Impurity Profiling

IMPURITY PROFILING
John Shinoda
Senior Support Specialist
Waters Corporation
November 5, 2009
©2007 Waters Corporation

Overview
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 2

Impurity Profiling
Importance 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
Impurity Profiling
Importance 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

NDA and ANDA Filing Considerations
Reporting, Control, and
Specification of Impurities
Drug Substance
Summarize actual and potential impurities
Provide documented evidence that analytical procedures are

validated and suitable for the detection and quantification
of impurities
Provide analytical results for all batches of drug substance used for
clinical, safety, stability testing; also for batches representative of
proposed commercial process
List impurities for specification based on impurities found in batch(es)

manufactured by the proposed commercial process
Guidance for Industry, Q3A Impurities in New Drug Substances, February 2003; \\CDS029\CDERGUID\4164fnl.doc
Draft Guidance for Industry, ANDAs: Impurities in Drug Substances, January 2005; J:!GUIDANC\6422dft.doc
Impurity Thresholds
Drug Substances
Threshold
Maximum
Daily Dose Reporting Identification Qualification
0.10% 0.15%
≤ 2 g/day
or or
0.05%
1.0 mg/day intake 1.0 mg/day intake
(lower) (lower)
> 2 g/day 0.03% 0.05% 0.05%
The quantitation limit for the analytical procedure

should not be more than the reporting threshold
Guidance for Industry, Q3A Impurities in New Drug Substances, February 2003; \\CDS029\CDERGUID\4164fnl.doc
Draft Guidance for Industry, ANDAs: Impurities in Drug Substances, January 2005; J:!GUIDANC\6422dft.doc
Analytical 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

Chromatographic 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

Simvastatin Impurity
UPLC Separation
1.419
3.246
3.700
0.006 Lovastatin Simvastatin
1.184
ACQUITY UPLC BEH C18

2.1 x 100 mm, 1.7 µm
Flow Rate: 0.65 mL/min
Mobile Phase A: 15 mM ammonium bicarbonate, pH 9.0
Mobile Phase B: Acetonitrile
Gradient: 40-100% B over 7 minutes
0.004
Injection Volume: 2.0 µL
Column Temperature: 35°C
UV at 238 nm
AU
4.609
4.713
3.443
0.002
6.786
5.187
5.372
1.309
2.855
1.758
0.000
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Minutes
Consistent Peak Detection & Integration
Importance 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

Effect of Sampling Rate on
Peak Shape
0.034
0.032 1 pt/s
0.030
2 pt/s
0.028
0.026
5 pt/s
0.024 10 pt/s
0.022 20 pt/s
0.020 40 pt/s
0.018
AU
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.000
0.565 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
Minutes
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
Detector Dynamic Range
3.5
Linearity of detector > linear 3 1.5% Deviation at 2.0 AU
range of impurity and parent 2.5
5.0% Deviation at 2.7 AU
Allows quantification and 2 1.3% Deviation at 2.0 AU

validation of low level 5.0% Deviation at 2.8 AU
1.5
impurities (<0.01%) and 1
Absorbance (AU)
large parent compounds 0.5 PDA - Analytical Flow Cell
PDA - High Sensitivity Flow Cell
simultaneously 0
0 10 20 30 40 50 60 70 80
3.5
3
Analytical flow cell 2.2% Deviation at 2.5 AU
2.5 5.0% Deviation at 3.0 AU
— 10mm pathlength
2 2.4% Deviation at 2.5 AU
High sensitivity cell 5.0% Deviation at 3.0 AU
1.5
— 25 mm pathlength 1
UV - Analytical Flow Cell
0.5
UV - High Sensitivity Flow Cell
0
0 10 20 30 40 50 60 70 80
% Absorber in Eluent

Detection of Low Level Components
Spectral Quality Maintained
Prilocaine Spectrum
Prilocaine
o-Toluidine
o-Toluidine Spectrum
0.01% Impurity
Expanded View

Integration of Low Level Impurities
with 2nd 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

Accurate Peak Detection
Shoulders Accurately Integrated
Traditional Integration
2nd derivative Integration

Photodiode Array Review

MS Review

Test Case: Budesonide Assay
Part I
Re-development of European Pharmacopoeial (EP) Assay for
Budesonide Using UPLC®
Goal
—Decrease raw material inventory costs by shortening the time to
accept new lots of budesonide drug substance
Analytical Needs
—Method development
o Explore column, pH, organic solvent, temperature
—Meet EP assay criteria
—MS compatible solvent system
o Single quadrupole mass detection for impurity confirmation

Current Situation
EP HPLC Method
0.040
R-epimer S-epimer
0.035
0.030
0.025
AU
0.020
0.015
0.010
0.005
0.000
2.00 4.00 6.00 8.00 10.00 12.00
Minutes
14.00 16.00 18.00 20.00 22.00 24.00
26.00
Isocratic separation using Acetonitrile and a

