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Analytical Method Validation

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Analytical method Validation
Analytical method Validation
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Analytical Method Validation

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Understanding of Analytical Method Validation Approach in Pharmaceutical Industry. Analytical method validation Verification is a wide chapter and a huge scope of applicability. In different types of methods, instrument, measurement approach all can effect the validation effort. However the basic fundamental will remains same, the parameters, acceptance criteria, functionality may vary depending upon the type of method, instrument etc.

Understanding of Analytical Method Validation Approach in Pharmaceutical Industry. Analytical method validation Verification is a wide chapter and a huge scope of applicability. In different types of methods, instrument, measurement approach all can effect the validation effort. However the basic fundamental will remains same, the parameters, acceptance criteria, functionality may vary depending upon the type of method, instrument etc.

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Analytical Method Validation

  1. 1. 1
  2. 2.  According FDA Validation is “Established documented evidence which provides a high degree of assurance that a specific process will continually produce a product meeting its predetermined specifications and quality attributes.”  The Validated method should be stability indicating method, which means the method that can detect the changes in properties of drug substance or drug product with time 2
  3. 3.  Verification  Compendial methods-Verification : • These methods are listed in pharmacopoeia’s • Use to evaluate defined characteristics of the drug substance or drug product • Suitability of Compendial methods must be verified under actual conditions • Should be partially validated  Validation  Non -Compendial methods-Validation • Proposed by the applicant for use instead of Regulatory Analytical Procedure • Equivalency: • Equivalency: required for an in-house method, when compendial standard is claimed 3
  4. 4. Validation Development Quality Control 4
  5. 5. ICH/USP GMPs FDA 5
  6. 6. Verification data is required.  The compendial methods as published are typically validated based on an API or an FPP originating from a specific manufacturer.  Different sources of the same API or FPP may contain impurities and/or degradation products that were not considered during the development of the monograph.  The FPP may also contain excipients not present in the FPP(s) for which the compendial method was developed.  API Assay: No validation generally required. Exception: specificity for any specified impurities not in the monograph. Purity: Full validation for specified impurities that are not included in the monograph (specificity, linearity, accuracy, repeatability, intermediate precision, LOD/LOQ).  FPP Assay: Specificity, accuracy and precision (repeatability). Eg. to rule out excipient interference Purity: Specificity, accuracy and precision (repeatability) for known imps.  Full validation for specified impurities that are not included in the monograph (specificity, linearity, accuracy, repeatability, intermediate precision, LOD/LOQ) 6
  7. 7.  Full validation is required for an in-house purity/assay method.  Equivalency must be demonstrated compared to the compendial method • Assay: same samples using the two methods; • Related substances: same samples spiking with specified impurities at the limits) using the two methods  If equivalency is not demonstrated, it can not be claimed as compendial standard 7
  8. 8. Equivalency is essentially two things:  Ensure the in-house method is not inferior (in which case they should adopt the compendial method)  Ensure the compendial test can be met, if applied.  Two samples are sufficient. Note that a compendial method becomes an in-house method when adjustments are made outside the allowable adjustments (Int.Ph, USP, EP general chapter Chromatography) 8
  9. 9.  Specificity  Precision  Accuracy  Linearity  Range  Quantitation Limit  Detection Limit  Robustness  Stability in Analytical Solution 9
  10. 10. USP ICH  Specificity  Linearity and Range  Accuracy  Precision  Limit of Detection  Limit of Quantitation  Ruggedness  Robustness  Specificity  Linearity  Range  Accuracy  Precision  Repeatability  Intermediate Precision  Reproducibility  Limit of Detection  Limit of Quantitation 10
  11. 11. 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 May be required, depending on the nature of the specific test 11
  12. 12. Testing a method to demonstrate it is suitable for its intended purpose and the results obtained are reliable, accurate and reproducible. Provides confidence that the method will perform properly under intended conditions.  Identification tests;  Quantitative or limit tests for the control of impurities;  Quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product. Including assay, Content Uniformity, dissolution, content of perseveratives. Validation of analytical procedures requires:  Qualified and calibrated instruments  Documented methods  Reliable reference standards  Qualified analysts  Sample integrity 12
  13. 13.  Ability of an analytical method to measure the analyte free from interference due to other components.  An investigation of specificity should be conducted during the validation of identification tests, the determination of impurities and assay  Should be able to discriminate between compounds closely related in structure.  Confirmed by obtaining negative results for samples with spiked related compounds and positive results for samples with analyte.  SELECTIVITY “A measure of the discriminating power of a given analytical procedure in differentiating between the analyte and other components in the test sample.” 13
  14. 14.  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.  Peak homogeneity tests should be performed using PDA or mass spectrometry to show that the analyte chromatographic peak is not attributable to more than one component. 14
