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Method Validation - ICH /USP Validation, Linearity and Repeatability

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Prepared by : Santram Rajput (Technical Manager)

Validation of analytical procedures reinforce the reliability and suitability of a methodology for providing accurate and precise results. This slide show elaborates in detail about the need for method validation with examples, along with that it also covers the factors to be evaluated prior to validation. This slide show further touches upon the characteristics which are of significance in context of the validation procedure.

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Method Validation - ICH /USP Validation, Linearity and Repeatability

  1. 1. 1 2016 Method Validation Prepared by : Santram Rajput (Technical Manager) Sigma Test & Research Centre
  2. 2. European and International regulatory bodies and their guidelines on different aspects of QA Body Full name Guidance on Eurachem Focus for Analytical Chemistry in Europe Method validation CITAC Cooperation of International Traceability in Analytical Chemistry Proficiency testing Quality Assurance EA European Cooperation for Accreditation Accreditation CEN European Committee for Normalization Standardization IUPAC International Union of Pure & Applied Chem. Method validation ISO International Standardization Organisation Standardisation AOAC ILAC Association of Official Analytical Chemists International Laboratory Accreditation Cooperat. Internal qual. Control Proficiency testing Accreditation FDA US Food and Drug Administration Method validation USP United States Pharmacopoeia Method validation ICH International Conference on Harmonization Method validation 22016
  3. 3. Method Validation  Validation of analytical procedures is the process of determining the suitability of a given methodology for providing useful analytical data. J. Guerra, Pharm. Tech. March 1986  Validation is the formal and systematic proof that a method compiles w i t h t h e r e q u i r e m e n t s f o r t e s t i n g a p r o d u c t w h e n observing a defined procedures. G. Maldener, Chromatographia, July 1989 32016
  4. 4.  Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, strength, quality, purity and potency of the substances in products.  Method validation is primarily concerned with: identification of the sources of potential errors quantification of the potential errors in the method  An method validation describes in mathematical and quantifiable t e r m s t h e p e r f o r m a n c e c h a r a c t e r i s t i c s o f a n a s s a y 42016
  5. 5. Examples of Methods That Require Validation Documentation  Chromatographic Methods - HPLC, GC, TLC, GC/MS, etc. Pharmaceutical Analysis - In support of CMC. Bioanalytical Analysis - In support of PK/PD/Clinical Studies.  Spectrophotometric Methods – UV/VIS, IR, AAS, XRD, ICP-MS, AAS, XRF, etc  Particle Size Analysis Methods - Laser, Microscopic, Sieving, SEC, etc.  Automated Analytical Methods - Robots, Automated Analysis. 52016
  6. 6. Considerations Prior to Method Validation Suitability of Instrument  Status of Qualification and Calibration Suitability of Materials  Status of Reference Standards, Reagents, Placebo Lots Suitability of Analyst  Status of Training and Qualification Records Suitability of Documentation  Written analytical procedure and proper approved protocol with pre-established acceptance criteria 62016
  7. 7. Validation Step  Define the application, purpose and scope of the method.  Analytes? Concentration? Sample matrices?  Develop a analytical method.  Develop a validation protocol.  Qualification of instrument.  Qualify/train operator  Qualification of material.  Perform pre-validation experiments.  Adjust method parameters and/or acceptance criteria if necessary.  Perform full validation experiments.  Develop SOP for executing the method in routine analysis.  Document validation experiments and results in the validation report. 72016
  8. 8. Purpose of Method Validation  Identification of Sources and Quantitation of Potential errors  Determination if Method is Acceptable for Intended Use  Establish Proof that a Method Can be Used for Decision Making  Satisfy Requirements 82016
  9. 9. What is not Analytical Method Validation?  Calibration The Process of Performing Tests on Individual System Components to Ensure Proper function For example) HPLC Detector calibration  Wavelength Accuracy/ Linear Range/ Noise Level/ Drift 92016
