonitrileGradient: 40-100% B over 7 minutesInjection Volume: 2.0 µLColumn Temperature: 35°CUV at 238 nm
©2007 Waters Corporation 10
Consistent Peak Detection & IntegrationConsistent Peak Detection & Integration
Importance of Sampling RateImportance of Sampling Rate
� Must ensure enough points are collected across a peak to
adequately define the peak shape
� Peak detection algorithms require a minimum number of
points across a peak to distinguish it from baseline noise and
correctly determine peak lift off and touch down
� A peak which does not have enough data points will be
difficult to integrate and therefore have irreproducible peak
areas and heights
©2007 Waters Corporation 11
Effect of Sampling Rate on Effect of Sampling Rate on
Peak ShapePeak Shape
Sampling Rate Points Across Peak Peak Area %RSD Peak Height %RSD
1 pt/s 2 2.436 15.515
2 pts/s 4 1.790 13.455
5 pts/s 7 0.971 3.962
10 pts/s 13 1.129 1.015
20 pts/s 25 0.603 1.156
40 pts/s 49 0.284 1.127
AU
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
0.022
0.024
0.026
0.028
0.030
0.032
0.034
Minutes0.565 0.570 0.575 0.580 0.585 0.590 0.595 0.600 0.605 0.610 0.615 0.620 0.625 0.630
1 pt/s1 pt/s2 pt/s2 pt/s5 pt/s5 pt/s
10 pt/s10 pt/s
20 pt/s20 pt/s40 pt/s40 pt/s
©2007 Waters Corporation 12
Detector Dynamic RangeDetector Dynamic Range
� Linearity of detector > linear
range of impurity and parent
� Allows quantification and
validation of low level
impurities (<0.01%) and
large parent compounds
simultaneously
� Analytical flow cell
— 10mm pathlength
� High sensitivity cell
— 25 mm pathlength
0
0.5
1
1.5
2
2.5
3
3.5
0 10 20 30 40 50 60 70 80
PDA - Analytical Flow Cell
PDA - High Sensitivity Flow Cell
1.5% Deviation at 2.0 AU
5.0% Deviation at 2.7 AU
1.3% Deviation at 2.0 AU
5.0% Deviation at 2.8 AU
0
0.5
1
1.5
2
2.5
3
3.5
40 50 60 80
% Absorber in Eluent
UV - Analytical Flow Cell
UV - High Sensitivity Flow Cell
2.2% Deviation at 2.5 AU
5.0% Deviation at 3.0 AU
2.4% Deviation at 2.5 AU
5.0% Deviation at 3.0 AU
Absorbance (AU)
0 10 20 30 70
©2007 Waters Corporation 13
Detection of Low Level ComponentsDetection of Low Level Components
Spectral Quality MaintainedSpectral Quality Maintained
o-Toluidine0.01% ImpurityExpanded View
Prilocaine Spectrum
o-Toluidine Spectrum
Prilocaine
©2007 Waters Corporation 14
Integration of Low Level ImpuritiesIntegration of Low Level Impurities
with 2with 2ndnd derivative Integration derivative Integration
� Automatic first pass integration using default parameters
—Auto Peak Width
—Auto Threshold
� Integrates negative peaks effectively
� Integrates small peaks in noisy or drifting baseline effectively
� Peak shoulders easily detected
� Gaussian skimming available for very complex
chromatograms
� Manual integration is minimized
©2007 Waters Corporation 15
�� Traditional IntegrationTraditional Integration
� 2nd derivative IntegrationIntegration
Accurate Peak DetectionAccurate Peak Detection
Shoulders Accurately Integrated Shoulders Accurately Integrated
©2007 Waters Corporation 16
Photodiode Array ReviewPhotodiode Array Review
©2007 Waters Corporation 17
MS ReviewMS Review
©2007 Waters Corporation 18
Test Case: Budesonide Assay Test Case: Budesonide Assay
Part IPart I
ReRe--development of European Pharmacopoeial (EP) Assay for development of European Pharmacopoeial (EP) Assay for
Budesonide Using UPLCBudesonide Using UPLC®®
� Goal
—Decrease raw material inventory costs by shortening the time to
