Phase Appropriate Method Validation Aryo Boston-Nitto 2

Phase Appropriate Method ValidationAryo Nikopour

The Southern California Pharmaceutical Discussion Group (SCPDG) of AAPSIrvine, CA

January 12, 2017

VALIDATION OF ANALYTICAL METHOD

DATA QUALITY TRIANGLE

METHOD LIFE CYCLEValidation

Development Optimization

METHOD VALIDATION

Skilled

Qualified

Reference Standards

Quality

Temperature

VibrationsIrradiations

Humidity

Time

Analysts’ Support

Documented

Supplies

Characterized

Calibrated

RobustMan

Machine

Methods

Management

Milieu

Material

Suitable

Quality of Analytical

Method

PUBLISHED VALIDATION GUIDELINES 1978 Current Good Manufacturing Practices (cGMP) 1987 FDA Validation Guideline 1989 Supplement 9 to USP XXI

1994 CDER Reviewer Guidance: Validation of Chromatographic Method 1995 ICH Validation Definitions: Q2A, Text on Validation of Analytical procedures 1997 ICH Validation Methodology: Q2B, Validation of Analytical Procedures: Methodology 1999 Supplement 10 to USP 23 <1225>: Validation of Compendial Methods 1999 CDER “Bioanalytical Method Validation for Human Studies” 2000 CDER Draft “Analytical Procedures and Method Validation” 2014 CDER/CBER Guidance for Industry: “Analytical Procedure and Method Validation for Drug

and Biologic” PDA Technical Report No. 57 : Analytical Method Validation and Transfer for Biotechnology

Products”

GUIDELINES FOR METHOD VALIDATION

www.ICH.orgwww.ICH.org

(Dates indicate ICH finalization)

ICH Q2(R1): Validation of Analytical Procedures: Methodology- Nov. 2005

ICH Q3A(R): Impurities in New Drug Substances - Feb. 2002ICH Q3B(R): Impurities in New Drug Products – Feb. 2003ICH Q3C: Impurities: Residual Solvents - July 1997ICH Q5A,D: Biotech/Biological Products - 1997ICH Q5B,C: Biotech/Biological Products - 1995

GUIDELINES FOR METHOD VALIDATION

www.ICH.orgwww.ICH.org

(Dates indicate ICH finalization)

ICH Q2(R1): Validation of Analytical Procedures: Methodology- Nov. 2005

ICH Q3A(R): Impurities in New Drug Substances - Feb. 2002ICH Q3B(R): Impurities in New Drug Products – Feb. 2003ICH Q3C: Impurities: Residual Solvents - July 1997ICH Q5A,D: Biotech/Biological Products - 1997ICH Q5B,C: Biotech/Biological Products - 1995

VALIDATION IN THE 21 CENTURY

METHOD VALIDATION

CFR• There are many reason to validate analytical methods:

– Regulatory Requirements

– Good Science

– Quality Control requirements.

• The Code of Federal Regulations (CFR) 311.165c explicitly states that the, “Accuracy, Sensitivity, Specificity, and Reproducibility of test methods employed by the firm shall be established and documented.”

ICH GUIDELINE Q2(R1)• The objective of validation of an analytical procedure is to

demonstrate that it is suitable for its intended purpose,

In practice, it is usually possible to design the experimental work such that the appropriate validation characteristics can be considered simultaneously, to provide a sound, overall knowledge of the capabilities of the analytical procedure, for instance; Specificity, Linearity, Range, Accuracy, and Precision.

Support the identity, strength, quality, purity, and potency of the drug substances and drug products.

WHAT IS METHOD VALIDATION? Validation is procedure dependent. Validation, “Proves” the procedure works as described. Validation is product specific. Procedures are instrument dependent.

VERIFICATION USP <1226>• Current USP <1226> Verification of Compendial Procedure

– The Analytical procedures in the current USP are legally recognized under section

501(b) of the Federal Food, Drug and Cosmetic Act as the regulatory analytical

procedures for the compendial items. The suitability of these procedures must be

verified under the actual conditions of use.

VERIFICATION

• When using USP analytical procedures, the guidance recommends

that information be provided for the following characteristics:

– Specificity of the procedure

– Stability of the sample solution

– Intermediate precision

METHOD TRANSFER, USP <1224>• Method Transfer is a documented process that qualifies a

laboratory (Receiving Lab) to use an analytical test procedure that is originated from the transferring laboratory.

