Validation of analytical methods Rutendo Kuwana Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

BACKGROUND-LAB METHOD FLOW Validation Transfer Development Approved

VALIDATION “ THE PROCESS OF PROVIDING DOCUMENTED EVIDENCE THAT SOMETHING DOES WHAT IT IS INTENDED TO DO”

WHY VALIDATE? To demonstrate that the method is suitable for its intended use Provides assurance of reliability

WHO GMP - 4.11 Analytical methods, computers and cleaning procedures “It is of critical importance that particular attention is paid to the validation of analytical test methods, automated systems and cleaning procedures.” Validation of analytical procedures used in the examination of pharmaceutical materials (WHO Expert Committee on Specifications for Pharmaceutical Preparations. 32nd Report. Geneva, WHO, 1992 (WHO Technical Report Series, No. 823) Text on Validation of Analytical Procedures Q2 (R1) Validation of Analytical Procedures: Text and Methodology. ICH Harmonized Tripartite Guidelines

ANALYTICAL METHOD VALIDATION PERFORMANCE CHARACTERISTICS

TYPES OF ANALYTICAL METHODS TO BE VALIDATED Identification tests Quantitative tests for impurities’ content Limit tests for the control of impurities Quantitative tests of the active in samples of the drug substance (raw material), finished product or other selected components in the drug

ACCURACY Expresses 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 i.e. individual observation or mean of measurements The closeness of test results to the true value obtained by the method (trueness). Established across the range

DETERMINATION OF ACCURACY Drug Substance Analysis of reference material Compare results to a second, well-characterized method Drug Product Analysis of synthetic mixtures spiked with known quantities of components Determined concurrently with precision, linearity and specificity

DETERMINATION OF ACCURACY cont’ Impurities (Quantitation) Analysis of samples (Drug substances/Drug product) spiked with known amounts of impurities If impurities are not available, see specificity Recommended Data Minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e.g. 3 concentrations/3 replicates each) Reported as % recovery of known added amount or difference between the mean and true value, with confidence intervals

PRECISION Precision The measure of the degree of agreement (degree of scatter) among test results when the method is applied repeatedly to multiple samplings of a homogeneous sample Expressed as %RSD for a statistically significant number of samples

Precision (of any process) Measured mean Real mean The precision (VARIABILITY) of an analytical procedure is usually expressed as the standard deviation (S), variance (S2), or coefficient of variation (= relative standard deviation, R.S.D.) of a series of measurements. The confidence interval should be reported for each type of precision investigated.

PRECISION cont’ May be considered at 3 levels: Repeatability, a measure of variability under the same operating conditions over a short interval (intra-assay precision). Minimum of 9 determinations covering specified range Intermediate precision, a measure of within-laboratory variations (different days, different analysts, different equipment) Reproducibility, expresses precision between laboratories (e.g. in collaborative studies), also applies to method transfer

Repeatability (of any process) Repeatability expresses the precision (spread of the data, variability) under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision. Measured mean

Intermediate Precision and Reproducibility Intermediate precision expresses within-laboratories variations. #1, #2 and #3: different days, different analysts, different (manufacturing) equipment, etc. Reproducibility expresses the precision between laboratories #1, #2 and #3 (collaborative studies, usually applied to standardization of methodology). (Transfer of technology) Measured means

ACCURACY & PRECISION Accurate and precise

SPECIFICITY/SELECTIVITY The ability to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the matrix The degree of interference Active Ingredients Excipients Impurities (synthetic precursors, enantiomers) Degradation Products Placebo Ingredients

SPECIFICITY/SELECTIVITY Combination of 2 or more analytical procedures may be required to achieve necessary level of discrimination Stability indicating analytical methods should always be specific Analysts should ascertain whether the peaks within a sample chromatogram are pure or consist of more than one compound. Therefore should know how many compounds are in the sample or use procedures to detect peak purity

LOD, LOQ and SNR Limit of Quantitation (LOQ) Limit of Detection (LOD) Signal to Noise Ratio (SNR) Peak B LOQ Peak A LOD noise Baseline

LIMIT OF DETECTION The lowest concentration of an analyte in a sample that can be detected, not quantified Expressed as a concentration at a specified signal:noise ratio

LIMIT OF QUANTIFICATION The lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under stated operational conditions Expressed as concentration of analyte

LINEARITY The Ability of the method to obtain test results that are directly proportional to concentration within a given range Method: dilution of stock solution/separate weighings Expressed as the variance of the slope of the regression line Correlation coefficient, y-intercept, slope of regression line and residual sum of squares should be presented together with plot of the data

Linearity Linearity expresses differences in precision at different points of a given range. The linearity of an analytical procedure is its ability (within a given range) to obtain test results, which are directly proportional to the concentration (amount) of analyte in the sample

RANGE Interval between upper and lower levels of analyte demonstrated by the method Confirms that the analytical procedure provides acceptable degree of linearity, accuracy and precision when applied to samples containing amounts of analyte within or at the extremes of the specified range Minimum 5 concentrations

