6 th November 2002

1. Method Validation in the Pharmaceutical IndustryKate Arnot Analytical Development, PAR&D (AW) 6th November 2002

2. Overview • The drug development process • General recommendations • Examples • AssayDissolution test for an immediate release tablet • IdentityIdentification test for a drug substance • Impurities Quantitative test – residual solvents in a drug substance Limit test – residual solvents in a tablet

3. DRUG DEVELOPMENT PROCESS Years 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Start Approval Phase 4 Clinical Trials Phase 1 Phase 2 Phase 3 50-100 people 100-200 people 500-5000 people Full Scale Manufacture Initial research Drug discovery Clinical Development Toxicology and Pharmacokinetics Chemical development Sales and Marketing Pharmaceutical development £150M £300M £2M Cost 10,000 compounds 1 new medicine

4. A phased approach to analytical validation • Phase I – Limited validation, focussing on key method attributes eg.specificity, limits of quantitation and linearity • Phase II – Starting to include accuracy and precision data to support specifications • Phase III – Full validation according to ICH guidelines will be completed for all analytical methods prior to submission of marketing applications

5. General recommendations • Design your validation studies to reflect the way that the method will be applied in routine use as far as possible • Ensure that you have considered the specification you want to achieve in designing your studies • Know your method before you start to validate!

6. Assay - Dissolution test for an immediate release tablet • Dissolution testing is used to evaluate the release of drug substance from tablets • Dissolution apparatus 2 (paddles), 50rpm, 37°C, aqueous surfactant as dissolution medium, UV analysis, sampling at a single 45 minute timepoint • Typical Specification: Q = 80 at 45 minutes (ie each of 6 units should achieve 85% dissolution)

7. Dissolution specification – USP acceptance table • S1, 6 unitsEach unit is not less that Q+5% • S2, 6 unitsAverage of 12 units (S1+S2) is equal to or greater than Q and no unit is less than Q-15% • S3, 12 unitsAverage of 24 units (S1+S2+S3) is equal to or greater than Q, not more than 2 units are less than Q-15%, and no unit is less than Q-25%

8. Dissolution apparatus

9. Typical dissolution profile % Dissolution Time (mins)

10. Specificity Linearity Accuracy and range Precision Repeatability Intermediate precision Robustness Validation parameters evaluated

11. Specificity • Demonstrate absence of interference from excipients and reagents at the monitoring wavelength • For example calculate the absorbance of a placebo solution as a percentage of the absorbance of a standard solution • Typically results of less that 2% would be considered acceptable • Specificity may also be inferred from linearity and accuracy data

12. Linearity • Range studied 0 to 150% of nominal (7 solutions, including a ‘blank’ solution) • Linearity experiments conducted in the presence and absence of excipients • Determine equation of the line, correlation coefficient, 95% confidence interval of the slope and the intercept. Verify that the 95% confidence interval of the intercept includes zero, or that the result is insignificant in the context of the experiment • Check that the response for both experiments is the same – calculate the slopes as a % of each other • Typically results of 98 to 102% would be considered acceptable

13. Linearity in the presence of placebo y = 42.983x + 0.004r = 0.9998995% CI of slope 0.073095% CI of intercept 0.016 Absorbance Concentration (g/100ml)

14. Linearity in the absence of placebo y = 43.033x + 0.006r = 0.9999095% CI of slope 0.69595% CI of intercept 0.009 Absorbance Comparison of slopes:(42.983/43.033) x 100= 99.9% Concentration (g/100ml)

15. Accuracy and Range • 3 samples at each of 5 different concentration levels were evaluated: 50%, 85%, 100%, 115% and 125% of nominal • Samples were analysed against standards as per the method and % recoveries calculated • Typically results in the range 95 to 105% would be considered acceptable • The overall standard deviation and relative standard deviation may be used as an estimate of precision

16. Experiment 1 50% 97.3 85% 96.8 100% 96.5 115% 96.0 125% 95.8 Mean 96.5 sd 0.7 Experiment 2 50% 99.3 85% 99.4 100% 99.6 115% 98.7 125% 98.9 Mean 99.2 sd 0.5 Example Accuracy Data

17. Example Accuracy Data - continued • Experiment 1 • Samples mixed, and allowed to stand overnight • Experiment 2 • Samples stirred continuously Time 1 Recovery 98.8% (hours) 4 99.1% 21 99.2% 27 99.3%

18. Accuracy/Linearity – how do you prepare your samples? • Prepare samples containing drug substance plus an appropriate quantity of ground placebo tablet • Prepare samples containing drug substance plus appropriate quantities of an excipient mix • Prepare samples by using different weights from a ground active tablet preparation, making a correction for nominal concentration • Make sure that you are confident in your sample extraction technique

19. Precision - Repeatability • One analyst conducting multiple replicates of the same sample, on the same day using the same equipment, and applying the method as written. • For dissolution experiments correcting for tablet weight may be appropriate • Determine the mean, the range, the standard deviation and the relative standard deviation.

