Supplementary T raining Workshop on Good Manufacturing Practices (GMP)

1. Supplementary Training Workshop on GoodManufacturing Practices (GMP) VALIDATION of ANALYTICAL TEST METHODS János Pogány, pharmacist, PhD, consultant to WHO Pretoria, South Africa, 28 June 2005 E-mail: pogany@t-online.hu Dr. Pogány

2. GMP - 4.11 Analytical methods, computers and cleaning procedures • „It is of critical importance that particular attention is paid to the validation ofanalytical 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 Q2A(1994) Validation of Analytical Procedures: Methodology Q2B(1996) ICH Harmonized Tripartite Guidelines Dr. Pogány

3. Characteristics of methods • Accuracy (also termedtrueness) • Precision • repeatability • intermediate precision (intra-laboratory variation) • reproducibility (inter-laboratory variation) • Robustness, ruggedness • Stability Dr. Pogány

4. Accuracy and precision Inaccurate and imprecise Accurate Accurate and precise Precise Dr. Pogány

5. The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true valueor an accepted reference valueand the value found(individual observation or mean of measurements). application of an analytical procedure to an analyte of known purity (e.g. reference material); comparison of the results of the proposed analytical procedure with those of a second well-characterized procedure, the accuracy of which is stated and/or defined accuracymay be inferred once precision, linearity and specificity have been established. Accuracy (of any process) Dr. Pogány

6. 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 (of any process) Measured meanReal mean PRECISION Dr. Pogány

7. 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. Repeatability (of any process) Measured mean REPEATABILITY Dr. Pogány

8. 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) Intermediate Precision and Reproducibility Measured means Intermediate precision or Reproducibility Dr. Pogány

9. Sensitivity and robustness Input-output relationship Dr. Pogány

10. Stability (of the analytical solution) expresses variation of the measured mean as a function of time. (Manufacturing machines) #1 … First mesurements #2 … Second mesurements of the same samplewithin a relatively short period of time. Stability Stability Measured means Dr. Pogány

11. Characteristics of analytical procedures • Linearity • Range • Specificity (selectivity) • Sensitivity (versus robustness) • Limit of detection • Limit of quantitation Dr. Pogány

12. 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.” Linearity Measured Real mean meanPrecision Dr. Pogány

13. 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 impurityto 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 Dr. Pogány

14. Specificity (selectivity) • Specificity is the ability to assess unequivocally the analyte in the presence of components, which may be expected to be present. Typically these might include impurities, degradants, excipients, etc. • Stability indicating analytical methods should always be specific. Dr. Pogány

15. LOD, LOQ and SNR • Limit of Quantitation (LOQ) • Limit of Detection (LOD) • Signal to Noise Ratio (SNR) Peak BLOQ Peak ALOD noise Baseline Dr. Pogány

16. 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 Dr. Pogány

17. Criteria for analytical classes Dr. Pogány

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

19. HPLC Method Development and Validation for Pharmaceutical Analysis by Ghulam A. Shabir Pharmaceutical Technology Europe, 1 March 2004

20. Prequalification requirements • Analytical method validation is required by WHO for the prequalification of product dossiers. Non-compendial ARV APIs and FPPs were/are tested with methods developed by the manufacturer. • Analytical methods should be used within GMP and GLP environments, and must be developed using the protocols andacceptance criteria set out in the ICH guidelines Q2A and Q2B. Dr. Pogány

21. HPLC system Dr. Pogány

22. Linearity and range Dr. Pogány

23. ICH requirements • Concentration range 0.025–0.15 mg/mL (25–150% of the theoretical concentration in the test preparation, n=3) • Regression equation was found by plotting the peak area (y) versus the analyte concentration (x) expressed in mg/mL: y = 3007.2x + 4250.1 (r2 = 1.000). • The regression coefficient demonstrates the excellent relationship between peak area and concentration of analyte. • The analyte response is linear across 80-120% of the target progesterone concentration. Dr. Pogány

24. Accuracy The data show that the recovery of analyte in spiked samplesmet the evaluation criterion for accuracy (100 ± 2.0% across 80–120% of target concentrations). Dr. Pogány

25. Specificity Dr. Pogány

26. Specificity • An example of specificity criterion for an assay method is that the analyte peak will have baseline chromatographic resolution of at least 2.0 minutes from all other sample components. • In this study, a weight of sample placebo equivalent to the amount present in a sample solution preparation was injected to demonstrate the absence of interference with progesterone elution. Former slide demonstrates specificity. Dr. Pogány

27. The repeatability precision obtained by one analyst in one laboratory was 1.25% RSD for the analyte and, therefore, meets the evaluation criterionof RSD ≤2%. Repeatability Dr. Pogány

28. Intermediate precision Dr. Pogány

29. Limit of detection • The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected, not quantified. It is expressed as a concentration at a specified signal:noise ratio (SNR), usuallybetween 3 and 2:1. • In this study, the LOD was determined to be 10ng/mL with a signal:noise ratio of 2.9:1. Dr. Pogány

30. Limit of quantitation • The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method. The ICH has recommended a signal:noise ratio (SNR) of10:1. • The LOQ was 20 ng/mL with a signal:noise ratio of 10.2. The RSDfor six injections of the LOQ solution was ≤2%. Dr. Pogány

31. Analytical solution stability Standard and sample solutions stored in a capped volumetric flask on a lab bench under normal lighting conditions for 24 h were shown to be stable with no significant change in progesterone concentration during this period. Dr. Pogány

32. 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. Dr. Pogány