Range (minimum requirements) Assay of an API or a FPP: ± 20% of the test concentration. Content uniformity: ± 30% of the test concentration (unless a wider more appropriate range, based on the nature of the dosage form (e.g., metered dose inhalers), is justified). Dissolution testing: ± 20 % over the specified range. Impurity: from the reporting level of an impurity to 120% of the specification. (Unusually potent or toxic impurities, LOD and LOQ should be commensurate with ICH requirement.) If assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the range from the reporting level of the impurities to 120% of the assay specification

Typical expected results for Analyte concentration vs Typical expected results for Analyte concentration vs. Precision (The AOAC manual for the Peer-Verified Methods program) Analyte Conc (%) Analyte Ratio Unit RSD% 100 1 100% 1.3 10 10-1 10% 2.8 10-2 1 % 2.7 0.1 10-3 0.1% 3.7 0.01 10-4 100 ppm 5.3 0.001 10-5 10 ppm 7.3 0.0001 10-6 1 ppm 11 0.00001 10-7 100 ppb 15 0.000001 10-8 10 ppb 21 0.0000001 10-9 1 ppb 30

RUGGEDNESS Ruggedness Degree of reproducibility of test results under a variety of conditions Different Analysts Different Laboratories Different Instruments Different Reagents Different Days Etc. Expressed as %RSD

ROBUSTNESS Measure of the capacity to remain unaffected by small (deliberate) variations in method parameters Indication of reliability during normal use

Sensitivity and robustness Input-output relationship

Stability of analytical solution Solutes may readily decompose prior to chromatographic investigations e.g. during sample preparation , extraction, cleanup, phase transfer or storage of prepared vials (refrigerators or automatic sampler). Method development should investigate the stability of the analytes AND standards. System stability stability of the samples being analyzed in a sample solution. Measure of the bias in assay results generated during a preselected time interval e.g. 1 – 48 hours using a single solution should be determined by replicate analysis of the sample solution. considered appropriate when the RSD, calculated on the assay results obtained at different time intervals, Less than 20 percent of the corresponding value of the system precision

SYSTEM SUITABILITY The checking of a system, before or during analysis of unknowns, to ensure system performance. “No sample analysis is acceptable unless the requirements for system suitability have been met.” (USP Chapter 621) Plate Count, Tailing, Resolution Determination of reproducibility (%RSD) For %RSD < 2.0%, Five replicates For %RSD > 2.0%, Six replicates System Suitability "Sample“ - A mixture of main components and expected by-products utilized to determine system suitability “Whenever There is a Significant change in Equipment or Reagents System Suitability Testing Should be Performed” (USP Chapter 621)

Classes of analytical tests “The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intented purpose.” Class A: To establish identity Class B: To detect (Bd) and quantitate (Bq) impurities Class C: To determine quantitatively the concentration, or assay Class D: To assess characteristics Other classes not covered in the guides

Criteria for analytical classes Bq Bd C D Accuracy X Precision Robustness Linearity and range Specificity X ? Limit of detection Limit of quantitation

General requirements Qualified and calibrated instruments Documented methods Reliable reference standards Qualified analysts Sample integrity Change control (e.g., synthesis, FPP composition)

General requirements (2) Analytical methods should be used within GMP and GLP environments, and must be developed using the protocols and acceptance criteria set out in the ICH guidelines Q2 (R1)

General requirements (3) Validation Protocol important Revalidation should accompany formulation changes (new samples with new compounds or new matrices) manufacturing batch changes new analysts with different skills, new instruments with different characteristics, new location with different environmental conditions, new chemicals and/or reference standards and modification of analytical parameters.

Validation Report  Objective and scope of the method (applicability, type).  Summary of methodology.  Type of compounds and matrix.  All chemicals, reagents, reference standards, QC samples with purity, grade, their source or detailed instructions on their preparation.  Procedures for quality checks of standards and chemicals used.  Method parameters.  Critical parameters taken from robustness testing.  Listing of equipment and its functional and performance requirements, e.g., cell dimensions, baseline noise and column temperature range.

Validation Report (2)  Detailed conditions on conduct of experiments, including sample preparation  Statistical procedures and representative calculations.  Procedures for QC in routine analyses, e.g., system suitability tests.  Representative plots, e.g., chromatograms, spectra and calibration curves.  Method acceptance limit performance data and expected uncertainty of measurement results. Criteria for revalidation. The person(s) who developed and validated the method. References (if any). Summary and conclusions.

Use and Validation of Pharmacopoeial methods The degree of validation of a pharmacopoeial method should be adequate for required purpose, and the laboratory be able to match any stated performance data. Following product – specific attributes should be considered the type of compounds to be analyzed, matrices, the type of information required (qualitative or quantitative), detection and quantitation limits, Concentration range for analysis based on own product precision and accuracy (sample preparation critical) as specified by the client of the analytical data and the type of equipment—its location and environmental conditions. Therefore PQP three parameters required for PQP – Specificity, Accuracy and Precision

Main Points Again Validation of analytical procedures is a critical requirement in risk assessment and management: establishment of product-specific acceptance criteria, and stability of APIs and FPPs. Validation should demonstrate that the analytical procedure is suitable for its intented purpose. HPLC systems and method validation deserves special attention during the inspection of QC laboratories.