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 Analyst Method

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 <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.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<1225> Method Validation Method Qualification Method Qualification USP<1226> Method Verification USP<1224> 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 <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. 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 <1225>ASSAY CATEGORIES Category Number I II II III IV Category Name Quantitative Impurities- Quantitative Impurities-Limit Performance Characteristics 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 II I 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. III IV

PHASE APPROPRIATE VALIDATION Pre- Clinical 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 Linearity System precision assessed by 6 replicate measurement/injections At least 5 Concentration over the range Assay: 50% to 125% of Specification limit QL-150% of specification limit %RSD 2% Detection Limit (DL) DL= 3.3 ( /S) S/N 3 Calibration Model is valid R 0.998 Report Intercept, Slope and %Bias Quantitation Limit (QL) DL= 10 ( /S) %RSD 15%

METHOD VALIDATION CHARACTERISTICS Validation Characteristics Experimental Details Acceptance Criteria Precision : Repeatability Intermediate Precision (Ruggedness) Reproducibility 6 replicates 6 replicates Comparative Precision/Partial Validation Accuracy At least 9 determination over 3 concentration level e.g. 70 to 120% for %RSD 2% Overall %RSD (two Analyst) 3 Sigma 3 Sigma For Assay Mean Recovery 97 to 103% for Impurities : 85% to 115% Range Solution Stability Robustness The range is defined by the results obtained for linearity, accuracy and precision Determine solution stability of Reference Standard Solution and Sample over 72 hours Deliberately change critical parameters of the method Linearity, accuracy and precision demonstrated over the range Assay: 98 to 102 % of control Impurities: 95 to 105% Must meet system suitability and selectivity requirements

VALIDATION: PHASE I Drug Product Assay I.D. Quantitative Impurities Limit Test Selectivity X X X X Repeatability X X Accuracy/Precision Recovery at 100% At 100% of Reporting Threshold 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 II Assay I.D Quantitative Impurities Limit Test Selectivity X X X X Specificity Repeatability X X X Accuracy Recovery at 3 levels X 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 III Assay I.D Quantitative Limit Test Impurities Selectivity X X X X Specificity X Repeatability X X X Intermediate Precision X 2 nd Analyst X X Accuracy X X Linearity X X DL/QL DL X QL or at Limit 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 system suitability?

SYSTEM SUITABILITY What parameters do you measure for system suitability? Selectivity Efficiency Capacity

SYSTEM SUITABILITY Date NB/Page 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% 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 2770 0.1685 3 Sigma 8311 0.5054 min 21418 2.88 max 38040 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% H 2 O 2 1 to 24 hours Light 600 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 24 to 72 hours 60 C 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!!

EXAMPLE: PHOTOLYTIC STRESS 3.80 2.50 1.25 0.00 1 - Sequence Name: Forced De Sample Name: Fresh 30 mg Sample 2 - Sequence Name: Forced De Sample Name: Light Stressed 3 Sample 3 - Sequence Name: Forced De Sample Name: Light Stressed 3 Sample 4 - Sequence Name: Forced De Sample Name: Light Stressed 3 Sample mau 4 3 2 Imp 1-3.397 Imp 3-6.097 Imp 5-7.873 Imp 6-8.320 DesMethyl-LVF - 8.980 Levofloxacin - 9.777 Imp 7-10.733 Imp 9-12.817 Imp 10-13.320 Imp 11-14.517 1 2.20 min 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 Imp 14-20.487 WVL:280 nm

MASS BALANCE 1 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 error 1.

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 <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: 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 ( /S) QL= 10 ( /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.

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 = m x + b Where: 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.

ACCURACY Determination 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.

ACCURACY Should 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.

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: Repeatability Intermediate Precision Reproducibility 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

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.

ROBUSTNESS Examples of typical RP-HPLC variations: ph of mobile phase mobile phase composition Ionic Strength Different columns Column temperature flow rate

ROBUSTNESS Parameter Nominal Procedure Condition Conditions Tested for Robustness Determination MPA*-Buffer constituent ph 4.0 3.9, 4.1 MPA*-Buffer salt concentration 10 mm Ammonium Formate 9 mm, 11 mm Column Temperature 30 C 25 C, 35 C Detector 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 Redevelopment of the method required due to change Revalidation required due to change Change is not covered by existing validation Development of the Method Validation of the Method Method in Routine use Change to Method: Evaluate the effect Change is covered by existing validation

REFERENCES 1. Bob Snider, CMC Group 2. ICH Q2 (R1) 3. Current USP <1224> 4. Current USP <1225> 5. Current USP <1226> 6. FDA Guidance for Industry 7. Miller, JM., Crowther, JB. 2000. Analytical Chemistry in a GMP Environment. John Wiley & Sons, Inc.

WHAT IS SUCCESS?

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