Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations Protea Hotel Victoria Junction, Wa

1. Pharmaceutical Development Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations Protea Hotel Victoria Junction, Waterfront Cape Town, South Africa Date: 16 to 20 April 2007

2. Pharmaceutical Development Analytical Method Development Presenter: János Pogány, pharmacist, PhD pogany.janos@chello.hu WHO expert

3. Analytical Method Development Outline and Objectives of presentation • Introduction, guidelines • Dossier requirements • Assay • Related substances • Other issues • Main points again

4. Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations Introduction, guidelines

5. Interchangeability (IC) INTERCHANGEABILITY (IC) OF MULTISOURCE FPPs = (ESSENTIAL SIMILARITY WITH INNOVATOR FPP) = PHARMACEUTICAL EQUIVALENCE (PE) + BIOEQUIVALENCE (BE) IC=PE+ BE

6. Pharmaceutical equivalence • FPPs meet same or comparable standards (e.g., marketing authorization, analytical methods) • Same API(chemical and physical equivalence) • Same dosage form and route of administration • Samestrength • Comparable labeling • Pharmaceutical development equivalence • Stability equivalence • WHO-GMP(manufacturing equivalence)

7. 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 Q2(R1)

8. Guidelines used in PQP • „WHO-GMP 4.11 „It is of critical importance that particular attention is paid to the validation of analytical test methods, automated systems and cleaning procedures.” • Appendix 4. Analytical method validation(in WHO Expert Committee on Specifications for Pharmaceutical Preparations. 40th Report. Geneva, WHO, 2006 (WHO Technical Report Series, No. 937). http://whqlibdoc.who.int/trs/WHO_TRS_937_eng.pdf • Validation of analytical procedures:text and methodology Q2(R1)ICH Harmonized Tripartite Guidelines, (2005) http://www.ich.org/LOB/media/MEDIA417.pdf

9. General requirements • Qualifiedandcalibrated instruments • Documented methods • Reliable reference standards • Qualified analysts • Sample selection and integrity • Change control

10. Measure of variation (spread of data) 68.26% 95.46%

11. Mean (average) chart Abnormal variation of process – special causes USL Upperspecification limit Normal variation due to common causes average = mean LSL Lower specification limit Abnormal variation of process – special causes

12. Almost all the measurements of a stable process fall inside the specification limits USL – LSL681012 Cp1.00 1.331.662.00 OoS results: .27%.6 ppm 64 ppm2 ppb Capable process http://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htm

13. NEVIRAPINE – Reference Standard System suitability requirement: RSD is NMT 0.85%

14. Training Workshop on Pharmaceutical Development with focus on Paediatric Formulations Dossier requirements

15. Use of analytical methods - generics

16. Analytical procedure characteristics

17. Accuracy - ISO 5725 1-6 Source: ISO. 1994. ISO 5725 1-6: Accuracy (Trueness and Precision) of Measurement Methods and Results. ISO, Geneva, Switzerland.

18. Accuracy and precision Inaccurate and imprecise Precise Accurate Accurate and precise

19. Percent accuracy (hypothetical figures) The data show that the recovery of analyte in spiked samplesmet theevaluation criterion for accuracy(100 ± 2.0% across 50–130% of target concentrations).

20. Percent accuracy (hypothetical figures) Red line: LA Green lines: USL and LSL

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

22. 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

23. Repeatability (hypothetical figures) 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%.

24. 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 (of any process) Measured means Intermediate precision or Reproducibility

25. Intermediate precision (ruggedness)

26. 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 and excipients. • 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 • Stability indicating analytical methods should always be specific.

27. Identification – a special case • Diethylene glycol (DEG) in paediatric dosage forms has been implicated as the causative agent in numerous deaths since 1937. The victims were mainly children. • Illustrative analytical issues of investigation • IR identity test was able to detect DEG at about 20 %w/w • Testing of DEG in Glycerol (and in Propylene Glycol) was recommended with a LOD (sensitivity) of NLT 0.1 %. For detecting DEG at low levels, GC seemed preferable. • The assay was the most relevant test (accurate within ± 0.2%) • Illustrative regulatory issues • Legislation • GMP • Specificity is an essential but not sufficient characteristic of identification

28. Specificity(hypothetical figures and data) HPLC chromatograms of (a) API reference standard, (b) FPP and (c) placebo

29. SPECIFICITY – degradants There were no peaks in the placebo chromatogram at the retention times of nevirapine (N), methylparaben (MP) and propylparaben (PP) peaks. *Sum of N, MP and PP peak areas. The three ingredients can be assessed in the presence of (non-expected) degradants. The peaks are homogeneous and pure. The method is selective, specific and stability-indicating.

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

31. LOD and LOQ (hypothetical figures)

32. LOD and LOQ • 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 0.086 μg/ml(Impurity 1) with a signal:noise ratio of 3.6 : 1 • 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 0.171 μg/ml(Impurity 1) with a signal:noise ratio of 11.3. The RSD for six injections of the LOQ solution was ≤2%.

33. 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 MeasuredReal meanmeanPrecision

34. Linearity and range Acceptance criterion: correlation coefficient should not be less than 0.9990

35. Linearity and range • Concentration range1.0–1.3 mg/ml (10–130% of the theoretical concentration in the test preparation, n=3) • Regression equation was found by plotting the means ofpeak area (y) against the analyte concentration (x) expressed in %: y = 36.124x -7.2984 (R2 = 0.9998). • The regression coefficient demonstrates an excellent relationship between peak area and concentration of analyte. • The analyte response is linear across 10-130% of the target nevirapine concentration.

36. 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

37. Stability (of the analytical solution) expressesvariationof the measured mean as a function of time. #1 … First measurements #2, #3, #4, …n Series of measurements of the same sample within a relatively short period of time. Stability of analytical solution Stability Measured means

38. Stability of test analytical solution An analytical solution prepared from Nevirapine 50mg/5ml Oral Suspension was spiked with Impurity-1 at specification level and stored in a capped volumetric flask on a laboratory bench at uncontrolled room temperature under normal lighting conditions for 25 hours. Conclusion: the stability of the analytical solution of Impurity-1 is not a source of variation.

39. Sensitivity and robustness

40. Robustness

41. Methods for cleaning validation • Method for assay and related substances used in stability studies of API and FPP • Specificity (in samples taken from a cleaning assessment) • Linearity of response (from 50% of the cleaning limit to 10x this concentration; R2≥0.9900; ) • Precision • Repeatability (RSD ≤5%), • intermediate precision [ruggedness (USP)], and • reproducibility • Limits of detection and quantitation • Accuracy or recovery from rinsate (≥80%), swabs (≥90%), and process surface (≥ 70%) • Range (lowest level is at least 2x higher than LOQ)

42. Main Points Again • Analytical procedures play a critical role in pharmaceutical equivalence and risk assessment/ 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 attentionduring the assessment of dossiers for prequalification.

43. THANK YOU