Pharmaceutical Development

1. Pharmaceutical Development Analytical Method Development Presented by: Birgit Schmauser, pharmacist, PhD

2. Analytical method development Objectives of the presentation • Originator and multisource generic FPPs • Equivalence (comparability) • Specifications • setting • Stability • assessment • Cleaning validation • Parallel development of analytical methods

3. Interchangeability (IC) Interchangeability (IC) of multisource generic FPPs (Essential similarity with Innovator FPP) Pharmaceutical + Bioequivalence Equivalence IC = PE + BE

4. Pharmaceutical equivalence • 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 stability • Equivalence in manufacture (WHO-GMP)

5. Prequalification requirements • 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 methodsto particular products must be demonstrated (verification) • Analytical methods must be developed and validated according to ICH Q2 (R1) • To be used within GLP and GMP environments

6. Use of analytical methods - generics

7. Prerequisites for validation Quality  fitness for use • Six “M”s • 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 suitablesurroundings

8. Validation characteristics

9. Accuracy and precision Accurate & precise Accurate & imprecise Inaccurate & precise Inaccurate & imprecise

10. Precision • 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(S2) or coefficient of variation(RSD) of a series of measurements • Precision may be considered at three levels • Repeatability(intra-assay precision) • Intermediate Precision(variability within a laboratory) • Reproducibility(precision between laboratories)

11. General consideration Mean ± 1SD ± 2SD ± 3SD • Spread of data • 68.26% of measured valueswithin mean ± 1 SD • 95.46% of measured valueswithin mean ± 2 SD • 99.73% of measured valueswithin mean ± 3 SD • An interval of ± 3 SD shouldbe calculated to fully cover variability

12. Repeatability • 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) Mean ± 3 SD =Confidence interval of 99.73% 98.96 ± 3x1.32% = 95% - 102.92%

13. Intermediate Precision • Expresses within-laboratories variations • Different days, different analysts, different equipment etc. Mean ± 3 SD: (177252  100%) 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%

14. Reproducibility • Expresses the precision between laboratories • Collaborative studies, usually applied to standardisation of methodology • Transfer of technology • Compendial methods

15. Accuracy • Expresses the closeness of agreementbetween the value which is accepted either as a conventional true valueor an accepted reference value and the value found • Sometimes referred to as „TRUENESS“ true mean

16. Accuracy To find out whether a method is accurate: • 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) • 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 matrix • Drug substance/Drug product (Impurities) • Application of the method to samples spiked with known amounts of impurities

17. Accuracy • Application of the method to synthetic mixtures of the drug product component to which known quantities of the analyte have been added • Recovery reducedby ~10 – 15% • Source: Analytical MethodValidation and InstrumentPerformance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. Lee,and Xue-Ming ZhangISBN 0-471-25953-5Wiley & Sons

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…) • 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. Separation of very closely related analytes • Specificity

20. Specificity • Overlay chromatogram of an impurity solution with a sample solution • Source: Analytical MethodValidation and InstrumentPerformance Verification, Edited by Chung ChowChan, Herman Lam,Y.C. Lee,and Xue-Ming ZhangISBN 0-471-25953-5Wiley & Sons

21. Specificity and stability • 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. 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 • Determination is usually based on • Signal to noise ratio (~3:1) (baseline noise) or • Standard deviation of response (s) and Slope (S) • 3.3 s/S

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 quantitation limit is used particularly for the determination of impurities and/or degradation products • Determination is usually based on • Signal to noise ratio (~10:1) (baseline noise) or • Standard deviation of response (s) and Slope (S) • 10 s/S

24. LOD/LOQ LOD, LOQ and Signal to Noise Ratio (SNR) LOQ Signal to Noise = 10:1 LOD Signal to Noise = 3:1 Noise

25. LOQ and impurities • 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 • Spiking of test concentration (API/FPP) with impurities at levels of their specification range

26. LOQ and impurities • Spiking • API test concentration (normalised) • 0.1 mg/ml (100%) • Impurity spiking concentrations • 0.001 mg/ml (1%) – specification limit • 0.0001 mg/ml (0.1%) – limit of quantitation (minimum requirement)

27. Linearity • The linearity of an analytical procedure is its ability(within a given range) to obtain test resultswhich 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 (R2) • Y-intercept • Slope of regression line • Residual sum of squares • PLOT OF THE DATA

28. Linearity • Usual acceptance criteria for a linear calibration curve • r > 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

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

30. Range • Assay • 80 to 120% of test concentration • Content uniformity • 70 to 130% of test concentration) • Dissolution • Q-20% to 120% • Impurities • Reporting level – 120% of specification limit (with respect to test concentration of API) • Assay & Impurities • Reporting level to 120% of assay specification

31. Range • Linearity is limited to 150% of shelf life specification of impurities • Test concentration can beused to determine impurities • To determine drug substance(assay) the test concentrationmust be diluted • The range is 0 – ~ 150% ofimpurity specification • 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

32. Robustness • Robustness of an analytical procedure should show the reliabilityof an analysis with respect to deliberate variations in method parameters • 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

33. Robustness • 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% • Missing: Acceptance criterion for maximal deviation of retention time should be defined unless justified

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 • System suitability test parameters are established for each analytical procedure individually • System suitability parameters depend on the type of analytical procedure

35. Method stability • System suitability over time • Stability of analytical solutions • Sample solution stability • A 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 thyminedoes not allow to clearly distinguish between degradation and spike due to the lower precision at impurity levels • Should be analysed immediately

36. Setting Specifications • Upper and lower specification limits • Process variability • Analytical variability • ± 3 SD and specification acceptance range • Given specification limits/ranges • Assay • Analytical variability Process variability • Impurities • LOQ and specification limit (e.g. qualification limits NMT 0.15%) • Response factors (LOQ modified by response factor)

37. 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)] • 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)

38. Summary • Analytical procedures play a critical role in pharmaceutical equivalence and risk assessment/management • Establishment of product-specificacceptance 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 deserves special attention during assessmentof dossiers for prequalification

39. THANK YOU