accept new lots of budesonide drug substance
� Analytical Needs
—Method development
oExplore column, pH, organic solvent, temperature
—Meet EP assay criteria
—MS compatible solvent system
oSingle quadrupole mass detection for impurity confirmation
©2007 Waters Corporation 19
Current SituationCurrent Situation
EP HPLC MethodEP HPLC Method
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
Minutes
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
R-epimer S-epimer
26.00
Isocratic separation using Acetonitrile and a Phosphate buffer
©2007 Waters Corporation 20
UPLC Method DevelopmentUPLC Method Development
Column Scout for BudesonideColumn Scout for Budesonide
ACQUITY TUV ChA - ACQUITY TUV ChA 240nm
AU
0.000
0.005
0.010
0.015
ACQUITY TUV ChA - ACQUITY TUV ChA 240nm
AU
0.000
0.005
0.010
0.015
ACQUITY TUV ChA - ACQUITY TUV ChA 240nm
AU
0.000
0.005
0.010
0.015
ACQUITY TUV ChA - ACQUITY TUV ChA 240nm
AU
0.000
0.005
0.010
0.015
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
BEH PhenylBEH Phenyl
BEH CBEH C1818
BEH ShieldBEH Shield
HSS T3 CHSS T3 C1818
©2007 Waters Corporation 21
Temperature ScoutTemperature Scout
240.0nm - PDA Spectrum - PDA Spectrum (230-350)nm
AU
0.000
0.005
0.010
0.015
0.020
240.0nm - PDA Spectrum - PDA Spectrum (230-350)nm
AU
0.000
0.005
0.010
0.015
0.020
240.0nm - PDA Spectrum - PDA Spectrum (230-350)nm
AU
0.000
0.005
0.010
0.015
0.020
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
30 30 °°CC
40 40 °°CC
60 60 °°CC
ACQUITY UPLC BEH C18
©2007 Waters Corporation 22
BudesonideBudesonide
Final UPLC MethodFinal UPLC Method
AU
0.000
0.007
0.014
0.021
0.028
Minutes
0.00 0.80 1.60 2.40 3.20 4.00 4.80 5.60 6.40 7.20 8.00
0.00
0.40
0.80
0.00 3.00 6.00
1
2
3 4
5
6
7
R-epimer S-epimer
8.00
ConditionsColumn: ACQUITY UPLC BEH C18Dimensions: 100 x 2.1, 1.7um Mobile Phase A: 20mM Ammonium
formate, pH 3.2Mobile Phase B: acetonitrileFlow Rate: 0.6 mL/minIsocratic: 68%A : 32%B Injection Volume: 5.0 µLTemperature: 40CDetection: UV @ 240 nmInstrument: ACQUITY UPLC w/ PDA
©2007 Waters Corporation 23
Budesonide EP System Suitability Budesonide EP System Suitability
SpecificationsSpecifications
� The EP requires the following system suitability specifications based on a 500 ug/ml budesonide test solution and reference solutions:
1) Resolution between the R-epimer and S-epimer is not less than 1.5
2) Run time 1.5x the retention of the S-epimer
3) The symmetry factor for the R-epimer peak is less than 1.5
4) The theoretical plates calculated for the R-epimer peak is at least 4000
5) After 6 injections of the 500 ug/ml reference solution, the RSD of the sum of the peak areas of the two epimers is at most 1.0%
©2007 Waters Corporation 24
Final UPLC MethodFinal UPLC Method
Meets EP Assay CriteriaMeets EP Assay CriteriaP
ea
k1
- 0
.64
7
Pe
ak2
- 1
.86
0P
ea
k3
- 2
.01
8
Pe
ak4
- 2
.59
8
Pe
ak5
- 3
.24
9
Pe
ak6
- 4
.11
1P
ea
k7
- 4
.29
1
R-e
pim
er
- 4
.81
9
S-e
pim
er
- 5
.19
4
Pe
ak1
0 -
5.7
53
AU
0.000
0.007
0.014
0.021
0.028
Minutes
0.00 0.80 1.60 2.40 3.20 4.00 4.80 5.60 6.40 7.20 8.00
1
2
Name RT ResolutionEP
Plate CountSymmetry
FactorSignal_to_Noise
R-epimer
S-epimer
4.819
5.194
3.74
2.40
16150
16456
1.27
1.22
7151.854
4628.869
1
2
3
4
5
6
7
8
9
10
Name RT % Area Area % Height HeightWidth(sec)
Signal_to_Noise
Peak1
Peak2
Peak3
Peak4
Peak5
Peak6
Peak7
R-epimer
S-epimer
Peak10
0.647
1.860
2.018
2.598
3.249
4.111
4.291
4.819
5.194
5.753
0.11
0.08
0.21
0.20
0.19
0.33
0.81
57.91
40.07
0.10
9450