• Types of Method Transfer:– Comparative Testing– Co -validation – Revalidation/Partial Validation– Transfer Waiver

CLASSIFICATION OF VALIDATED ANALYTICAL METHODS

Compendial (USP 39/ NF 34):• Legally recognized under section 501 (b) of the Federal Food, Drug, and Cosmetic Act.• Recommends information be provided for; Specificity, Sample Solution Stability, and

Intermediate Precision.

Noncompendial:• Submitted with the NDA/ BLA or ANDA application.• If the compendial procedure is not stability-indicating, perform an alternative analytical

procedure with complete validation.

USP <1225>ASSAY CATEGORIESCategory Category NumberNumber Category NameCategory Name Description of AssayDescription of Assay

I QuantitativeQuantitation of major components/active ingredients present at high concentrations.

II Impurities-Quantitative Determination of impurities or

degradation products.II Impurities-Limit

III Performance Characteristics

Parameters to be tested depend on the nature of the test; includes dissolution testing.

IV Identity

METHOD VALIDATION REQUIREMENTS

USP Assay Category

I II

III IV Parameter: Quantitative Limit Tests

Accuracy Y Y Y N Precision Y Y N Y N Intermediate Precision Y Y N Y N Specificity N Y Y N Y Detection Limit N Y Y N Quantitation Limit N Y N N Linearity Y Y N N Range Y Y N Robustness Y Y N N N Selectivity Y Y N Y Y System Suitability Y Y N Y N Solution Stability Y Y N Y N May be required, depending on the nature of the specific test.

PHASE APPROPRIATE VALIDATION

Pre-clinical

PM

METHOD VALIDATION READINESS

Define the application, purpose and scope of the method.

Define Analytes, Dosage Strength and Sample Matrix.

Review Method Development Summary Report.

Evaluate method validation parameters during development.

METHOD VALIDATION CHARACTERISTICSValidation Characteristics Experimental Details Acceptance CriteriaSpecificity Stress Studies 5-10% Degradation

Selectivity Determine Chromatographic non-interference

No inference , minimum resolution between peaks of interest and impurities should be >1.5

System Suitability System precision assessed by 6 replicate measurement/injections

%RSD ≤2%

Linearity At least 5 Concentration over the rangeAssay: 50% to 125% of Specification limitQL-150% of specification limit

Calibration Model is validR ≥0.998Report Intercept, Slope and %Bias

Detection Limit (DL) DL= 3.3 (DL= 3.3 (/S)/S) S/N≥ 3S/N≥ 3

Quantitation Limit (QL) DL= 10 (DL= 10 (/S)/S) %RSD≤ 15%%RSD≤ 15%

METHOD VALIDATION CHARACTERISTICSValidation Characteristics Experimental Details Acceptance CriteriaPrecision :RepeatabilityIntermediate Precision (Ruggedness)Reproducibility

6 replicates6 replicates

%RSD≤ 2%Overall %RSD (two Analyst)

Accuracy At least 9 determination over 3 concentration levele.g. 70 to 120% for

For Assay Mean Recovery 97 to 103% for Impurities : 85% to 115%

Range The range is defined by the results obtained for linearity, accuracy and precision

Linearity, accuracy and precision demonstrated over the range

Solution Stability Determine solution stability of Reference Standard Solution and Sample over 72 hours

98 to 102 % of control

Robustness Deliberately change critical parameters of the method

Must meet system suitability and selectivity requirements

VALIDATION: PHASE IDrug Product Assay I.D. Quantitative

Impurities Limit Test

Selectivity X X X X

Repeatability X X

Accuracy/Precision Recovery at 100%

At 100% of ReportingThreshold

Linearity X QL to 200% of Limit

Range Defined by ALP Defined by ALP

DL/QL DL QL QL or at Limit

System Suitability X X X X

Solution Stability X X X

VALIDATION: PHASE IIAssay I.D Quantitative

ImpuritiesLimit Test

Selectivity X X X X

Specificity X

Repeatability X X X

Accuracy Recovery at 3 levels

At 100% of Reporting Threshold

Linearity X X X

DL/QL DL X QL

Range Define by ALP Defined by ALP

System Suitability X X X X

Solution Stability X X X

VALIDATION: PHASE IIIAssay I.D Quantitative

ImpuritiesLimit Test

Selectivity X X X X

Specificity X

Repeatability X X X

Intermediate Precision X 2nd Analyst X X

Accuracy X X

Linearity X X

DL/QL DL X QL

Range Defined by ALP Defined by ALP

Solution Stability X X X

System Suitability X X X X

Robustness X X X

METHOD VALIDATION

SYSTEM SUITABILITY Based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such.