Phosphate buffer
UPLC Method Development
Column Scout for Budesonide
ACQUITY TUV ChA - ACQUITY TUV ChA 240nm

0.015
0.010 BEH C18

AU
0.005
0.000

0.015
0.010 HSS T3 C18

AU
0.005
0.000

0.015
0.010
BEH Phenyl
AU
0.005
0.000

0.015
0.010
BEH Shield
AU
0.005
0.000
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes

Temperature Scout
ACQUITY UPLC BEH C18

240.0nm - PD A Spectrum - PD A Spectrum (230-350)nm
0.020
0.015 30 °C
0.010
AU
0.005
0.000

0.020
0.015 40 °C
0.010
AU
0.005
0.000

0.020
0.015
60 °C
0.010
AU
0.005
0.000
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes ©2007 Waters Corporation 21
Budesonide
Final UPLC Method
0.028 0.80
R-epimer S-epimer
Conditions
0.40 Column: ACQUITY UPLC BEH C18
Dimensions: 100 x 2.1, 1.7um
Mobile Phase A: 20mM Ammonium
0.021 0.00 formate, pH 3.2
Mobile Phase B: acetonitrile
0.00 3.00 6.00 Flow Rate: 0.6 mL/min
Isocratic: 68%A : 32%B
Injection Volume: 5.0 µL
Temperature: 40C
7 Detection: UV @ 240 nm
AU
0.014 Instrument: ACQUITY UPLC w/ PDA
0.007 3 4 6
2 5
0.000
0.00 0.80 1.60 2.40 3.20 4.00 4.80 5.60 6.40 7.20 8.00
Minutes 8.00
Budesonide EP System Suitability
Specifications
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%

Final UPLC Method
Meets EP Assay Criteria
EP Symmetry
Name RT Resolution Signal_to_Noise
Plate Count Factor
1 R-epimer 4.819 3.74 16150 1.27 7151.854
2 S-epimer 5.194 2.40 16456 1.22 4628.869
R-epimer - 4.819
S-epimer - 5.194
0.028 Peak Results
Width
Name RT % Area Area % Height Height Signal_to_Noise
(sec)
1 Peak1 0.647 0.11 9450 0.60 9007 3.450 72.755
2 Peak2 1.860 0.08 7109 0.18 2733 8.900 22.079
3 Peak3 2.018 0.21 18639 0.40 5960 11.100 48.137
0.021 4 Peak4 2.598 0.20 17675 0.38 5712 10.250 46.141
Peak7 - 4.291
5 Peak5 3.249 0.19 16603 0.19 2793 12.150 22.556
6 Peak6 4.111 0.33 28804 0.39 5908 12.850 47.718
7 Peak7 4.291 0.81 70719 0.93 14019 15.500 113.234
8 R-epimer 4.819 57.91 5081008 58.80 885434 19.200 7151.854
Peak1 - 0.647
9 S-epimer 5.194 40.07 3515396 38.06 573076 20.200 4628.869
0.014 10 Peak10 5.753 0.10 8469 0.08 1251 17.750 10.101

AU
Peak3 - 2.018
Peak6 - 4.111
Peak4 - 2.598
Peak2 - 1.860
Peak5 - 3.249
Peak10 - 5.753
0.007
0.000
0.00 0.80 1.60 2.40 3.20 4.00 4.80 5.60 6.40 7.20 8.00
Minutes
Original method was 26.0

min long
Test Case: Budesonide Assay
Part II
Part II
Qualification of Multiple Batch Lots of Budesonide
from 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 25
EP 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)
Required 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)

Supplier Comparisons
Representative PDA Chromatograms
Supplier A
Supplier B
Supplier C1
Supplier C2

Supplier Comparisons
Representative MS TIC Chromatograms
Supplier A
Supplier B
Supplier C1
Supplier C2

Confirmation of Impurities
Mass Spectra

EP Related Substances Test
Supplier A Supplier C2
European Pharmacopoeia Supplier B Supplier C1
>99% EP grade
Related Substances Test 100.2% R&D grade
purity 98% -
Specification (no spec)
102%
Individual Impurities
(x < 2.5 µg/mL ∑ of epimers Fail Pass Fail Fail
areas)
Total Impurities
(x < 7.5µg/mL ∑ of epimers Fail Pass Fail Fail
areas)
R-epimer/S-epimer Ratio
50.49%/ 51.38%/ 58.66%/ 59.24%/
(S-epimer is 40.0% to 51%
49.51 48.62 41.34 40.76%
∑ of epimers areas)
Purity 97.99% 99.52% 98.07% 98.24%

Leachables
©2007 Waters Corporation

Where do Leachables come from?
Plastic containers
Metal container coatings
Elastomeric closures or septa
Label Adhesives
Printing inks

What do Leachables have in
common with the objects below?

