1. PHARM 462 PART-2 2009 1/31

2. 2009 2/31 Good Manufacturing Practices (GMP) VALIDATION of ANALYTICAL TEST METHODS

3. 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 Q2A (1994) Validation of Analytical Procedures: Methodology Q2B (1996) ICH Harmonized Tripartite Guidelines 2009 3/32

4. Characteristics of methods  Accuracy (also termed trueness)  Precision  repeatability  intermediate precision (intra-laboratory variation)  reproducibility (inter-laboratory variation)  Robustness, ruggedness  Stability 2009 4/31

5. Accuracy and precision Accurate and precise Precise Accurate Inaccurate and imprecise 2009 5/31

6. Accuracy (of any process) The accuracy of an analytical procedure 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 (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  accuracy may be inferred once precision, linearity and specificity have been established. 2009 6/31

7. Precision (of any process) The precision (VARIABILITY) of an analytical procedure is usually expressed as the standard deviation (S), variance (S 2 ), 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. Measured mean Real mean PRECISION 2009 7/31

8. 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. REPEATABILITY Measured mean 2009 8/31

9. 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 Intermediate precision or Reproducibility 2009 9/31

10. Sensitivity and robustness Input-output relationship 2009 10/31

11. Stability 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 sample within a relatively short period of time. Stability Measured means 2009 11/31

12. Characteristics of analytical procedures  Linearity  Range  Specificity (selectivity)  Sensitivity (versus robustness)  Limit of detection  Limit of quantitation 2009 12/31

13. 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.” Measured Real mean mean Precision 2009 13/31

14. 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 2009 14/31

15. 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. 2009 15/31

16.  Limit of Quantitation (LOQ)  Limit of Detection (LOD)  Signal to Noise Ratio (SNR) noise Peak A LOD Peak B LOQ Baseline LOD, LOQ and SNR 2009 16/31

17. 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 (B d ) and quantitate (B q ) impurities  Class C: To determine quantitatively the concentration, or assay  Class D: To assess characteristics  Other classes not covered in the guides 2009 17/31

18. Criteria for analytical classes CriteriaABqBq BdBd CD AccuracyXXX PrecisionXXX RobustnessXXXXX Linearity and rangeXXX SpecificityX ?XXXX Limit of detectionX Limit of quantitationX 2009 18/31

19. General requirements  Qualified and calibrated instruments  Documented methods  Reliable reference standards  Qualified analysts  Sample integrity  Change control (e.g., synthesis, FPP composition) 2009 19/31

20. HPLC Method Development and Validation for Pharmaceutical Analysis Pharmaceutical Technology Europe, 1 March 2004 200920/34

21. 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 and acceptance criteria set out in the ICH guidelines Q2A and Q2B. 2009 21/31

22. HPLC system 2009 22/31

23. Linearity and range 2009 23/31

24. 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 (r 2 = 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. 2009 24/31

25. Accuracy The data show that the recovery of analyte in spiked samples met the evaluation criterion for accuracy (100 ± 2.0% across 80–120% of target concentrations). 2009 25/31

26. Specificity 2009 26/31

27. 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. 2009 27/31

28. Repeatability The repeatability precision obtained by one analyst in one laboratory was 1.25% RSD for the analyte and, therefore, meets the evaluation criterion of RSD ≤2%. 2009 28/31

29. Intermediate precision 2009 29/31

30. 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), usually between 3 and 2:1.  In this study, the LOD was determined to be 10ng/mL with a signal:noise ratio of 2.9:1. 2009 30/31

31. 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) of 10:1.  The LOQ was 20 ng/mL with a signal:noise ratio of 10.2. The RSD for six injections of the LOQ solution was ≤2%. 2009 31/31

32. 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. 2009 32/31

33. 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. 2009 33/31