7109
18639
17675
16603
28804
70719
5081008
3515396
8469
0.60
0.18
0.40
0.38
0.19
0.39
0.93
58.80
38.06
0.08
9007
2733
5960
5712
2793
5908
14019
885434
573076
1251
3.450
8.900
11.100
10.250
12.150
12.850
15.500
19.200
20.200
17.750
72.755
22.079
48.137
46.141
22.556
47.718
113.234
7151.854
4628.869
10.101
Peak Results
Original method was 26.0 min long
©2007 Waters Corporation 25
Test Case: Budesonide AssayTest Case: Budesonide Assay
Part IIPart II
Part IIPart II
Qualification of Multiple Batch Lots of Budesonide Qualification of Multiple Batch Lots of Budesonide from Different Suppliersfrom Different Suppliers
� Goal
— To determine most consistent and cost-effective supplier of
budesonide
� Analytical needs
—Quality of each manufacturer’s batch lot of budesonide need to be
evaluated
— Perform the EP related substances test on each lot
o Individual impurities
o Total impurities
o R/S epimer ratio
o % purity
— Confirmation of detected impurities
©2007 Waters Corporation 26
EP Related Substances TestEP Related Substances Test
The EP related substances test as described in the Budesonide EP
monograph was performed on four different batch lots of budesonide
which were purchased from three different suppliers
1. Supplier A
2. Supplier B
3. Supplier C [R&D grade (C1) and EP grade (C2)]
Procedure:
1. Test solutions (500 µg/mL) were prepared for each batch lot
Each test solution was diluted to yield 2 reference solutions each with
concentrations of:
• 2.5 µg/mL representing 0.5% of the 500 µg/mL solution.
• 7.5 µg/mL representing 1.5% of the 500 µg/mL solution.
*There should be 3 standard preps for each lot of substance (12 total)
©2007 Waters Corporation 27
Required TestsRequired Tests
1. Individual Impurities
x < 2.5µg/mL ∑ of epimers areas in the 500 µg/mL test
solution
2. Total Impurities
x < 7.5µg/mL ∑ of epimers areas in the 500 µg/mL test
solution
3. R-epimer/S-epimer Ratio
S-epimer is 40.0% to 51% of the ∑ of epimers areas in the
500 µg/mL test solution
4. Purity
98% to 102% (as the case for most raw material qualification for use as an authentic reference standard)
©2007 Waters Corporation 28
Supplier ComparisonsSupplier Comparisons
Representative PDA ChromatogramsRepresentative PDA Chromatograms
Supplier ASupplier A
Supplier BSupplier B
Supplier C1Supplier C1
Supplier C2Supplier C2
©2007 Waters Corporation 29
Supplier ComparisonsSupplier Comparisons
Representative MS TIC ChromatogramsRepresentative MS TIC Chromatograms
Supplier ASupplier A
Supplier BSupplier B
Supplier C1Supplier C1
Supplier C2Supplier C2
©2007 Waters Corporation 30
Confirmation of ImpuritiesConfirmation of Impurities
Mass SpectraMass Spectra
©2007 Waters Corporation 31
EP Related Substances Test EP Related Substances Test
European Pharmacopoeia Related Substances Test
Specification
Supplier A>99% purity
Supplier B100.2%
Supplier C1R&D grade (no spec)
Supplier C2 EP grade98% -102%
Individual Impurities(x < 2.5 µg/mL ∑ of epimers
areas)Fail Pass Fail Fail
Total Impurities(x < 7.5µg/mL ∑ of epimers
areas)Fail Pass Fail Fail
R-epimer/S-epimer Ratio(S-epimer is 40.0% to 51%
∑ of epimers areas)
50.49%/49.51
51.38%/48.62
58.66%/41.34
59.24%/40.76%
Purity 97.99% 99.52% 98.07% 98.24%
©2007 Waters Corporation
LeachablesLeachables
©2007 Waters Corporation 33
� Plastic containers
� Metal container coatings
� Elastomeric closures or septa
� Label Adhesives
� Printing inks
Where do Leachables come from? Where do Leachables come from?
