Phase Appropriate Method Validation
Phase Appropriate Method Validation Aryo Nikopour Irvine, California | January 12, 2017 The Southern California Pharmaceutical Discussion Group (SCPDG) of AAPS
OUTLINE • • • • • • •
What is Validation Guidelines Method Verification Method Transfer Phase Approporiate Method Validation Method Validation Characteristics Mass Balance
VALIDATION OF ANALYTICAL METHOD Calibration
System Suitability
Validation
Method
Analyst
DATA QUALITY TRIANGLE QC Checks
System Suitability Tests Analytical Method Validation Analytical Instrument Qualification
METHOD LIFE CYCLE Validation
Development
Optimization
METHOD VALIDATION
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 : 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.org ICH Q2(R1):
Validation of Analytical Procedures: Methodology‐ Nov. 2005
ICH Q3A(R):
Impurities in New Drug Substances ‐ Feb. 2002
ICH Q3B(R):
Impurities in New Drug Products – Feb. 2003
ICH Q3C:
Impurities: Residual Solvents ‐ July 1997
ICH Q5A,D:
Biotech/Biological Products ‐ 1997
ICH Q5B,C:
Biotech/Biological Products ‐ 1995
(Dates indicate ICH finalization)
VALIDATION IN THE 21 CENTURY ICH FDA USP
METHOD VALIDATION USP
Method Qualification
• Method Validation • Method Qualification
USP
• Method Verification
USP
• Method Transfer
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 • Current USP 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 • 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.
Non-Compendial: • •
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 ASSAY CATEGORIES Category Number
Category Name
I
Quantitative
II
ImpuritiesQuantitative
II
Impurities-Limit
III
Performance Characteristics
IV
Identity
Description of Assay Quantitation of major components/active ingredients present at high concentrations. Determination of impurities or degradation products. Parameters to be tested depend on the nature of the test; includes dissolution testing.
METHOD VALIDATION REQUIREMENTS USP Assay Category I
II
III
Quantitative Limit Tests Parameter: Accuracy Y Y Y Precision Y Y N Y Intermediate Y Y N Y Precision Specificity N Y Y N Detection Limit N Y Y Quantitation Limit N Y N Linearity Y Y N Range Y Y Robustness Y Y N N Selectivity Y Y N Y System Suitability Y Y N Y Solution Stability Y Y N Y May be required, depending on the nature of the specific test.
IV
N N N Y N N N N N Y N N
PHASE APPROPRIATE VALIDATION
PreClinical
Phase I
Phase II
Phase III
PM
LCM
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 CHARACTERISTICS Validation Characteristics
Experimental Details
Acceptance Criteria
Specificity
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 range Assay: 50% to 125% of Specification limit QL-150% of specification limit
Calibration Model is valid R ≥0.998 Report Intercept, Slope and %Bias
Detection Limit (DL)
DL= 3.3 (/S)
S/N≥ 3
Quantitation Limit (QL)
DL= 10 (/S)
%RSD≤ 15%
METHOD VALIDATION CHARACTERISTICS Validation Characteristics
Experimental Details
Acceptance Criteria
6 replicates 6 replicates Comparative Precision/Partial Validation
%RSD≤ 2% Overall %RSD (two Analyst) 3 Sigma 3 Sigma
Accuracy
At least 9 determination over 3 concentration level e.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
Assay: 98 to 102 % of control Impurities: 95 to 105%
Robustness
Deliberately change critical parameters of the method
Must meet system suitability and selectivity requirements
Precision : Repeatability Intermediate Precision (Ruggedness) Reproducibility
VALIDATION: PHASE I Assay
I.D.
Quantitative Impurities
Limit Test
Selectivity
X
X
X
X
Repeatability
X
Drug Product
Accuracy/Precision
Linearity Range
X
Recovery at 100%
At 100% of Reporting Threshold
X
QL to 200% of Limit
Defined by ALP
Defined by ALP
DL/QL System Suitability
X
Solution Stability
X
DL
QL
QL or at Limit
X
X
X
X
X
VALIDATION: PHASE II Selectivity
Assay
I.D
Quantitative Impurities
Limit Test
X
X
X
X
Specificity Repeatability
X X
X
Accuracy
Recovery at 3 levels
At 100% of Reporting Threshold
Linearity
X
X
X
X
QL
DL/QL Range
DL Define by ALP
System Suitability
X
Solution Stability
X
X
Defined by ALP X
X
X
X
X
VALIDATION: PHASE III Selectivity
Assay
I.D
Quantitative Impurities
Limit Test
X
X
X
X
Specificity
X
Repeatability
X
Intermediate Precision
X
Accuracy
X
X
Linearity
X
X
DL/QL Range
2nd Analyst
DL
X
X
X
X
X
QL or at Limit
Defined by ALP
Defined by ALP
Solution Stability
X
X
X
System Suitability
X
X
X
Robustness
X
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 system suitability?