  15. 15.  SELECTIVITY & SPECIFICITY 15 Selectivity: Specificity: If an analytical procedure is able to separate and resolve the various components of a mixture and detect the analyte qualitatively If the method determines or measures quantitatively the component of interest in the sample matrix without separation Restricted to qualitative detection of the components of a sample Restricted to quantitative measurement of one or more analyte Blank solution - to show no interference Placebo - to demonstrate the lack of interference from excipients Spiked samples - to show that all known related substances are resolved from each other Stressed sample of about 5% to 20% degradation is used to demonstrate the resolution between degradants and the analyte of interest Check peak purity of drug substance by photodiode array detector (PDA) Representative chromatograms should be provided
  16. 16. Temperature  Humidity  Acid Hydrolysis  Base Hydrolysis  Oxidation  Light (UV/Vis/Fl) Intent is to create 10 to 30 % Degradation 16
  17. 17.  The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample. Precision… Considered at 3 Levels  Repeatability  Intermediate Precision  Reproducibility 17
  18. 18.  Repeatability A minimum of nine determinations covering the specified range for the procedure, e.g.  three concentrations/three replicates each, or a minimum of six determinations at 100% of the test concentration  Intermediate Precision Within-laboratory variations  usually on different days, different analysts and different equipment.  Reproducibility Precision between laboratories 18
  19. 19.  Closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found  Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range  Accuracy is usually reported as percent recovery by the assay (using the proposed analytical procedure) of known added amount of analyte in the sample or as the difference between the mean and the accepted true value together with the confidence intervals. The range for the accuracy limit should be within the linear range 19
  20. 20.  Drug Substance For Assay  Accuracy can be inferred once precision, linearity and specificity have been established For Related Substances/ Residual Solvents  Accuracy should be assessed on samples spiked with known amount of impurities Drug Product For Assay  Known quantities of drug substance to be analysed have been added to synthetic mixture of drug product components. For Related Substances/ Residual Solvents  Accuracy should be assessed on samples spiked with known amount of impurities 20
  21. 21. Accuracy range….. Across the range of LOQ-150% of the target concentration (shelf life limit), 3-5 concentrations, in triplicate each. (LOQ, 50%, 100%, 150%) Per cent recovery: in general, within 80-120%, depends on the level of limit Accurate & precise Accurate & imprecise Inaccurate & precise Inaccurate & imprecise 21
  22. 22.  Linearity of an analytical procedure is its ability to obtain test results which are directly proportional to concentration of analyte in the sample within a specified range.  A series of samples should be prepared in which the analyte concentrations span the claimed range of the procedure. If there is a linear relationship, test results should be evaluated by appropriate statistical methods  Minimum of 5 concentration is recommended within the range Range: This is an expression of the lowest and highest levels of analyte that have been demonstrated to be determinable for the product The specified range is normally derived from linearity studies 22
  23. 23.  Assay : 80-120% of the theoretical content of active  Content Uniformity: 70-130%  Dissolution: ±20% of limits; eg if limits cover from 20% to 90% l.c. (controlled release), linearity should cover 0-110% of l.c.  Impurities: reporting level to 120% of shelf life limit  Assay/Purity by a single method: reporting level of the impurities to 120% of assay limit 23
  24. 24.  Is the amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy  Approaches (instrumental or non-instrumental):  visual evaluation (Used for non-instrumental methods)  signal to noise ratio (10:1 for Quantitation Limit)  standard deviation of the response and the slope Quantitation Limit = 10 S  : SD of Response (The residual SD of regression line or SD of y intercept of regression lines can be used as SD) S : Slope of calibration curve 24
  25. 25. Signal Noise Peak A LOD Peak B LOQ Baseline Ybl LOD LOQ Statistical estimate of LOD & LOQ LOD = 3.3 Sbl / b LOQ = 10 Sbl / b Y = b X + a 25
  26. 26.  Is the amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value  Approaches (instrumental or non-instrumental):  visual evaluation (Used for non-instrumental methods)  signal to noise ratio (3:1 for Detection Limit)  standard deviation of the response and the slope Quantitation Limit = 3.3 S  : SD of Response (The residual SD of regression line or SD of y intercept of regression lines can be used as SD) S : Slope of calibration curve 26
  27. 27.  Not required for assay/dissolution methods.  Applicant should provide  The method of determination  The limits  Chromatograms  Visually identified  Established by Repeatability 27
  28. 28.  Measure of its capacity to remain unaffected by small , but deliberate variations in method parameters and provide an indication of its reliability during normal usage  If measurements are susceptible to variations in analytical conditions, than it should be suitably controlled or a precautionary statement should be included in procedure  System suitability parameters should be evaluated to ensure that validity of analytical procedure is maintained when ever used 28
  29. 29.  For Liquid Chromatography, typical variations are  Influence of pH in a mobile phase ( ± 0.2 units)  Influence of mobile phase composition (± 2.0% Organic phase)  Different columns[ Lots/Suppliers]  Temperature [Column Oven] (± 5° C)  Flow rate ( ± 0.2ml/min)  System Suitability  Evaluate the System suitability parameters  Validation of analytical method must be focused on critical separation of components which can alter during change of conditions  These critically separated components should be part of system suitability 29