What parameters do you measure for What parameters do you measure for system suitabilitysystem suitability

SYSTEM SUITABILITYWhat parameters do you measure for system suitability?What parameters do you measure for system suitability?

CapacityCapacityEfficiencyEfficiencySelectivitySelectivity

Date NB/Page

Standard B1 (n=6) Injections SST Solution B1 and B2

Area %RSDAverage Tailing

Factor %RSD Retention Time Average Theoretical PlatesResolution LVF and

DesMethyl-LVF)Response Factor %

Difference

1% 0.8 Tf 1.4 1% >15000 NLT 2.5 2%

8/18/2006 1494/18 0.1 1.03 0.1 29834 3.27 0.2

8/18/2006 1494/31 0.1 1.03 0 32177 3.28 0.1

8/20/2006 1494/52 0.1 1.03 0.1 27792 3.3 0.7

8/22/2006 1494/72 0.1 1.03 0 26567 3.31 2.7

8/23/2006 1504/1 0 1.03 0.2 27228 3.29 0.8

8/24/2006 1504/8 0.2 1.02 0.1 26535 3.32 1.2

8/25/2006 1504/17 0.1 1.02 0.1 26903 3.31 3.6

9/5/2006 1494/129 0.1 1.03 0.1 27894 3.31 0.5

9/13/2006 1494/171 0.2 1.02 0.1 26916 3.31 0

9/15/2006 1494/181 0.1 1.03 0.1 29553 3.29 0.2

9/15/2006 1494/187 0.1 1.12 0.1 32361 3.47 0.1

9/15/2006 1494/193 0.1 1.11 0.1 27303 3.12 0.2

9/15/2006 1494/199 0.2 1.02 0.1 29424 3.3 0

9/18/2006 1504/37 0.2 1.02 0.1 28020 3.27 0.2

9/18/2006 1504/42 0.6 1.03 0 27627 3.29 1.9

9/18/2006 1461/40 0.1 1.03 0.1 31109 3.66 0.2

10/5/2006 1504/65 0.1 1.04 0.1 36973 3.85 0.2

Average 0.1 1.04 0.1 29729 3.39 0.8

Min 0 1.02 0 26535 3.12 0

Max 0.6 1.12 0.2 37049 3.86 3.6

STDEV 0.128051 2770 0.168485904

3 Sigma 0.384153 8311 0.505457713

min -0.28 21418 2.88

max 0.48 38040 3.90

GAUSSIAN DISTRIBUTION

C.I. =

CONTROL CHART

SELECTIVITY AND SPECIFICITY

SelectivitySelectivity vs. vs. SpecificitySpecificity

SELECTIVITY AND SPECIFICITYSelectivity:

A method’s ability to separate the analyte from other components that may be present in the sample.

Definition of Selectivity from IUPAC: Selectivity of a method, refers to the extent to which it can determine particular analytes under given conditions in mixtures or matrices, simple or complex, without interferences from other components.

SELECTIVITY AND SPECIFICITY

SELECTIVITY AND SPECIFICITY

Specificity:A method’s ability to identify and measure absolutely and unequivocally the analyte in the presence of the other components in the sample, such as; impurities, degradation products, and excipients.

There must be inarguable supporting data for a method to be considered specific. Specificity implies identification, purity tests, and assay (content or potency).

SELECTIVITY AND SPECIFICITY

Regulatory Requirements: Stability indicating methods are not specified, but implied in 21 CFR Part 211.165 and 211.166 (3):

•211.165 (e) States that the accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented.

•211.166 (a) (3) Requires that test methods be reliable, meaningful, and specific.

STABILITY INDICATING METHOD (SIM) VS.

STABILITY SPECIFIC METHODS (SSM)

• Stability indicating assays accurately quantitate active ingredients without interference from:

– Degradation products

– Process impurities

– Excipients

• A stability-specific method is one that meets all of the criteria above but, in addition, the degradation components are detected and quantitated.

StressStudies

“Absence of evidence is not evidence of absence” - Carl Sagan,

The Dragons of Eden: Speculations on the Evolution of Human Intelligence

WHY DO WE PERFORM STRESS STUDIES?