  10. 10.  System Suitability Test to verify the proper functioning of the operating system, i.e., the electronics, the equipment, the specimens and the analytical operations.  Minimum Resolution of 3.0 between the analyte peak and internal standard peaks  Relative Standard Deviation of replicate standard injections of not more than 10.0% 102016
  11. 11. 11 System Suitability Sample Validation MethodAnalyst Calibration Pump Detector Injector Data System 2016
  12. 12. Method Life Cycle 12 Validation Development Optimization 2016
  13. 13. Verification vs. Validation  Compendial vs. Non-compendial Methods  Compendial methods-Verification  Non-compendial methods-Validation requirement 132016
  14. 14. Published Validation Guidelines  1978 Current Good Manufacturing Practices (cGMPs)  1987 FDA Validation Guideline  1989 Supplement 9 to USP XXI  1994 CDER Reviewer Guidance: Validation of Chromatographic Method  1995 ICH Validation Definitions: Q2A, Text on Validation of Analytical procedures  1997 ICH Validation Methodology: Q2B, Validation of Analytical Procedures: Methodology  1999 Supplement 10 to USP 23 <1225>: Validation of Compendial Methods  1999 CDER “Bioanalytical Method Validation for Human Studies”  2000 CDER Draft “Analytical Procedures and Method Validation” 142016
  15. 15.  The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose 15 ICH Guideline for Industry Q2A, Text on Validation of Analytical Procedures March 1995 2016
  16. 16.  In practice, it is usually possible to design the experimental work such that the appropriate validation characteristics can be considered simultaneously to provide a sound, overall knowledge of the capabilities of the analytical procedure, for instance: Specificity, Linearity, Range, Accuracy, and Precision. 16 ICH Guideline for Industry Q2B, Validation of Analytical Procedures: Methodology 2016
  17. 17. Today’s Validation Requirements 17 ICH/USP GMPs (legal) FDA 2016
  18. 18. ICH/USP Validation Requirements & Parameters  Specificity  Linearity  Range  Accuracy  Precision  Repeatability  Intermediate Precision  Reproducibility  Limit of Detection  Limit of Quantitation 18 ICH  Specificity  Linearity and Range  Accuracy  Precision  Limit of Detection  Limit of Quantitation  Ruggedness  Robustness USP 2016
  19. 19. USP Data Elements Required For Assay Validation 19 Analytical Performance Parameter Assay Category 1 Assay Category 2 Assay Category 3Quantitative Limit Tests Accuracy Yes Yes * * Precision Yes Yes No Yes Specificity Yes Yes Yes * LOD No No Yes * LOQ No Yes No * Linearity Yes Yes No * Range Yes Yes * * Ruggedness Yes Yes Yes Yes * May be required, depending on the nature of the specific test. 2016
  20. 20. ICH Validation Characteristics vs. Type of Analytical Procedure 20 Type of Analytical Procedure Identification Impurity testing Assay Quantitative Limit Tests Accuracy No Yes No Yes Precision Repeatability No Yes No Yes Interm. Prec. No Yes No Yes Specificity Yes Yes Yes Yes LOD No No Yes No LOQ No Yes No No Linearity No Yes No Yes Range No Yes No Yes 2016
  21. 21. Specificity/Selectivity  Ability of an analytical method to measure the analyte free from interference due to other components. 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, matrix, etc. Purity Tests: to ensure that all the analytical procedures performed allow an accurate statement of the content of impurities of an analyte, i.e. related substances test, heavy metals, residual solvents content, etc. Assay (content or potency): to provide an exact result which allows an accurate statement on the content or potency of the analyte in a sample.  Selectivity describes the ability of an analytical method to differentiate various substances in a sample 212016
  22. 22. Specificity: Impurities Assay  Chromatographic Methods  Demonstrate Resolution  Impurities/Degradants Available  Spike with impurities/degradants  Show resolution and a lack of interference  Impurities/Degradants Not Available  Stress Samples  For assay, Stressed and Unstressed Samples should be compared.  For impurity test, impurity profiles should be compared. 222016
  23. 23. Forced Degradation Studies  Temperature (50-60℃)  Humidity (70-80%)  Acid Hydrolysis (0.1 N HCl)  Base Hydrolysis (0.1 N NaOH)  Oxidation (3-30%)  Light (UV/Vis/Fl) Intent is to create 10 to 30 % Degradation 232016
  24. 24. Linearity  Ability of an assay to elicit a direct and proportional response to changes in analyte concentration. 242016