  30. 30.  Should be established  By keeping the sample at RT or below 10°C and injecting it at regular intervals  No significant variations or trend should be observed  Any significant variation in particular area / absorbance should be compared or calculated against the initial value.  In case of impurity the % of impurity present should be verified against the initial .  The outcome of the result should be mentioned in the particular regular test procedure. 30
  31. 31. When Re- validation is required !!!  Method parameters have been changed  The scope of the method has been changed  Synthetic methods have been changed  Impurity profile has been changed (Due to change in excipient).  Changes in equipment or suppliers of critical supplies of the API or final drug product will have the potential to change their degradation profile and may require the method to be redeveloped and revalidated. 31
  32. 32.  Integral part of many analytical procedures  Based on the concept that:  The equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such  Determination (in General): Repeatability, Tailing factor (T), Capacity factor (k’), Resolution (R), and Theoretical Plates (N) 32
  33. 33. Parameters Recommendations K’ In general k’ ≥ 2.0 R R > 2, between the peak of interest and the closest potential interferent (degradant, internal STD, impurity, excipient, etc…..) T T ≤ 2 N In general N > 2000 Repeatability RSD ≤ 2.0% (n ≥ 5) Assay: RSD ≤1% (API) or ≤ 2% (FPP), n ≥ 5 Impurities: in general, RSD ≤ 5% at the limit level, up to 10% or higher at LOQ, n ≥ 6 33
  34. 34. Allowed HPLC Adjustment USP (Ref: General Chapter <621>) EP (Ref: General Chapter 2.2.46) Column Length ±70% ±70% Internal Diameter Can be adjusted if linear velocity is kept constant ±25% Particle Size Reduction of 50%, no increase Reduction of 50%, no increase Flow Rate ±50% or more as long as the linear velocity is kept contant ±50% Column Temperature ±10C ±10% Max 60C Injection Volume Change allowed as long as SST criteria are met May be decreased (if LOD and repeatability ok) PH ±0.2 Units ±0.2 Units (±1% for neutral substances) UV wavelength <±3 nm (in condition) <±3 nm ((in condition)) Conc. Salts in Buffer ±10% ±10% Composition of mobile phase Minor Components (<50%) ±30% or ±10% absolute whichever is smaller Minor Components ±30% or ±2% absolute whichever is larger 34
  35. 35.  Some 483 Observations  Analytical methods are not adequately validated  There was no inadequate method validation specificity data to demonstrate that each method was capable of distinguishing the active ingredient from its impurities and degradation products.  Specificity studies did not include the minimum stress conditions of acid and base hydrolysis, oxidation, thermal degradation and photolysis, degradation schematic for the active ingredient that identifies the major degradation products was not included for each product. 35
  36. 36.  Some 483 Observations  Stress studies conducted as part of method validation do not target a minimum amount of degradation. … a standard period of two hours as commonly used for stress studies with no justification…  Spreadsheets used to calculate linearity, percent recovery, and final assay results for the cleaning validation of …were not validated and the data transcribed from chromatographs to the spreadsheets were not checked for accuracy. 36
  37. 37.  FDA Waning Letter  On addition to the example of modifying both Compendial methods and customer supplied methods, we also observed the use of un-validated in-house methods as well as un-validated modifications to in-house methods.  A statement indicating that the method has not been validated in the particular formulation was included in the certificate of analysis for…use of this statement does not absolve…from using valid, accurate, and reproducible methods.  OOS accuracy results reported by analyst 3 were never submitted in the final report. Repeat analysis performed in a different system passed specifications and these results were submitted in the report. 37
  38. 38.  FDA Warning Letter  There is no assurance that qualification or maintenance of the laboratory equipment can consistently produce valid and accurate analytical results in that numerous examples of test data were invalidated due to instrument malfunction.  Attempts to corroborate data in the validation report with supporting raw data in the laboratory were difficult and frustrating for the FDA personnel conducting the inspection  Raw data and calculations were not checked by a second responsible individuals required by your procedures. Inaccurate calculations were noted in the report 38
  39. 39.  FDA Warning Letter  The process validation samples were assays using an HPLC method that had not been validated. The method validation used for both products, did not include a protocol that included specification and acceptance criteria. … The method validation was not reviewed and approved until during the current inspection. Lots of both products were released for distribution prior to completion of the method validation.  Method validation for the product one product is inadequate in that the data does not assess all variables, such as different mobile phase concentrations and analytes, to demonstrate that the method can sustain variance. 39
  40. 40.  Data for acceptance, release, stability will only be trustworthy if the methods used are reliable  More efficient use of resources in the R&D process, as a consequence  Validation should be Protocol driven  Document each and every information with regard to std/sample/reagent/impurities/process/ preparation etc.  Document the details of each experiment with suitable way.  Review each experiment after its completion and relate its outcome with other experiments  Identification and elimination of the need to duplicate studies to meet different regulatory requirements 40
  41. 41.  From My Side……  Why is analytical monitoring necessary?  What is the purpose of analytical validation? 41
  42. 42. 42

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