Safety and Efficacy

Forced degradation or stress testing is undertaken to demonstrate specificity when developing stability-indicating methods, particularly when little information is available about potential degradation products.

WHY DO WE PERFORM STRESS STUDIES?• Development and validation of stability-indicating methodology.

• Determination of degradation pathways of drug substances and drug products.

• Discernment of degradation products in formulations that are related to drug substances versus those that are related to non-drug substances (excipients).

• Structure elucidation of degradation products.

• Determination of intrinsic stability of drug substance molecule.

WHY DO WE PERFORM STRESS STUDIES?

Defining characteristics of degradation studies:

• Carry out in solution and/or in the solid state.

• Involve conditions more severe than accelerated testing.

• Typically carry out on placebo, drug product, and API.

• Not part of formal stability program.

FORCED DEGRADATION (STRESS STUDIES)

Steps to Approaching Stress Studies in the Lab:

• Investigate the chemical structure and functional group.

• Study chemical and physical properties.

• Study synthetic route.

• Predict stress pathways based on storage conditions and manufacturing process.

• Identify suitable separation method and detection.

• Design study based on the formulation (feed, tablet, ointment, etc.).

FORCED DEGRADATION (STRESS STUDIES)

Chemical Physical Environmental

Acid Agitation Heat

BaseDenaturation, aggregation, adsorption and precipitation

Light (ICH Option I or II)

Oxidation RH

Deamidation Freeze/Thaw

Disulfide Bond Exchange

STRESS STUDY PATHWAYS

Pharmaceutical Biologics

Hydrolytic Hydrolytic

Oxidative Oxidative

Photolytic Aggregation

Thermolytic Deamidation

Disulfide Bond Exchange

FORCED DEGRADATION (STRESS STUDIES)

Stress Pathway Condition Time

Acid 0.01N 1 to 24 hours

Base 0.01N 1 o 24 hours

Oxidation 0.3% H2O2 1 to 24 hours

Light600 to 800 foot candles (sources include metal halides, Hg, Xe lamp, or UVB fluorescence)

Option II: 74Hours

Option I: 2-4 Hours

Heat/RH 40 °C/ 75% RH and 60 °C

24 to 72 hours

Freeze/Thaw -20 °C to 25 °C 3 Cycle of 24 hours

WHAT IS ADEQUATE STRESS?

Overstressing a molecule can lead to degradation profiles that are not representative of primary degradation and are irrelevant to the stability of the product.

Stress-testing conditions should be realistic, not excessive (5 – 10%).

FORCED DEGRADATION (STRESS STUDIES)

Optimize detector setting Stress blank, placebo,

standard and sample Inject controls Extend run time Orthogonal Method

Overstress!!Overstress!!

EXAMPLE: PHOTOLYTIC STRESS

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0-2.20

0.00

1.25

2.50

3.80

1 - Sequence Name: Forced Degradation Light Sample Name: Fresh 30 mg/mL Control, Sample #122 - Sequence Name: Forced Degradation Light Sample Name: Light Stressed 30 mg/mL Set 1, Sample #173 - Sequence Name: Forced Degradation Light Sample Name: Light Stressed 30 mg/mL Set 2, Sample #264 - Sequence Name: Forced Degradation Light Sample Name: Light Stressed 30 mg/mL Set 3, Sample #35mAU

min

4

3

2

1

Imp

1 - 3

.397

Imp

3 - 6

.097

Imp

5 - 7

.873 Im

p 6

- 8.3

20De

sMet

hyl-L

VF -

8.98

0

Levo

floxa

cin -

9.77

7

Imp

7 - 1

0.73

3

Imp

9 - 1

2.81

7Im

p 10

- 13

.320

Imp

11 -

14.5

17

Imp

14 -

20.4

87

WVL:280 nm

EXAMPLE: ACID STRESSEXAMPLE: ACID STRESS

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0-3.40

-2.00

0.00

2.60

1 - Sequence Name: Forced Degradation Acid_Base Sample Name: Fresh 30 mg/mL Control, Sample #172 - Sequence Name: Forced Degradation Acid_Base Sample Name: 2 hrs Acid 30 mg/mL, Sample #243 - Sequence Name: Forced Degradation Acid_Base Sample Name: 4 hrs Acid 30 mg/mL, Sample #384 - Sequence Name: Forced Degradation Acid_Base Sample Name: 8 hrs Acid 30 mg/mL, Sample #505 - Sequence Name: Forced Degradation Acid_Base Sample Name: 24 hrs Acid 30 mg/mL, Sample #64mAU