SYSTEM SUITABILITY What parameters do you measure for system suitability?
Selectivity
Efficiency
Capacity
SYSTEM SUITABILITY Standard B1 (n=6) Injections SST Solution B1 and B2 Average Tailing %RSD Average Resolution LVF and Response Factor Area %RSD Factor Retention Time Theoretical Plates DesMethyl-LVF) % Difference 1% 1% 2% 0.8 Tf 1.4 >15000 NLT 2.5
Date
NB/Page
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 3 Sigma min max
2770 8311 21418 38040
0.1685 0.5054 2.88 3.90
GAUSSIAN DISTRIBUTION
C.I. =
CONTROL CHART
SELECTIVITY AND SPECIFICITY Selectivity vs. Specificity
SELECTIVITY AND SPECIFICITY Selectivity: 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.
Stress Studies
“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 Active Moiety.
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 stability studies.
•
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. • Orthogonal Approach : develop MS compatible method • Design study based on the formulation (feed, tablet, ointment, etc.).
FORCED DEGRADATION (STRESS STUDIES) Chemical
Physical
Environmental
Acid
Agitation
Heat
Base
Denaturation, 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
600 to 800 foot candles (sources include metal halides, Hg, Xe lamp, or UVB fluorescence)
Option II: 74Hours
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
Light
Option I: 2-4 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!!
EXAMPLE: PHOTOLYTIC STRESS
4
Sample Sample Sample Sample WVL:280 nm Imp 14 - 20.487
Imp 11 - 14.517
Imp 9 - 12.817 Imp 10 - 13.320
Imp 1 - 3.397
1.25 0.00
Imp 3 - 6.097
2.50
Sample Name: Fresh 30 mg Sample Name: Light Stressed 3 Sample Name: Light Stressed 3 Sample Name: Light Stressed 3
Imp 5 - 7.873 Imp 6 - 8.320 DesMethyl-LVF - 8.980 Levofloxacin - 9.777 Imp 7 - 10.733
1 - Sequence Name: Forced De 2 - Sequence Name: Forced De 3 - Sequence Name: Forced De 4 - Sequence Name: Forced De 3.80 mAU
3 2 1
2.20 0.0
min 2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
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 STABILITY Purpose: 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: 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:
lowest concentration for which RSD is
OUTLINE • • • • • • •
What is Validation Guidelines Method Verification Method Transfer Phase Approporiate Method Validation Method Validation Characteristics Mass Balance
VALIDATION OF ANALYTICAL METHOD Calibration
System Suitability
Validation
Method
Analyst
DATA QUALITY TRIANGLE QC Checks
System Suitability Tests Analytical Method Validation Analytical Instrument Qualification
METHOD LIFE CYCLE Validation
Development
Optimization
METHOD VALIDATION
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 : 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.org ICH Q2(R1):
Validation of Analytical Procedures: Methodology‐ Nov. 2005
ICH Q3A(R):
Impurities in New Drug Substances ‐ Feb. 2002
ICH Q3B(R):
Impurities in New Drug Products – Feb. 2003
ICH Q3C:
Impurities: Residual Solvents ‐ July 1997
ICH Q5A,D:
Biotech/Biological Products ‐ 1997
ICH Q5B,C:
Biotech/Biological Products ‐ 1995
(Dates indicate ICH finalization)
VALIDATION IN THE 21 CENTURY ICH FDA USP
METHOD VALIDATION USP
Method Qualification
• Method Validation • Method Qualification
USP
• Method Verification
USP
• Method Transfer
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 • Current USP 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 • 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.
Non-Compendial: • •
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 ASSAY CATEGORIES Category Number
Category Name
I
Quantitative
II
ImpuritiesQuantitative
II
Impurities-Limit
III
Performance Characteristics
IV
Identity
Description of Assay Quantitation of major components/active ingredients present at high concentrations. Determination of impurities or degradation products. Parameters to be tested depend on the nature of the test; includes dissolution testing.
METHOD VALIDATION REQUIREMENTS USP Assay Category I
II
III
Quantitative Limit Tests Parameter: Accuracy Y Y Y Precision Y Y N Y Intermediate Y Y N Y Precision Specificity N Y Y N Detection Limit N Y Y Quantitation Limit N Y N Linearity Y Y N Range Y Y Robustness Y Y N N Selectivity Y Y N Y System Suitability Y Y N Y Solution Stability Y Y N Y May be required, depending on the nature of the specific test.
