Birth of Drug Stages of new drug discovery from the initial synthesis to Phase I in humans Analytical Method Validation in Drug Development The Importance of Analytical Methodology in the Drug Development Process After the registration of drug product to regulatory authority, the test methods are used in the pharmaceutical quality control laboratory to do the following. 1) Identify the drug substance and product 2) Quantitate the pharmaceutical active ingredient 3) Determine level of purity 4) Guarantee the overall quality of the product 5 “Stakeholders” in Quality Analytical Testing During Drug Development Process development /Characterizati on/Controls Support to Clinical Trials Stability / Release Analytical test Product development Regulatory Filings Toxicology Pharmacokinetics 6 Requirements of Analytical methods During the Drug Development Process “Depending on the stage of development, these analytical methods are standard screening methods at the start of the development process, which over time are gradually upgraded to thoroughly validated methods for NDA application. The filed methods must be simple, robust, and reliable- that is, easy to use and perform without deviations when appropriately applied in a qualified laboratory.” Note 1. 2. Screening methods are typically not optimized for speed and robustness. The filed methods referred to as VTR2AP methods (validated, transferable, robust, rapid, accurate, and precise) James M. Miller and Jonathan B. Crowther, “Analytical Chemistry in a GMP envirinment: A practical guide, John Wiely & Sons,New York” (2000) Laboratory Analytical Methods Flow During the Drug Development Process Method Development Method Validation Method Transfer Approved 8 What’s validation? The FDA defines the term as : “Established documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications and quality attributes.” “General Principles of Validation” (1987) ICH guideline defines the term as : “A documented program that provides a high degree of assurance that a specific process, method, or system will consistently produce a result meeting pre-determined acceptance criteria.” “Q7A-GMP for active phamarceutical ingredients” (2000) 9 Why Method Validation? To minimize analytical and instrumental errors To give reliable and reproducible results in accordance with the given specifications of the test method To ensure the quality of the test results To meet accreditation requirement Objective evidence for defense against challenges To be assured of the correctness of results Specification “A specification is defined as A list of tests, references to analytical procedures, and appropriate acceptance criteria that are numerical limits, ranges, or other criteria for the tests described. It establishes the set of criteria to which a drug substance or drug product should confirm to be considered acceptable for its intended use” “Test procedures and acceptance criteria for New drug substances and New drug products: Chemical products” (1997) “A list of detailed requirements with which the products or materials used or obtained during manufacture have to conform. They serve as a basis for quality evaluation.” “GMPs for pharmaceutical products: main principles” WHO TRS 908 Annex 4 (2003) 11 Acceptance criteria ICH guideline define the term as : “Numerical limits, ranges, or other suitable measures for acceptance of test results.” “Q7A-GMP for active phamarceutical ingredients” (2000) “Acceptance criteria means The product specifications and acceptance / rejection criteria, such as acceptable quality and unacceptable quality level, with an associated sampling plan, that are necessary for making a decision to accept or reject a lot or batch (or any other convenient subgroups of manufactured units)” “CFR Part 210.3 Definitions (2000)” 12 Prerequisites for analytical method validation –Six “M”s Man Machine qualified Methods characterised calibrated robust documented suitable skilled qualified Reference standards Quality of the analytical method Vibrations Temperature Irradiations Humidity Time Analysts´ support Quality Supplies Material Milieu Management Analytical Method Validation • Initial Method Validation Guidance Issued in 1987 – Guideline for submitting samples and analytical data for methods validation. Food and Drug Administration, February 1987. US Government Printing Office:1990-281794:20818. • Updated in August 2000 (Draft Guidance!) – Analytical Procedures and Method Validation. Fed. Reg. 65(169), 52,776-52,777, 30 August 2000 • CFR Part 211.165 Testing and release for distribution (e) – “ The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented.” • ICH guideline Q7 “GMP guide for active pharmaceutical ingredients” • ICH guideline Q2(R1) “ Validation of analytical Procedure : Text and Methodology” ICH & USP Method Validation Analytical Performance Characteristics Validation parameter Accuracy Repeatibility Precision Interm. precision ICH O O O USP O O - Reproducibility Specificity of Selectivity Detection limit Quantitation limit Linearity Range Robustness Ruggedness O O O O O O O - O O O O O O O Definitions Precision: The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Accuracy: The closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is some times called Trueness. 16 To find out whether a method is accurate: • Drug substance (assay) – Application of the method to an analyte of known purity (e.g. reference substance) – Comparison of the results of one method with those of a second wellcharacterised method (accuracy known) • Drug product (assay) – Application of the method to synthetic mixtures of the drug product component to which known quantities of the analyte have been added • Drug product may exceptionally be used as matrix • Drug substance/Drug product (Impurities) – Application of the method to samples spiked with known amounts of impurities Recommended Data • Accuracy should be assessed using a min. of 9 determinations over a min. of 3 concentration levels covering the specified range (e.g. 3 concentrations/3 replicates each of the total analytical procedure). • Accuracy should be reported as: – % recovery by the assay of known added amount of analyte in the sample. Example: • Nine solutions containing different concentrations of ketotifen fumarate reference standard added to ketotifen tablet batch no. 2506VAMG were assayed Example (continued): Conc. of ketotifen fumarate mg/ml 0.280 0.320 0.360 0.380 0.400 0.420 0.440 0.480 0.520 % 70 80 90 95 100 105 110 120 130 Area detected 1473566 1677013 1904848 1905862 2091215 2180374 2293647 2518976 2670144 Recovery (%) 99.32 99.48 100.94 