min

5

4

3

2

1

Imp 1 - 2

.670

Imp 3 - 3

.387

Imp 7 - 8

.430

DesM

ethy

l-LVF

- 9.09

7

Levo

floxa

cin - 9

.890

WVL:280 nm

EXAMPLE: HEAT STRESSEXAMPLE: HEAT STRESS

0.3 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0-1.50

0.00

1.50

1 - Sequence Name: Forced Degradation Heat Sample Name: Fresh 50 mg/mL Control, Sample #172 - Sequence Name: Forced Degradation Heat Sample Name: 4 hr Heat, 50 mg/mL Assay, Sample #243 - Sequence Name: Forced Degradation Heat Sample Name: 8 hr Heat, 50 mg/mL Assay, Sample #314 - Sequence Name: Forced Degradation Heat Sample Name: 24 hr Heat, 50 mg/mL Assay, Sample #38mAU

min

4

3

2

1

Imp

1 - 2

.660

Imp

3 - 4

.327

Imp

4 - 6

.070

Imp

6 - 8

.287

DesM

ethy

l-LVF

- 8.

960

Levo

floxa

cin -

9.75

3

WVL:280 nm

MASS BALANCE1

From ICH Q1 A “Stability Testing of New Drug Substance and Product”• The process of adding together the assay value and levels of degradation

products to see how closely these add up to 100 percent of the initial value, with due consideration of the margin of analytical error1.

MASS BALANCE• Uncertainty in potency

• Loss of volatiles

• Diffusive losses

• Loss of UV chromophore

• Lack of universal detection

• Design of calculation

SOLUTION STABILITYPurpose:: To determine stability of sample and standard Test solutions to support duration of run sequence and potential investigation studies.

Procedure:To evaluate several time intervals; (0, 24, 48, 72 hours), for both stock and evaluated solution.

ESTABLISHING RANGE• Range:

– Definition– Criteria

• Limits of Detection and Quantitation• Linearity• Accuracy• Precision• Repeatability

DL & QL VERSUS SENSITIVITY

Sensitivity is measured by the slope of the calibration curve:Sensitivity is measured by the slope of the calibration curve: More sensitive method, steeper slope: Results in a larger change in the measured

response versus the controlled variable

DL & QL are measured by one of the four methods:DL & QL are measured by one of the four methods: lowest concentration for which RSD is <5.0% plot of standard deviation versus concentration 95% CI of a best fit signal to noise ratio

DETERMINING DL AND QL:

Per ICH-Q2A:Per ICH-Q2A:

DL & QL can be calculated based on the standard deviation of the response () and the slope of the calibration curve (S) at levels approximating the limits according to the following formulas:

DL= 3.3 (DL= 3.3 (/S)/S)

QL= 10 (QL= 10 (/S)/S)

The can be determined based on the of the blank, the residual of the regression line, or the of y-intercepts of regression lines.

DETECTION LIMIT (DL)

4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0-0.100

-0.000

0.100

0.200

0.300

1 - LOD_LOQ #1 Diluent Blank UV_VIS_12 - LOD_LOQ #11 LVF LOD (0.004 ug/mL) UV_VIS_13 - LOD_LOQ #12 LVF LOD (0.004 ug/mL) UV_VIS_14 - LOD_LOQ #13 LVF LOD (0.004 ug/mL) UV_VIS_1mAU

min

4

3

21

WVL:280 nm

Quantitation Limit (QL)

4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0-0.10

0.50

1.00

1.50

1 - LOD_LOQ #1 Diluent Blank UV_VIS_12 - LOD_LOQ #14 LVF LOQ (0.009 ug/mL) UV_VIS_13 - LOD_LOQ #15 LVF LOQ (0.009 ug/mL) UV_VIS_14 - LOD_LOQ #16 LVF LOQ (0.009 ug/mL) UV_VIS_15 - LOD_LOQ #17 LVF LOQ (0.009 ug/mL) UV_VIS_16 - LOD_LOQ #18 LVF LOQ (0.009 ug/mL) UV_VIS_17 - LOD_LOQ #19 LVF LOQ (0.009 ug/mL) UV_VIS_1mAU

min

7

6

5

4

3

2

1

WVL:280 nm

LINEARITY

The ability of an analytical procedure (within a given range), to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

LINEARITY CALCULATIONS

y = my = mx + bx + bWhere: y = response, x = concentration, m = slope, and

b = y intercept

Percent Bias = b

(x m) + b 100%

ACCURACY

• The measure of how close the experimental value is to the true value.