  25. 25. Linearity Should be Evaluated  By Visual Inspection of plot of signals vs. analyte concentration  By Appropriate statistical methods  Linear Regression (y = mx + b)  Correlation Coefficient, y-intercept (b), slope (m)  Acceptance criteria: Linear regression r2 > 0.95 Requires a minimum of 5 concentration levels 252016
  26. 26. Range  The specified range is normally derived from linearity studies and depends on the intended application of the procedure. It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples containing amounts of analyte within or at the extremes of the specified range of the analytical procedure.  Acceptable range having linearity, accuracy, precision.  For Drug Substance & Drug product Assay  80 to 120% of test Concentration  For Content Uniformity Assay  70 to 130% of test Concentration  For Dissolution Test Method  +/- 20% over entire Specification Range  For Impurity  From MDL to 100% of Impurity Specification Limit 262016
  27. 27. Accuracy  Closeness of the test results obtained by the method to the true value. 27
  28. 28. Accuracy  Should be established across specified range of analytical procedure.  Should be assessed using a minimum of 3 concentration levels, each in triplicate (total of 9 determinations)  Should be reported as:  Percent recovery of known amount added or  The difference between the mean assay result and the accepted value 282016
  29. 29. Accuracy Data Set (1 of 3) 29 Amount Added (mg) Amount Found (mg) Percent Recovery 0.0 0.0 --- 50.2 50.4 100.5 79.6 80.1 100.6 99.9 100.7 100.8 120.2 119.8 99.7 150.4 149.7 99.5 2016
  30. 30. Precision  The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample. Should be investigated using homogeneous, authentic samples. 302016
  31. 31. Precision… Considered at 3 Levels  Repeatability  Intermediate Precision  Reproducibility 312016
  32. 32. Repeatability  Express the precision under the same operating conditions over a short interval of time.  Also referred to as Intra-assay precision 32 Should be assessed using minimum of 9 determinations (3 concentrations/ 3 replicates) or Minimum of 6 determinations at the 100% level. 2016
  33. 33. Intermediate Precision 33 Express within-laboratory variations. Expressed in terms of standard deviation, relative standard deviation (coefficient of variation) and confidence interval. Depends on the circumstances under which the procedure is intended to be used. Studies should include varying days, analysts, equipment, etc. 2016
  34. 34. Repeatability & Intermediate Precision Day 1 Day 2 100.6 99.5 100.8 99.9 100.1 98.9 100.3 99.2 100.5 99.7 100.4 99.6 34 Grand Mean = 100.0 RSD = 0.59% Mean = 100.5 RSD = 0.24% Mean = 99.5 RSD = 0.36% 2016
  35. 35. Reproducibility  Definition: Ability reproduce data within the predefined precision  Determination: SD, RSD and confidence interval  Repeatability test at two different labs. Note: Data not required for BLA/NDA Lab 1 Lab 2 Lab 3 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Man 1 Man 2 Man 1 Man 2 Man 1 Man 2 3 Prep 3 Prep 3 Prep 3 Prep 3 Prep 3 Prep 35
  36. 36. Detection Limit (LOD)/ Quantitation Limit (LOQ)  LOD Lowest amount of analyte in a sample that can be detected but not necessarily quantitated. Estimated by Signal to Noise Ratio of 3:1. 36 LOQ Lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision. Estimated by Signal to Noise Ratio of 10:1. 2016
  37. 37. 1. Based in Visual Evaluations - Used for non-instrumental methods 2. Based on Signal-to Noise-Ratio - 3:1 for Detection Limit - 10:1 for Quantitation Limit 3. Based on Standard Deviation of the Response and the Slope 37 LOD and LOQ Estimated by 2016
  38. 38.  S = slope of calibration curve  s = standard deviation of blank readings or standard deviation of regression line Validated by assaying samples at DL or QL 38 DL = 3.3s QL = 10s S S LOD and LOQ Estimated by 2016
  39. 39. 39 Ybl LOD LOQ Statistical estimate of LOD & LOQ LOD = 3.3 Sbl / b LOQ = 10 Sbl / b Y = b X + a 2016
  40. 40.  Definition: Capacity to remain unaffected by small but deliberate variations in method parameters  Determination: Comparison results under differing conditions with precision under normal conditions  Examples of typical variations in LC  Influence of variations of pH in a mobile phase  Influence of variations in mobile phase composition  Different columns (different lots and/or suppliers)  Temperature  Flow rate 40 Robustness 2016
  41. 41. Ruggedness  Degree of reproducibility of test results under a variety of conditions  Different Laboratories  Different Analysts  Different Instruments  Different Reagents  Different Days  Etc.  Expressed as %RSD 412016
  42. 42. 422016 Reference Sites       
  43. 43. Thankyou 432016