20. Precision – Intermediate precision • A second operator conducts the ‘repeatability experiment’ on a different day using different equipment where possible. • Determine the individual data and overall mean, range, standard deviation and relative standard deviation. • Consider testing statistically that there is no significant difference between operators before combining the data set.

21. Robustness • A number of attributes were evaluated prior to validation: • Standard and sample solution stability • Effect of pH and the requirement to buffer and/or ‘degass’ the dissolution medium • Reagents from different suppliers were evaluated • Sampling tubes, filters and sampling and filtration speeds were evaluated • Single point vs profile testing

22. Identification – Infrared method for a drug substance • Infrared spectroscopy is frequently used in identification testing • Sample is dispersed in KBr, analysed using the DRIFTS technique, and the spectrum obtained visually compared with that of a fully characterised reference standard analysed under the same conditions. • Typical Specification: Result consistent with reference standard

23. Specificity Identification testing should optimally be able to discriminate between compounds of closely related structure which are likely to be present (ICH Q6A) The choice of such potentially interfering materials should be based on sound scientific judgement with a consideration of the interferences that could occur (ICH Q2B) Validation parameters evaluated

24. Specificity • Demonstrate specificity between drug substance and other potentially interfering compounds • eg. starting materials, isolated intermediates and other materials manufactured or formulated in the same plant • Consideration should also be given to enantiomers, counter ions/salts, solvates and polymorphs if necessary.

25. Drug substance Developmentcompound Compound formulatedin same plant Isolatedintermediate

26. Impurities – Quantitative test for residual solvents in drug substance • A range of solvents are used in the manufacture of a drug substance. It is necessary to quantify these, and demonstrate that they comply with ICH Q3C requirements • Capillary GC method with flame ionisation detection. • Typical Specification: 0.5% w/w maximum

27. Specificity Linearity Accuracy Precision Reproducibility Intermediate precision Range Limits of detection and quantitation Robustness Validation parameters evaluated

28. Specificity • Demonstrate method is specific for all the solvents used in the registered synthetic process • Can use linearity data for solvents in the presence and absence of the drug substance to show that the drug substance does not interfere

29. Specificity chromatogram

30. Linearity • Range studied 0 to 150% of nominal (9 solutions, including a ‘blank’ solution and a solution at the LOQ) • Drug substance required without solvent, or low levels of solvent and then make a correction • Determine equation of the line, correlation coefficient, 95% confidence interval of the slope and the intercept. Verify that the 95% confidence interval of the intercept includes zero, or that the result is insignificant in the context of the experiment

31. Accuracy • Calculate recoveries from the linearity experiments by analysing the solutions against an external standard as per the method. • Typically results in the range 80 to 120% would be considered acceptable • The overall standard deviation and relative standard deviation may be used as an estimate of precision

33. Intermediate precision - Japanese style Expt No 1 2 3 4 5 6 7 8 Operator 1 1 1 1 2 2 2 2 GC 1 2 2 1 1 2 2 1 Column 1 2 1 2 1 2 1 2 Experiments are conducted on different days with each analyst using each instrument/column combination in turn Report results, overall mean, overall standard and relative standard deviation, and 95% CI of sd

34. Range • Determined from the linearity, accuracy and precision data

35. Limit of Quantitation and Detection • Limit of quantitation established as 0.05% w/w • Samples at this concentration included in linearity and accuracy experiments • Limit of detection estimated as 0.02% w/w by visual inspection of chromatograms

36. LOQ chromatogram

37. Robustness • A number of attributes were evaluated prior to validation using an experimental design approach: • Injector temperature • Detector temperature • Initial temperature • Ramp rate • Hold time • Split flow • Column head pressure

38. Impurities – Limit test for residual solvent in a tablet • An alcohol is used as a preservative in a purchased film coating concentrate. It is removed during the manufacturing process, but this needs to be demonstrated • Fast GC method with flame ionisation detection. • Need to demonstrate that tablets comply with requirements for Class 3 solvents ie less than 0.5% w/w

39. Specificity Limit of quantitation Linearity For solvents arising from the drug substance and tablet manufacturing process 0.05% w/w 0.05% to 0.5% w/w Validation parameters evaluated

40. References • ICH – www.ICH.org • International Pharmaceutical Product Registration – Aspects of Quality, Safety and Efficacy, eds Cartwright and Matthews, Ellis Horwood, 1994, Chapter 8, Analytical validation • The validation of analytical methods for drug substances and drug products in UK pharmaceutical laboratories, G S Clarke, J. Pharm. Biomed. Anal., 12. 643 – 652 (1994) • FDA – www.FDA.gov • Technical review guide: Validation of chromatographic methods