IV
N N N Y N N N N N Y N N
PHASE APPROPRIATE VALIDATION
PreClinical
Phase I
Phase II
Phase III
PM
LCM
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 CHARACTERISTICS Validation Characteristics
Experimental Details
Acceptance Criteria
Specificity
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 range Assay: 50% to 125% of Specification limit QL-150% of specification limit
Calibration Model is valid R ≥0.998 Report Intercept, Slope and %Bias
Detection Limit (DL)
DL= 3.3 (/S)
S/N≥ 3
Quantitation Limit (QL)
DL= 10 (/S)
%RSD≤ 15%
METHOD VALIDATION CHARACTERISTICS Validation Characteristics
Experimental Details
Acceptance Criteria
6 replicates 6 replicates Comparative Precision/Partial Validation
%RSD≤ 2% Overall %RSD (two Analyst) 3 Sigma 3 Sigma
Accuracy
At least 9 determination over 3 concentration level e.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
Assay: 98 to 102 % of control Impurities: 95 to 105%
Robustness
Deliberately change critical parameters of the method
Must meet system suitability and selectivity requirements
Precision : Repeatability Intermediate Precision (Ruggedness) Reproducibility
VALIDATION: PHASE I Assay
I.D.
Quantitative Impurities
Limit Test
Selectivity
X
X
X
X
Repeatability
X
Drug Product
Accuracy/Precision
Linearity Range
X
Recovery at 100%
At 100% of Reporting Threshold
X
QL to 200% of Limit
Defined by ALP
Defined by ALP
DL/QL System Suitability
X
Solution Stability
X
DL
QL
QL or at Limit
X
X
X
X
X
VALIDATION: PHASE II Selectivity
Assay
I.D
Quantitative Impurities
Limit Test
X
X
X
X
Specificity Repeatability
X X
X
Accuracy
Recovery at 3 levels
At 100% of Reporting Threshold
Linearity
X
X
X
X
QL
DL/QL Range
DL Define by ALP
System Suitability
X
Solution Stability
X
X
Defined by ALP X
X
X
X
X
VALIDATION: PHASE III Selectivity
Assay
I.D
Quantitative Impurities
Limit Test
X
X
X
X
Specificity
X
Repeatability
X
Intermediate Precision
X
Accuracy
X
X
Linearity
X
X
DL/QL Range
2nd Analyst
DL
X
X
X
X
X
QL or at Limit
Defined by ALP
Defined by ALP
Solution Stability
X
X
X
System Suitability
X
X
X
Robustness
X
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 system suitability?
SYSTEM SUITABILITY What parameters do you measure for system suitability?
Selectivity
Efficiency
Capacity
SYSTEM SUITABILITY Standard B1 (n=6) Injections SST Solution B1 and B2 Average Tailing %RSD Average Resolution LVF and Response Factor Area %RSD Factor Retention Time Theoretical Plates DesMethyl-LVF) % Difference 1% 1% 2% 0.8 Tf 1.4 >15000 NLT 2.5
Date
NB/Page
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 3 Sigma min max
2770 8311 21418 38040
0.1685 0.5054 2.88 3.90
GAUSSIAN DISTRIBUTION
C.I. =
CONTROL CHART
SELECTIVITY AND SPECIFICITY Selectivity vs. Specificity
SELECTIVITY AND SPECIFICITY Selectivity: 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.
Stress Studies
“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 Active Moiety.
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 stability studies.
•
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. • Orthogonal Approach : develop MS compatible method • Design study based on the formulation (feed, tablet, ointment, etc.).
FORCED DEGRADATION (STRESS STUDIES) Chemical
Physical
Environmental
Acid
Agitation
Heat
Base
Denaturation, 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
600 to 800 foot candles (sources include metal halides, Hg, Xe lamp, or UVB fluorescence)
Option II: 74Hours
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
Light
Option I: 2-4 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!!
EXAMPLE: PHOTOLYTIC STRESS
4
Sample Sample Sample Sample WVL:280 nm Imp 14 - 20.487
Imp 11 - 14.517
Imp 9 - 12.817 Imp 10 - 13.320
Imp 1 - 3.397
1.25 0.00
Imp 3 - 6.097
2.50
Sample Name: Fresh 30 mg Sample Name: Light Stressed 3 Sample Name: Light Stressed 3 Sample Name: Light Stressed 3
Imp 5 - 7.873 Imp 6 - 8.320 DesMethyl-LVF - 8.980 Levofloxacin - 9.777 Imp 7 - 10.733
1 - Sequence Name: Forced De 2 - Sequence Name: Forced De 3 - Sequence Name: Forced De 4 - Sequence Name: Forced De 3.80 mAU
3 2 1
2.20 0.0
min 2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
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 STABILITY Purpose: 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: 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:
lowest concentration for which RSD is