©2007 Waters Corporation 34
What do Leachables have in What do Leachables have in
common with the objects below? common with the objects below?
©2007 Waters Corporation 35
Types of Polymer AdditivesTypes of Polymer Additives
� Polymer Additives are small molecules that are added to plastics to give them desired properties
—Biocides
—UV absorbers & light stabilizers
—Plasticizers
—Lubricants & mold release agents
—Dyes
—Antioxidants & heat stabilizers
—Flame retardants
—Anti-static & conductive agents
©2007 Waters Corporation 36
10 Common Polymer Additives10 Common Polymer Additives
O
O OH
N
N
N
Cl
HO
N
N
N
Cl
HO
NN
N OH OH
NN
N
S
OHHO
OH
O
S
O
O O
OH
O S O
O O
1
5
7
10
2
4
9
O
OOH
3
NH2
O
O
OH
8
Lowilite 20
Lowinox TBM6
Chimassorb 81
Irganox 1035
Tinuvin 326
6
Erucamide
Lowilite 27 Vitamine E
Irganox PS 800
Lowilite 36
©2007 Waters Corporation 37
Typical Sample Preparation uses Typical Sample Preparation uses
SPESPE
� Leachables are typically
present at low
concentration
� Solid phase extraction
removes interferences and
concentrates the sample
� A typical SPE method is
shown on the right
� At the end of the
extraction, the sample is in
mobile phase
©2007 Waters Corporation 38
UPLCUPLC™™/Photodiode Array/Photodiode Array
PDA Timed wavelength chromatogram (0 min, 320 nm; 0.6 min, 275 nm)
Overlay 7 Replicate Injections
1
2
3
45
7
8
10
AU
0.00
0.15
0.30
Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
Impurity in 8
1 Lowilite 202 Lowinox TBM63 Chimasorb 814 Irganox 10355 Tinuvin 3266 Erucamide (?)7 Lowilite 278 Vitamin E9 Irganox PS 800 (?)10 Lowilite 36
©2007 Waters Corporation 39
Lowilite 20
Lowinox TBM6
Chimassorb 81
Irganox 1035
Tinuvin 326
Lowilite 27
Vitamin E
Lowilite 36
UV Library Spectra UV Library Spectra
Library Matching & Peak Purity:•Confirm peak identity•Assure non-coelution of spectrally dissimilar components
UV spectra extracted from PDA chromatograms
©2007 Waters Corporation 40
UPLCUPLC™™/Evaporative Light Scattering/Evaporative Light Scattering
ELS Chromatogram of 40 ppm polymer additives
2
3
45
6
7
8
9
10
LSU
0.00
60.00
120.00
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
Irganox PS 800
Erucamide
1
Since there are many polymer additives that do not contain a UV chromophore dual detection is ideal for the analysis of polymer additives
©2007 Waters Corporation 41
ELS Calibration Curves ELS Calibration Curves
Calibration Plot
Name: 6; Equation Y = 5.54e+001 X 2̂ + 2.27e+002 X + 1.42e+003; R 2̂: 0.998102Name: 9; Equation Y = 1.42e+001 X 2̂ + 4.17e+002 X - 2.91e+003; R 2̂: 0.997246
Are
a
0.0
25000.0
50000.0
75000.0
100000.0
Amount0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Erucamide (6)
Irganox PS 800 (9)
Quadratic fit calibration curves
©2007 Waters Corporation 42
Questions?Questions?
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