Prequalification programme: Priority essential medicines

1. Prequalification programme:Priority essential medicines Training Workshop for evaluators from National Medicines Regulatory Authorities in the East African Community: Evaluation of quality and interchangeability of medicinal products. Dar Es Salaam United Republic of Tanzania 10 – 14 September 2007

2. Training Workshop on Evaluation of quality and interchangeability of medicinal products. ANALYTICAL VALIDATION Presenter: Drs. J. Welink Senior pharmacokineticist Medicines Evaluation Board, NL WHO adviser E-mail: j.welink@cbg-meb.nl

3. History Development pharmacokinetics: computers separation technics analytical methods

4. History '30 ‘50 ‘60 ‘70 ‘90 pg/ml mass spectrometry ng/ml liquid chromatography gas chromatrography μg/ml chromatography mg/ml spectrometry

5. Methods ANALYTICAL METHODS LC-MS/MS immunological methods GC-MS GLC HPLC

6. Principle

7. Guidance • FDA Guidance for Industry • Bioanalytical method validation, May 2001 • ICH Guidance for industry • Validation of analytical methods: definitions and terminology, June 1995 • Validation of analytical procedures: methodology, November 1996

8. GCP/GLP • GCP/GLP compliance • Clinical studies have to be performed under conditions complying with the principles of Good Clinical Practice, and for analytical methods and sample data handling conditions complying with the principles of Good Laboratory Practice are required. • For older studies without statement of complinace with the above mentioned principles, the assessor should rely on the quality of the submitted report.

9. Choices of methods • LC-MS-MS • GC-MS • HPLC • GLC • Immunological methods

10. Choices of methods • Method used for the determination of drugs and/or metabolites should be: Sensitive Accurate Discriminative Precise

11. Sensitivity • Method should be able to quantify the drug in the sampled specimen at least 10 % of the maximum concentration reached after dosing. Limit of Quantification (LOQ): 1/10 Cmax

12. Discriminative • The method should be able to discriminate between the selected analyte and interfering compounds from the environment or from other compounds administered simultaneously

13. Accuracy • The method must be accurate enough to measure the true value (concentration) of the analyte in a relative small sample

14. Precision • The analytical method should be presice enough to reveal identical results when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of the biological matrix

15. Validation To measure is to know!

16. Validation • Range • Accuracy • Precision • Robustness • Specificity • Detection limit (LOD) • Quantification limit (LOQ) • Linearity

17. Validation-specificity • Investigation of specificity should be conducted during the validation phase of the assay • The procedures used to demonstrate specificity should be clearly reported • Must be applied with structurally similar materials • Choices base on scientific judgements

18. Validation-specificity

19. Validation-LOD • Various methods possible visual evaluation • minimum level at which the analyte can be detected reliably signal-to noise • 3:1 ratio is acceptable standard deviation of the slope and response • LOD = 3.3 σ / S • σ = standard deviation of the response • S = slope of the calibration curve

20. Validation-LOQ • Based on signal-to noise • Reliable quantification is a 10:1 ratio • Based on SD of the response and the slope • LOQ = 10 σ / S • σ = standard deviation of the response • S = slope of the calibration curve

21. Validation-LOD/LOQ Recommended data: • The LOD and LOQ and the method used for the LOQ should be presented • The limits should be validated by the analyses of a suitable number of samples prepared at the LOD and LOQ limits

22. Validation-LOD

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

24. Validation-linearity • Should be evaluated across the range of concentrations expected during the study • A minimum of five concentrations used in the range is recommended • The correlation coefficient, y-intercept slope of the regression and residual sum of squares should be submitted • Deviations from the regression line should be analysed for evaluating linearity

25. Validation-linearity

26. Validation-range • The specified range is derived from linearity studies and should cover the extremes of the concentrations probably reached during the study • The range should be justified in the report based on scientific information

