1. | Slide 1 of 43 April 2007 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 Pharmaceutical Development

2. | Slide 2 of 43 April 2007 Pharmaceutical Development Analytical Method Development Presenter: János Pogány, pharmacist, PhD pogany.janos@chello.hu WHO expert

3. | Slide 3 of 43 April 2007 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. | Slide 5 of 43 April 2007 Interchangeability (IC) INTERCHANGEABILITYIC WITH INNOVATOR FPP INTERCHANGEABILITY (IC) OF MULTISOURCE FPPs = (ESSENTIAL SIMILARITY WITH INNOVATOR FPP) = PHARMACEUTICAL EQUIVALENCEPE PHARMACEUTICAL EQUIVALENCE (PE) + BIOEQUIVALENCE (BE) ICPE IC = PE + BE

6. | Slide 6 of 43 April 2007 Pharmaceutical equivalence same or comparable standards analytical methods  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 –Same strength –Comparable labeling  Pharmaceutical development equivalence  Stability equivalence  WHO-GMP (manufacturing equivalence)

7. | Slide 7 of 43 April 2007 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. | Slide 8 of 43 April 2007 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 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 http://www.ich.org/LOB/media/MEDIA417.pdf

9. | Slide 9 of 43 April 2007 General requirements  Qualified and calibrated instruments  Documented methods  Reliable reference standards  Qualified analysts  Sample selection and integrity  Change control

10. | Slide 10 of 43 April 2007 Measure of variation (spread of data) 95.46% 68.26%

11. | Slide 11 of 43 April 2007 Mean (average) chart Normal variation due to common causes USL Upper specification limit LSLLower specification limit Abnormal variation of process – special causes average = mean

12. | Slide 12 of 43 April 2007 Capable process Almost all the measurements of a stable process fall inside the specification limits USL – LSL 6 8 10 12 Cp 1.00 1.33 1.66 2.00 OoS results:.27%. 6 ppm 64 ppm 2 ppb http://www.itl.nist.gov/div898/handbook/pmc/section1/pmc16.htm

13. | Slide 13 of 43 April 2007 NEVIRAPINE – Reference Standard AtRtR Injection 124666.1601 123116.1672 124326.1663 125306.1724 124576.1655 124796.1686 124466.166Mean 740.004STD 0.59%0.06%RSD System suitability requirement: RSD is NMT 0.85%

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

15. | Slide 15 of 43 April 2007 Use of analytical methods - generics MethodsPharmaceuticalClinical At initial phase of pharmaceutical development -To understand the profile of related substances and to study stability and start measuring the impact of key product and manufacturing process parameters on consistent FPP quality To develop a stable and reproducible formulation for the manufacture of bioequivalence, dissolution, stability and pilot-scale validation batches To determine bioavailability in healthy volunteers At advanced phase of pharmaceutical development To be robust, transferable, accurate, and precise for specification setting, stability assessment, and QC release of prequalified batches To optimize, scale-up, and transfer a stable and controlled manufacturing process for the prequalification product To prove bioequivalence after critical variations to the prequalified dossier

16. | Slide 16 of 43 April 2007 Analytical procedure characteristics Type of characteristic IdentificationImpurities QuantitativeLimit Assay Accuracy-+-+ Precision-+-+ Specificity++++ Detection limit--+- Quantitation limit-+-- Linearity-+-+ Range-+-+ Robustness++++

17. | Slide 17 of 43 April 2007 Accuracy - ISO 5725 1-6 Accuracy Trueness Systematic errors Precision (Random errors) Intra-assay variability Repeatability Intra-laboratory variability Intermediate precison Inter-laboratory variability Reproducibility Source: ISO. 1994. ISO 5725 1-6: Accuracy (Trueness and Precision) of Measurement Methods and Results. ISO, Geneva, Switzerland.