− Established across a specified range.

− Also called trueness.

ACCURACYDetermination of Accuracy:

• 9 determinations over 3 concentrations in triplicate preparation.

• The mean is an estimate of accuracy.

• RSD is an estimate of sample analysis precision.

ACCURACYShould be reported as:

• The percent recovery by the assay of known added amount of analyte in the sample.

• The difference between the mean and the accepted true value together with the confidence intervals.

DETERMINING ACCURACY FOR DRUG SUBSTANCES

• Use reference material.

• Compare procedure results with those of a second, well-characterized procedure.

• Infer from precision, linearity and specificity; 80,100 and 120% levels of label claim.

DETERMINING ACCURACY FOR DRUG PRODUCTS

• Spike suitable Reference Materials into a Placebo.

• Add known quantities of analyte to the drug product.

• Compare procedure results with those of a second, well-characterized procedure.

• Infer from precision, linearity and specificity; 80, 100 and 120% levels of label claim.

DETERMINING ACCURACY FOR DRUG PRODUCT RELATED SUBSTANCES Add known quantities of impurities to the sample.

Compare procedure results with those of a second, well-characterized procedure.

If impurities are not available, how do you perform accuracy?

PRECISION

The closeness of agreement between a series of measurements, obtained from a multiple sampling of the same homogeneous sample, under the prescribed conditions.

PRECISION

Includes:Includes:

RepeatabilityIntermediate Precision

Reproducibility

Report:Report:

Standard Deviation, Relative Standard Deviation, Confidence Interval

REPRODUCIBILITY

• Expresses the precision between laboratories.

• Recommended parameters to be evaluated at the second laboratory include:

– Selectivity– DL/QL – Repeatability– System Suitability

RUGGEDNESS• Degree of reproducibility of test results under a variety of conditions:

−Different Laboratories

−Different Analysts

−Different Instruments

−Different Reagents

−Different Days

Ruggedness ≠ Robustness

MINIMUM SPECIFIED RANGES: DRUG SUBSTANCES

Impurity Reporting ThresholdsMaximum Daily Dose

Qualification and ID Threshold

Reporting Threshold

< 2g/day

0.1% or 1mg/day (choose the lower)

0.05%

> 2g/day 0.05% 0.03%

MINIMUM SPECIFIED RANGES: DRUG PRODUCTS

Degradation Product Reporting Thresholds In New Drug Products:

Maximum Daily Dose

Reporting Threshold

1 g/day 0.1% > 1 g/day 0.05%

ROBUSTNESS

• A measure of a method’s capacity to remain unaffected by small, deliberate variations in method parameters.

• Provides an indication of a method’s reliability during normal usage.

• Assessed by making small, deliberate changes to the method and evaluating the results.

ROBUSTNESSExamples of typical RP-HPLC variations:Examples of typical RP-HPLC variations:

pH of mobile phasemobile phase composition

Ionic StrengthDifferent columns

Column temperatureflow rate

ROBUSTNESS

ParameterNominal Procedure Condition

Conditions Tested for Robustness Determination

MPA*-Buffer constituent pH 4.0 3.9, 4.1

MPA*-Buffer salt concentration

10 mM Ammonium

Formate9 mM, 11 mM

Column Temperature 30°C 25°C, 35°CDetector Wavelength 290 nm 288 nm, 292 nm

Flow Rate 1.0 mL/min 0.9 mL/min, 1.1 mL/min

Injection Volume 20 µL 15 µL, 25 µL*MPA = Mobile Phase A

METHOD REVALIDATION

Revalidate due to changes in:

Synthesis of the drug substance. Composition of the drug product. Analytical procedure.

ANALYTICAL METHOD LIFE CYCLE

Change to Method: Evaluate the effect

Development of the Method

Validation of the Method

Method in Routine use

Redevelopment of the method required due to change

Revalidation required due to change

Change is not covered by existing validation

Change is covered by existing validation

REFERENCES1. Bob Snider, CMC Group2. ICH Q2 (R1)3. Current USP <1224>4. Current USP <1225>5. Current USP <1226>6. FDA Guidance for Industry7. Miller, JM., Crowther, JB. 2000. Analytical Chemistry in a GMP

Environment. John Wiley & Sons, Inc.

WHAT IS SUCCESS?

Questions? Comments?

THANK YOU