1. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 1 |1 | Pharmaceutical Development Analytical Method Development Presented by: Birgit Schmauser, pharmacist, PhD Presented by: Birgit Schmauser, pharmacist, PhD

2. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 2 |2 | Analytical method development Objectives of the presentation Originator and multisource generic FPPs Originator and multisource generic FPPs –Equivalence (comparability) Specifications Specifications –setting Stability Stability –assessment Cleaning validation Cleaning validation –Parallel development of analytical methods

3. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 3 |3 | Interchangeability (IC) Interchangeability (IC) of multisource generic FPPs (Essential similarity with Innovator FPP) Pharmaceutical + Bioequivalence Equivalence IC = PE + BE IC = PE + BE

4. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 4 |4 | Pharmaceutical equivalence FPPs meet the same or comparable standards FPPs meet the same or comparable standards –Same API (chemical and physical equivalence) –Same dosage form and route of administration –Same strength –Comparable labeling Equivalence in pharmaceutical development Equivalence in pharmaceutical development Equivalence in stability Equivalence in stability Equivalence in manufacture (WHO-GMP) Equivalence in manufacture (WHO-GMP)

5. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 5 |5 | Prequalification requirements Validation of analytical methods is a prerequisite for prequalification of product dossiers Validation of analytical methods is a prerequisite for prequalification of product dossiers –Non-compendial APIs and FPPs are tested with methods developed by the manufacturer –For compendial APIs and FPPs the „applicability“ of methods to particular products must be demonstrated (verification) Analytical methods must be developed and validated according to ICH Q2 (R1) Analytical methods must be developed and validated according to ICH Q2 (R1) –To be used within GLP and GMP environments

6. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 6 |6 | Use of analytical methods - generics METHODSPHARMACEUTICALCLINICAL At initial phase of pharmaceutical development To understand the profile of related substances and to study stability To 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 product batches To optimise, scale-up and transfer a stable and controlled manufacturing process for the prequalification product To prove bioequivalence after critical variations to the prequalified dossier

7. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 7 |7 | Prerequisites for validation Six “M”s Six “M”s –Man(Qualified personel) –Man (Qualified personel) –Machine (Qualified, calibrated robust instruments) –Methods (Suitable, characterised & documented) –Material (sufficient quality, & Reference standards) –Milieu (Laboratory conditions) –Management ensure by validation enable by suitable surroundings Quality  fitness for use

8. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 8 |8 | Validation characteristics AssayImpuritiesIdentification limitquantitative + -+ -Accuracy + -+ -Precision + ++ +Specificity - +- - Detection Limit - -+ - Quantitation Limit + -+ -Linearity + -+ -Range + ++ +Robustness

9. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 9 |9 | Accuracy and precision Accurate & precise Accurate & imprecise Inaccurate & precise Inaccurate & imprecise

10. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 10 | Precision Expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample Expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample Is usually expressed as the standard deviation (S), variance (S 2 ) or coefficient of variation (RSD) of a series of measurements Is usually expressed as the standard deviation (S), variance (S 2 ) or coefficient of variation (RSD) of a series of measurements Precision may be considered at three levels Precision may be considered at three levels –Repeatability (intra-assay precision) –Intermediate Precision (variability within a laboratory) –Reproducibility (precision between laboratories)

11. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 11 | General consideration Spread of data Spread of data –68.26% of measured values within mean ± 1 SD –95.46% of measured values within mean ± 2 SD –99.73% of measured values within mean ± 3 SD An interval of ± 3 SD should be calculated to fully cover variability ± 3SD Mean ± 1SD ± 2SD

12. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 12 | Repeatability Determination of the API in a FPP (tablet): Determination of the API in a FPP (tablet): –Six replicate sample preparation steps from a homogenously prepared tablet mixture (nominal value of API 150 mg) Assay Peak area Injection 147.10 mg/98.06% 1738651 148.00 mg/98.66% 1749262 146.32 mg/97.54% 1729333 148.08 mg/98.72% 1750114 151.95 mg/101.30% 1795575 149.28 mg/99.52% 1764256 148.45 mg/98.96% 175453Mean 1.98 mg/1.32% 2329SD 1.32%1.32%RSD Mean ± 3 SD = Confidence interval of 99.73% 98.96 ± 3x1.32% = 95% - 102.92%

13. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 13 | Intermediate Precision Expresses within-laboratories variations Expresses within-laboratories variations –Different days, different analysts, different equipment etc. Peak area analyst 3 Peak area analyst 2 Peak area analyst 1 Injection 1779651756561738651 1785561758781749262 1773421760041729333 1780111763441750114 1794661753321795575 1796881749591764256 178504175695175453Mean 9184952329SD 0.51%0.28%1.32%RSD Analyst 1: 98.96% ± 3 x 1.32% Analyst 2: 99.12% ± 3 x 0.28 Analyst 3: 100.70% ± 3 x 0.51 Average of 3 analysts ± 3SD: 95% - 102.23% Mean ± 3 SD: (177252  100%)

14. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 14 | Reproducibility Expresses the precision between laboratories Expresses the precision between laboratories –Collaborative studies, usually applied to standardisation of methodology Transfer of technologyTransfer of technology Compendial methodsCompendial methods

15. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 15 | 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 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 –Sometimes referred to as „TRUENESS“ true mean

16. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 16 | Accuracy To find out whether a method is accurate: Drug substance (assay) Drug substance (assay) –Application of the method to an analyte of known purity (e.g. reference substance) –Comparison of the results of one method with those of a second well- characterised method (accuracy known) Drug product (assay) Drug product (assay) –Application of the method to synthetic mixtures of the drug product component to which known quantities of the analyte have been added Drug product may exceptionally be used as matrixDrug product may exceptionally be used as matrix Drug substance/Drug product (Impurities) Drug substance/Drug product (Impurities) –Application of the method to samples spiked with known amounts of impurities

17. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 17 | Accuracy Application of the method to synthetic mixtures of the drug product component to which known quantities of the analyte have been added Application of the method to synthetic mixtures of the drug product component to which known quantities of the analyte have been added Recovery reduced by ~10 – 15% Source: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan, Herman Lam, Y.C. Lee, and Xue-Ming Zhang ISBN 0-471-25953-5 Wiley & Sons Source: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan, Herman Lam, Y.C. Lee, and Xue-Ming Zhang ISBN 0-471-25953-5 Wiley & Sons

18. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 18 | Specificity Is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present (impurities, degradants, matrix…) Is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present (impurities, degradants, matrix…) Identity testing –To ensure the identity of an analyte Purity testing –To ensure accurate statement on the content of impurities of an analyte Assay –To allow an accurate statement on the content of an analyte in a sample

19. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 19 | Separation of very closely related analytes Specificity Specificity

20. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 20 | Specificity Overlay chromatogram of an impurity solution with a sample solution Source: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan, Herman Lam, Y.C. Lee, and Xue-Ming Zhang ISBN 0-471-25953-5 Wiley & Sons Source: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan, Herman Lam, Y.C. Lee, and Xue-Ming Zhang ISBN 0-471-25953-5 Wiley & Sons

21. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 21 | Specificity and stability Stress stability testing to ensure the stability indicating potential of an analytical method Stress stability testing to ensure the stability indicating potential of an analytical method –Apply diverse stress factors to the API –Apply diverse stress factors to the FPP Stress conditions: e.g. Supplement 2 of Generic Guideline; TRS 929, Annex 5 Assure that the API can be assessed specifically in the presence of known and unknown (generated by stress) impurities Assure that known impurities/degradants can be specifically assessed in the presence of further degradants By peak purity assessment and (overlay of) chromatograms

22. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 22 | Limit of Detection (LOD, DL) The LOD of an analytical procedure is the lowest amount of analyte in sample which can be detected but not necessarily quantitated as an exact value The LOD of an analytical procedure is the lowest amount of analyte in sample which can be detected but not necessarily quantitated as an exact value Determination is usually based on Determination is usually based on –Signal to noise ratio (~3:1) (baseline noise) or –Standard deviation of response (  ) and Slope (S) 3.3  S3.3  S

23. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 23 | Limit of Quantitation (LOQ, QL) The LOQ is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The LOQ is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. –The quantitation limit is used particularly for the determination of impurities and/or degradation products Determination is usually based on Determination is usually based on –Signal to noise ratio (~10:1) (baseline noise) or –Standard deviation of response (  ) and Slope (S) 10  S10  S

24. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 24 | LOD/LOQ Noise LOD Signal to Noise = 3:1 LOQ Signal to Noise = 10:1 LOD, LOQ and Signal to Noise Ratio (SNR)

25. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 25 | LOQ and impurities In determination of impurities in APIs and FPPs the LOQ should be determined in the presence of API In determination of impurities in APIs and FPPs the LOQ should be determined in the presence of API –LOQ should be NMT reporting level –LOQ should be given relative to the test concentration of API Specificity of impurity determination should always be demonstrated in the presence of API at API specification levels Specificity of impurity determination should always be demonstrated in the presence of API at API specification levels –Spiking of test concentration (API/FPP) with impurities at levels of their specification range

26. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 26 | LOQ and impurities Spiking Spiking –API test concentration (normalised) 0.1 mg/ml (100%) 0.1 mg/ml (100%) –Impurity spiking concentrations 0.001 mg/ml (1%) – specification limit 0.001 mg/ml (1%) – specification limit 0.0001 mg/ml (0.1%) – limit of quantitation (minimum requirement) 0.0001 mg/ml (0.1%) – limit of quantitation (minimum requirement)

27. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 27 | Linearity 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 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 –If there is a linear relationship test results should be evaluated by appropriate statistical methods Correlation coefficient (R 2 ) Correlation coefficient (R 2 ) Y-intercept Y-intercept Slope of regression line Slope of regression line Residual sum of squares Residual sum of squares PLOT OF THE DATA PLOT OF THE DATA

28. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 28 | Linearity Usual acceptance criteria for a linear calibration curve Usual acceptance criteria for a linear calibration curve –r > 0.999; y-intercept a 0.999; y-intercept a < 0 to 5% of target concentration; RSD response < 1.5-2% Source:Analytical Method Validation and Instrument Performance Verification, Edited by Chung ChowChan, Herman Lam, Y.C. Lee,and Xue-Ming ZhangISBN 0-471-25953-5 Wiley & Sons Source: Analytical Method Validation and Instrument Performance Verification, Edited by Chung ChowChan, Herman Lam, Y.C. Lee,and Xue-Ming ZhangISBN 0-471-25953-5 Wiley & Sons

29. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 29 | Range The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity

30. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 30 | Range Assay Assay –80 to 120% of test concentration Content uniformity Content uniformity –70 to 130% of test concentration) Dissolution Dissolution –Q-20% to 120% Impurities Impurities –Reporting level – 120% of specification limit (with respect to test concentration of API) Assay & Impurities Assay & Impurities –Reporting level to 120% of assay specification

31. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 31 | Range Linearity is limited to 150% of shelf life specification of impurities Linearity is limited to 150% of shelf life specification of impurities –Test concentration can be used to determine impurities To determine drug substance (assay) the test concentration must be diluted The range is 0 – ~ 150% of impurity specification Source:Analytical Method Validation and Instrument Performance Verification, Edited by Chung ChowChan, Herman Lam, Y.C. Lee, and Xue-Ming Zhang ISBN 0-471-25953-5 Wiley & Sons Source: Analytical Method Validation and Instrument Performance Verification, Edited by Chung ChowChan, Herman Lam, Y.C. Lee, and Xue-Ming Zhang ISBN 0-471-25953-5 Wiley & Sons

32. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 32 | Robustness Robustness of an analytical procedure should show the reliability of an analysis with respect to deliberate variations in method parameters Robustness of an analytical procedure should show the reliability of an analysis with respect to deliberate variations in method parameters The evaluation of robustness should be considered during the development phase The evaluation of robustness should be considered during the development phase If measurements are susceptible to variations in analytical conditions the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure If measurements are susceptible to variations in analytical conditions the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure

33. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 33 | Robustness Influence of buffer pH and buffer concentration in mobile phase on retention times of API and impurities Influence of buffer pH and buffer concentration in mobile phase on retention times of API and impurities Conclusion: The buffer composition should be maintained in a range of 85 ± 0.5% Conclusion: The buffer composition should be maintained in a range of 85 ± 0.5% –Missing: Acceptance criterion for maximal deviation of retention time should be defined unless justified Impurity C Impurity B Impurity A API 8.267.433.8610.46 As is 8.387.513.9410.45 buffer pH 5.9 8.347.493.9410.46 buffer pH 6.9 6.666.163.437.84 Buffer conc. 83% 11.189.614.7715.26 Buffer conc. 87%

34. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 34 | System suitability Testing Based on the concept that equipment, electronics, analytical operations and samples to be analysed constitute an integral system that can be evaluated as such Based on the concept that equipment, electronics, analytical operations and samples to be analysed constitute an integral system that can be evaluated as such System suitability test parameters are established for each analytical procedure individually System suitability test parameters are established for each analytical procedure individually –System suitability parameters depend on the type of analytical procedure

35. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 35 | Method stability System suitability over time System suitability over time Stability of analytical solutions Stability of analytical solutions –Sample solution stability A solution of stavudine is stable for ~ 2 h, then it starts to degrade to thymineA solution of stavudine is stable for ~ 2 h, then it starts to degrade to thymine –Impurity-spiked sample solution stability Cave: A solution containing stavudine spiked with its impurity thymine does not allow to clearly distinguish between degradation and spike due to the lower precision at impurity levels Cave: A solution containing stavudine spiked with its impurity thymine does not allow to clearly distinguish between degradation and spike due to the lower precision at impurity levels –Should be analysed immediately

36. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 36 | Setting Specifications Upper and lower specification limits Upper and lower specification limits –Process variability –Analytical variability ± 3 SD and specification acceptance range Given specification limits/ranges Given specification limits/ranges –Assay Analytical variability Process variabilityAnalytical variability Process variability –Impurities LOQ and specification limit (e.g. qualification limits NMT 0.15%)LOQ and specification limit (e.g. qualification limits NMT 0.15%) –Response factors (LOQ modified by response factor)

37. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 37 | Methods for cleaning validation Method for assay and related substances used in stability studies of API and FPP 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%)Repeatability (RSD ≤ 5%) intermediate precision [ruggedness (USP)]intermediate precision [ruggedness (USP)] reproducibilityreproducibility –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)

38. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 38 | Summary Analytical procedures play a critical role in pharmaceutical equivalence and risk assessment/management Analytical procedures play a critical role in pharmaceutical equivalence and risk assessment/management –Establishment of product-specific acceptance criteria –Assessment of stability of APIs and FPPs Validation of analytical procedures should demonstrate that they are suitable for their intended use Validation of analytical procedures should demonstrate that they are suitable for their intended use Validation of analytical procedures deserves special attention during assessment of dossiers for prequalification Validation of analytical procedures deserves special attention during assessment of dossiers for prequalification

39. Training Workshop on Pharmaceutical Development with a Focus on Paediatric Medicines / 15-19 October 2007 39 | THANK YOU