18. | Slide 18 of 43 April 2007 Accuracy and precision Accurate and precise Precise Accurate Inaccurate and imprecise

19. | Slide 19 of 43 April 2007 Percent accuracy (hypothetical figures) The data show that the recovery of analyte in spiked samples met the evaluation criterion for accuracy (100 ± 2.0% across 50–130% of target concentrations). RSD % Recovery % Nevirapine, mg LA, %Sample RecoveredAdded 0.6499.20.4950.499501 0.3199.80.7010.703702 0.2799.90.7950.796803 1.88100.41.0051.0011004 0.3899.81.2091.2111205 1.1299.81.2961.2991306 0.7799.80.9170.918Mean

20. | Slide 20 of 43 April 2007 Percent accuracy (hypothetical figures) Red line: LA Green lines: USL and LSL

21. | Slide 21 of 43 April 2007 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, RSD%.) of a series of measurements. The confidence interval (CI) should be reported for each type of precision investigated. Measured meanReal mean Measured mean Real mean PRECISION

22. | Slide 22 of 43 April 2007 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

23. | Slide 23 of 43 April 2007 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 criterion of RSD ≤2%. Imp1Peak areaInjection 0.301579351 0.301578332 0.299574973 0.300576174 0.301577785 0.298572316 0.30057649Mean 0.0013257STD 0.4% RSD 0.001427095% CI

24. | Slide 24 of 43 April 2007 Intermediate Precision and Reproducibility ( of any process ) 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

25. | Slide 25 of 43 April 2007 Intermediate precision (ruggedness) Assay (mg/5ml)Sample 52.651.71 52.151.92 52.353.03 52.952.54 53.252.35 53.152.76 52.4Mean 0.440.49STD 0.8%0.9%RSD 0.460.5195% CI Combined values 52.5Mean 0.48STD 0.9%RSD 0.3195% CI

26. | Slide 26 of 43 April 2007 Specificity (selectivity) impurities, degradants and excipients  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. | Slide 27 of 43 April 2007 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. | Slide 28 of 43 April 2007 Specificity (hypothetical figures and data) HPLC chromatograms of (a) API reference standard, (b) FPP and (c) placebo

29. | Slide 29 of 43 April 2007 SPECIFICITY – degradants Purity thresholdPurity angleA (%) *Stress 0.2800.040 100.0Initial 0.3800.105 1.6300.725 0.2800.045 99.3Acid 0.4100.120 1.6101.040 0.2700.060 99.8Peroxide 0.3600.110 1.2500.690 NA 100.0All others 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. | Slide 30 of 43 April 2007  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

31. | Slide 31 of 43 April 2007 LOD and LOQ (hypothetical figures) Impurity 2Impurity 1 Injection LOQLODLOQLOD 78923497723541761 77914258809936082 82923275795041963 80503464816643034 83684008784739325 82844702841552386 8113386779524242Mean 238551402548STD 2.9%14.3%5.1%12.9%RSD 0.2140.1070.1710.086Conc. (μg/ml) 0.0390.0190.0330.017Conc. (%w/w)

32. | Slide 32 of 43 April 2007 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), usually between 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. | Slide 33 of 43 April 2007 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 Measured Real mean mean mean Precision

34. | Slide 34 of 43 April 2007 Linearity and range Acceptance criterion: correlation coefficient should not be less than 0.9990

35. | Slide 35 of 43 April 2007 Linearity and range  Concentration range 1.0–1.3 mg/ml (10–130% of the theoretical concentration in the test preparation, n=3)  Regression equation was found by plotting the means of peak area (y) against the analyte concentration (x) expressed in %: y = 36.124x - 7.2984 (R 2 = 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. | Slide 36 of 43 April 2007 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

37. | Slide 37 of 43 April 2007 Stability of analytical solution variation Stability (of the analytical solution) expresses variation of the measured mean as a function of time. #1 … First measurements same sample within a relatively short period of time. #2, #3, #4, …n Series of measurements of the same sample within a relatively short period of time. Measured means Stability Measured means

38. | Slide 38 of 43 April 2007 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. Impurity-1 Time in hours DifferenceArea 720790 0.7%715741 0.5%717402 0.2%719603 -0.4%723524 0.7%715735 -0.3%7232210 -0.3%7231015 -0.3%7231220 -0.8%7267025

39. | Slide 39 of 43 April 2007 Sensitivity and robustness

40. | Slide 40 of 43 April 2007 Robustness tRtR VariationMethod parameter Impurity 2Impurity 1 1.800.83- STP 1.810.83-10% Flow 1.820.8410% 1.810.82-5nm Wavelength 1.810.83+5nm 1.890.80-2% Variation of mobile phase 1.760.84+2% 1.800.82-5 o C Column temperature 1.810.83+5 o C 1.810.83-0.3 pH 1.800.83+0.3

41. | Slide 41 of 43 April 2007 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; R 2 ≥ 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. | Slide 42 of 43 April 2007 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 attention during the assessment of dossiers for prequalification.

43. | Slide 43 of 43 April 2007 THANK YOU