impurity profiling

©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


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


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


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


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


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


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


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


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


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


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




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





Absorbance (AU)

0 10 20 30 70


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


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


�� Traditional IntegrationTraditional Integration

� 2nd derivative IntegrationIntegration

Accurate Peak DetectionAccurate Peak Detection

Shoulders Accurately Integrated Shoulders Accurately Integrated


Photodiode Array ReviewPhotodiode Array Review


MS ReviewMS Review


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


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


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


AU

0.000

0.005

0.010

0.015


AU

0.000

0.005

0.010

0.015


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


Temperature ScoutTemperature Scout

240.0nm - PDA Spectrum - PDA Spectrum (230-350)nm

AU

0.000

0.005

0.010

0.015

0.020


AU

0.000

0.005

0.010

0.015

0.020


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


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


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%


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


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


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)


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)


Supplier ComparisonsSupplier Comparisons

Representative PDA ChromatogramsRepresentative PDA Chromatograms

Supplier ASupplier A

Supplier BSupplier B

Supplier C1Supplier C1



Supplier ComparisonsSupplier Comparisons

Representative MS TIC ChromatogramsRepresentative MS TIC Chromatograms

Supplier ASupplier A

Supplier BSupplier B




Confirmation of ImpuritiesConfirmation of Impurities

Mass SpectraMass Spectra


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%


� Plastic containers

� Metal container coatings

� Elastomeric closures or septa

� Label Adhesives

� Printing inks

Where do Leachables come from? Where do Leachables come from?


What do Leachables have in What do Leachables have in

common with the objects below? common with the objects below?


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


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


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


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


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


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


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


Questions?Questions?

impurity profiling

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