Types 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

10 Common Polymer Additives
O OH
OH O
S
1 O HO OH
2 O 3
Lowilite 20
Chimassorb 81
Lowinox TBM6
OH OH
NH 2
HO
O
4 N
N 6
O
Irganox 1035 O Cl N
S
5 Erucamide
O O
Tinuvin 326
HO
OH
N
N
O
Cl N
7 8
Lowilite 27 Vitamine E
O S O
O O
Irganox PS 800 9
N N
N N
N OH OH N
10
Lowilite 36
Typical Sample Preparation uses
SPE
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

UPLC™/Photodiode Array
Overlay 7 Replicate Injections
3
0.30 1 Lowilite 20
1
2 Lowinox TBM6
3 Chimasorb 81
4 Irganox 1035
5 Tinuvin 326
6 Erucamide (?)
7 Lowilite 27
8 Vitamin E
2 9 Irganox PS 800 (?)
AU
0.15 Impurity in 8 10 Lowilite 36
10
4
8
0.00
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
Minutes
PDA Timed wavelength chromatogram (0 min, 320 nm; 0.6 min, 275 nm)

UV Library Spectra
Lowilite 20
Lowinox TBM6 Library Matching & Peak

Purity:
Chimassorb •Confirm peak identity
81
•Assure non-coelution of
Irganox
1035 spectrally dissimilar
Tinuvin 326 components
Lowilite 27
Vitamin E
Lowilite 36
UV spectra extracted from PDA chromatograms

UPLC™/Evaporative Light Scattering
3
Erucamide
7
4
5
120.00
6
Irganox PS 800
8
LSU
60.00
10
9
1
0.00
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
Minutes
ELS Chromatogram of 40 ppm polymer additives
Since there are many polymer additives that do not contain a

UV chromophore dual detection is ideal for the analysis of
polymer additives

ELS Calibration Curves
Calibration Plot
100000.0
Erucamide (6)
75000.0
Area
50000.0
Irganox PS 800 (9)

25000.0
0.0
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Amount
Name: 6; Equation Y = 5.54e+001 X^2 + 2.27e+002 X + 1.42e+003; R^2: 0.998102
Name: 9; Equation Y = 1.42e+001 X^2 + 4.17e+002 X - 2.91e+003; R^2: 0.997246
Quadratic fit calibration curves ©2007 Waters Corporation 41

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Impurity Profiling

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IMPURITY PROFILING

©2007 Waters Corporation

©2007 Waters Corporation 2

Accept or reject raw material for API or drug product

Support process development and scale up

Sign off of finished API or drug product for release

Adhere to CGMPs and CMC regulatory requirements

©2007 Waters Corporation 4

Provide documented evidence that analytical procedures are

List impurities for specification based on impurities found in batch(es)

> 2 g/day 0.03% 0.05% 0.05%

The quantitation limit for the analytical procedure

Accurate and precise quantification of low-level impurities

Developing impurity profile methods that support potential

Accessing current and historical results when needed for

Expedient response and investigation into OOS events

©2007 Waters Corporation 7

High resolution separations

©2007 Waters Corporation 8

ACQUITY UPLC BEH C18

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Must ensure enough points are collected across a peak to

Peak detection algorithms require a minimum number of

A peak which does not have enough data points will be

©2007 Waters Corporation 10

Allows quantification and 2 1.3% Deviation at 2.0 AU

impurities (<0.01%) and 1

©2007 Waters Corporation 12

©2007 Waters Corporation 13

Automatic first pass integration using default parameters

©2007 Waters Corporation 14

2nd derivative Integration

©2007 Waters Corporation 15

©2007 Waters Corporation 16

©2007 Waters Corporation 17

©2007 Waters Corporation 18

Isocratic separation using Acetonitrile and a

ACQUITY TUV ChA - ACQUITY TUV ChA 240nm

0.010 BEH C18

ACQUITY TUV ChA - ACQUITY TUV ChA 240nm

0.010 HSS T3 C18

ACQUITY TUV ChA - ACQUITY TUV ChA 240nm

ACQUITY TUV ChA - ACQUITY TUV ChA 240nm

©2007 Waters Corporation 20

ACQUITY UPLC BEH C18

240.0nm - PD A Spectrum - PD A Spectrum (230-350)nm

240.0nm - PD A Spectrum - PD A Spectrum (230-350)nm

0.014 Instrument: ACQUITY UPLC w/ PDA

The EP requires the following system suitability

1) Resolution between the R-epimer and S-epimer is not less

©2007 Waters Corporation 23

0.021 4 Peak4 2.598 0.20 17675 0.38 5712 10.250 46.141

9 S-epimer 5.194 40.07 3515396 38.06 573076 20.200 4628.869

0.014 10 Peak10 5.753 0.10 8469 0.08 1251 17.750 10.101

Original method was 26.0

The EP related substances test as described in the Budesonide EP

©2007 Waters Corporation 27

©2007 Waters Corporation 28

©2007 Waters Corporation 29

©2007 Waters Corporation 30

Purity 97.99% 99.52% 98.07% 98.24%

©2007 Waters Corporation 31