27. Validation-accuracy • Accuracy should be assessed on samples spiked with known amounts of the analyte • Accuracy should be assessed using determinations over a minimum of 3 concentration levels (low, medium and high) • Accuracy should be reported as percent recovery from the added amount and with confidence intervals

28. Validation-accuracy HQC MQC LQC

29. Validation-precision • Repeatability • concentrations covering the specified range • Intermediate precision • Like days, analysts, equipment • Reproducibility • Determined if analyses take place in separate periods • Recommended data • SD, Coefficient of variations, and confidence intervals should be reported on each type of precision

30. Validation-accuracy/precision Accuracy/precision:

31. Validation-accuracy/precision Between-day: Intra-day:

32. Validation-accuracy/precision: Accuracy/precision calibrators:

33. Validation-accuracy/precision FDA Accuracy Precision within-run between-run within-run between-run normally: <15% LLOQ: <20% normally: <15% LLOQ: <20%

34. Validation-robustness • Robustness should be considered during development phase • Shows the reliability of the analytical method with respect to variations in the method parameters • In case variations occur they should be suitably controlled and if present adequately tested and documented

35. Validation-robustness Typical examples: • Stability of the analytical solutions • Influence of variations of pH of the mobile phase • Influence of variations of mobile phase composition • Influence of temperature and flow rate • Extraction conditions • pH and extraction time

36. Validation-robustness

37. Validation-recovery Recovery: • Extraction efficiency analytical method • consistent • precise • reproducible Recovery: 80% 75% 91% 97% 65% 73% Recovery: 15% 16% 13% 15% 16% 14% mean: 81.1% CV: 14.7% mean: 14.8% CV: 7.9%

38. Validation-stability Stability assessed prior sample analysis! • Required data • Freeze and thaw stability • Short term temperature stability • Long term stability • Stock solution stability • Post preparation stability

39. Analysis clinical samples • The analytical method should be validated before the start of obtaining clinical samples. • Each analytical run should contain sufficient QC samples at the beginning, middle and end at at least 3 levels (LQC, MQC and HQC). QC QC QC QC QC QC

40. Analysis clinical samples • Acceptation or rejection of a run should be predefined before the actual start of the analysis of the clinical samples. FDA criteria QC QC QC QC QC QC

41. Analysis clinical samples • All samples of 1 subject in 1 run • Subject sample reanalysis should be predefined before the actual start of the analysis of the clinical samples. Reasons: - improper sample injection - mailfunction - concentration > HLOQC - unexpected value - PK reason QC QC QC QC QC QC

42. Analysis clinical samples - unexpected value - PK reason

43. Report • All methods should be covered by adequate Standard Operating Procedures (SOP’s) for general and analysis specific procedures • Before the start of an analytical procedure an adequate study plan has to be written or be incorporated in the study protocol

44. Report • A specific detailed description of the bioanalytical method should be written • All experiments used to make claims or draw conclusions should be presented in the report • GLP compliance/inspections/audits

45. Report • The following data are required on the report: • 1) Author(s) and their affiliation • 2) Name of the institute or company where the investigations have been performed. • 3) Date of publication analytical study • 4) Identification number of the report. • N.B. The report should preferably be printed on original marked paper of the applicant or of the institute where the analysis has been performed.

46. Report • * For which compounds are the samples analysed (active substance, active and/or quantitatively important metabolite) • * Sample pre-treatment and extraction. • * Analytical method is used. • * Source of the analytical method • - references from literature • - modifications in the procedure. • * Validation of the analytical method • - minimal detectable concentration, stability, reproducibility • - linearity, precision, accuracy, selectivity, sensitivity • - inter- and intraday variability • All individual measurements have to be presented in the report!

47. Report • Analysis of subject samples in a separate report • * Reference to validation report • * Handling samples • * Set up analytical run • * Within study validation results • * Re-analysis • * Chromatograms • * Identification results • All individual measurements have to be presented in the report!

48. Example Accuracy/precision: normally: <15% LLOQ: <20%

49. End Be organised!