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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