100.51 100.06 100.03 100.07 101.01 98.99 Acceptance Criteria Mean (recovery) : 100.04 Standard deviation : 0.699 Relative standard deviation (RSD) : 0.699 % 98.0–102.0 % <2% • When to expect Accuracy problems – Insufficient selectivity of the method • Impurity peaks are not resolved and account for assay value – Recovery is < 100% • Irreversible adsorption of analyte to surfaces of the system – Incorrect assay value of a reference standard • Due to decomposition of reference standard – Incorrect assay value due to change in matrix • Analytical laboratory still uses the preceding matrix as standard • Precision – Expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample – Is usually expressed as the standard deviation (S), variance (S2) or coefficient of variation (RSD) of a series of measurements – Precision may be considered at three levels • Repeatability (intra-assay precision) • Intermediate Precision (variability within a laboratory) • Reproducibility (precision between laboratories) Example • The active ingredient, ketotifen fumarate, in tablets (batch no. 2506VAMG) was assayed seven times using HPLC. Example (continued) Sample no. 1 2 3 4 5 6 7 Concentration (mg/ml) 0.4 0.4 0.4 0.4 0.4 0.4 0.4 : : : Area detected 1902803 1928083 1911457 1915897 1913312 1897702 1907019 1910896 9841.78 0.515 % Mean Standard deviation Relative standard deviation (RSD) Acceptance criteria: Relative standard deviation (RSD): not more than 2 % Detection limit vs Quantitation limit ‘Know that it’s there’ vs ‘Know how much is there’ Detection limit Is any of it present? (means) Is it there? How much of it is present??? Quantitation limit How much of it is there? DETECTION LIMIT • The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value • Several approaches for determining the detection limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Based on Visual Evaluation • Visual evaluation may be used for noninstrumental methods but may also be used with instrumental methods. • The detection limit is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected . Based on Signal-to-Noise • This approach can only be applied to analytical procedures which exhibit baseline noise. • Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. • A signal-to-noise ratio between 3:1 or 2:1 is generally considered acceptable for estimating the detection limit. LOQ, LOD and SNR • Limit of Quantitation • Limit of Detection • Signal to Noise Ratio Peak B LOQ Peak A LOD Baseline noise Based on the Standard Deviation of the Response and the Slope The detection limit (DL) may be expressed as: DL = 3.3 s/S where s = the standard deviation of the response S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte. Estimate of s • Based on the Standard Deviation of the Blank – Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses • Based on the Calibration Curve – A specific calibration curve should be studied using samples containing an analyte in the range of DL. – The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation. Quantitation limit • Based on visual evaluation – The quantitation limit is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision. • • Based on signal-to-noise – A typical signal-to-noise ratio is 10:1. Based on the Standard Deviation of the Response and the Slope – The quantitation limit (QL) may be expressed as: – QL =10σ/S where σ= the standard deviation of response S = the slope of the calibration curve – The slope S may be estimated from the calibration curve of the standard. • Specificity – Is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present (impurities, degradants, matrix…) Identity testing – To ensure the identity of an analyte Purity testing – To ensure accurate statement on the content of impurities of an analyte Assay – To allow an accurate statement on the content of an analyte in a sample • Specificity: Overlay chromatogram of an impurity solution with a sample solution Specificity and stability • Stress stability testing to ensure the stability indicating potential of an analytical method – Apply diverse stress factors to the API – Apply diverse stress factors to the FPP Stress conditions: e.g. Supplement 2 of Generic Guideline; TRS 929, Annex 5 Assure that the API can be assessed specifically in the presence of known and unknown (generated by stress) impurities Assure that known impurities/degradants can be specifically assessed in the presence of further degradants By peak purity assessment and (overlay of) chromatograms Linearity and Range ‘Know that it’s a straight line’ vs ‘For what concentrations is it a straight line’ Linearity: Its ability to obtain test results which are directly proportional to the concentration (Amount) of analyte in sample within given range. Range: The Interval between the upper and lower concentration (amounts) of analyte in the sample ( including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. r > 0.997 r < 0.997 Example • Seven solutions containing different concentrations (0.280 – 0.520) mg/ml of ketotifen fumarate in tablet “batch no. 2506 VAMG were assayed using HPLC • The results were evaluated statistically and the results shown on the following slide Example (continued) Concentration of ketotifen fumarate mg/ml Area detected Acceptance criteria 0.280 0.320 0.360 0.400 0.440 0.480 0.520 1473566 1677013 1904848 2091215 2293647 2518976 2670144 0.998 – 1.002 Regression: y = ax + b a = 5055766.964 b = 67608.786 r2 = 0.9984 Robustness Small changes do not affect the results of the assay Robustness: A measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal use. (ICH) Typical Variations Liquid chromatography: • influence of variations of pH in a mobile phase, • influence of variations in mobile phase composition, • different columns (different lots and/or suppliers), • temperature, • flow rate. Gas chromatography: • different columns (different lots and/or suppliers), • temperature, • flow rate. Ruggedness • Ruggedness is defined by U.S. Pharmacoepia as the degree of reproducibility of results obtained under a variety of conditions, such as different laboratories, analysts, instruments, environmental conditions, operators and materials. • Ruggedness is a measure of reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst. • Ruggedness is determined by the analysis of aliquots from homogeneous lots in different laboratories. • Refer to the examples of “Ruggedness Trial” in the AOAC Requirements for Single Laboratory Validation of Chemical Methods (DRAFT 2002-11-07). 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Report "Validation in Pharmaceutical Manufacturing Industry"