Analytical mehod validation explained sadasiva
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The document discusses analytical method validation. It describes the key steps in analytical method validation including method development, qualification, transfer to quality control, periodic monitoring and revalidation if changes are made. It outlines the key parameters that are evaluated in validation including accuracy, precision, specificity, linearity, range, limit of detection, limit of quantitation and robustness. Approaches for evaluating each parameter are provided with examples. The importance of validation for ensuring reliable test methods as required by regulatory agencies is also highlighted.
Analytical mehod validation explained sadasiva
- 1. By Sada Siva,Scientist, Analytical Development, Aurobindo Pharma Ltd (Biologics) . Analytical Method Validation
- 2. Analytical Method life cycle 4.Method Validation To prove method has the ability to perform for its intendted use complete Documentation 3. Method Transfer to QC Method transfer to end user /clients 5. Periodically Monitoring / Review of Methods in Control Labs 1. Analytical method development To optimize a method for intended use 2. Qualification of method To prove method has the ability to perform for its intended use Intial Documentation 6. Method Revalidation If any changes in method / method perfomance changes with time
- 3. Validation-Introduction What Why Where Who When How Requirements Case study
- 4. What is a validation? It is a process of demonstrating that method is suitable their intended use with documentation. Validation should verify the suitability of an analytical method for its intended purpose Validation may be performed in different ways (individual purpose) according to common standards/guidelines
- 5. Why do we need to do validation? Regulatory bodies require safety and quality information of our product for approvals. For this one should need to have reliable test methods. That reliability of method is provided by doing a validation exercise ., and which is documented. *It is also essential part of GMP practices
- 6. Where , when & who will perform Validation ? Where: Generally validation is performed in a GxP laboratory or Equivalent. mostly it should be performed in End user (QC) laboratory where the method going to be used regularly. Who: A qualified analyst (GxP trained, qualified and skilled ) When: Before commercialization (during CT3 time- biosimilars ) Before CT or IND application ( for New Drugs)
- 7. Pre-requisites of validation? Prerequisites for analytical method validation Six “M”s Ready to start Validation Man Machine qualified calibrated robust qualified Methods suitable characterised documented MilieuMaterial Management Quality Reference standards Tempe- rature Analysts´ support skilled Humidity Vibrations Time Supplies Irradi- ations
- 8. How to perform validation? Many regulatory bodies have guideline how to perform validation of analytical method. Some of them are ICH Q2, FDA, USP, EMEA
- 9. Validation characteristics (parameters) Accuracy Specificity Precision Repeatability Intermediate precision Reproducibility Linearity Limit of detection Limit of quantitation Range Robustness
- 10. Degree of validation depends on Test
- 11. System suitability and Acceptance criteria Integral part of each method/procedure to ensure that all analytical operations, electronics and equipment's are working properly at the time of analysis. Appropriate system suitability and its acceptance criteria should be defined. Resolution ,tailing factor, RRT , capacity factor, no . of theoretical plates between 2 peaks in chromatography methods Minimum height /response of control samples Spike recovery in sample processing (extraction) is there. BC recovery of known amount of control sample In every parameter it should comply or pass if not the test will be considered (not used)
- 12. Specificity 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
- 13. Specificity-Approaches Selectivity: the extent to which a method can determine particular analyses in mixtures in matrix without interference from other compounds (used in chemistry but in specificity is the ultimate of selectivity-IUPAC) SAMPLE PURPOSE SYSTEM SUITABILITY SAMPLE SYSTEM SUITABILITY FORMULATION BUFFER (SEC,CEX) TO EVALUATE MATRIX INTERFERENCE OR TO EVALUATE SEPARATION SELECTIVITY TEST SAMPLE DILUTED IN FB, WFI (OD 280,SEC) TEST SAMPLE TEST SAMPLE SPIKED WITH IMPURITY (HCD) IMPURITY SAMPLE NEAT (HCP,HCD) UNRELATED SAMPLES (GLYCAN, SDS- PAGE,HCD) SYSTEM SUITABILITY SAMPLE SYSTEM SUITABILITY
- 14. Precision • Expresses the closeness of agreement between a series of measurements (replicates) obtained from multiple sampling(different vials) of the same homogenous sample (same batch) • Is usually expressed as the standard deviation (S), variance (S2) or relative standard deviation (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)
- 15. Repeatability-Approaches Repeating the analysis under same operating conditions in a short time interval Same samples analysis(different vials from same batch) in same sequence. Independent sample preparations (6) will be done but analysed in a single assay. Sample Name HMW Peak RT Area % of Area System suitability sample 3.24 58481 0.88 Test sample Replicate 1 3.24 109382 1.59 Test sample Replicate 2 3.24 109560 1.58 Test sample Replicate 3 3.24 110185 1.58 Test sample Replicate 4 3.24 108539 1.58 Test sample Replicate 5 3.24 109996 1.59 Test sample Replicate 6 3.24 110362 1.59 Test sample con 1(triplicates) concentration based precision (3 replicate / 3 different concentration) Test sample con 2 (triplicates) Test sample con 3 (triplicates) %RSD 0.00 1 0.17
- 16. Intermediate Precision- Approaches Same test sample is analysed varying any one test condition like analyst , Day, Instrument, reagents/consumables of different lots etc. Sample Name HMW Impurity RT Area % of Area Test sample _Ctrl 3.24 109670 1.58 Test sample_Inter instrument 3.24 109851 1.31 Test sample_Inter analyst 3.24 120053 1.49 Test sample_Interday 3.24 116089 1.53 P2_Test sample_Inter column 3.26 113579 1.57 %RSD 0.21 4 7.39
- 17. Reproducibility Precision between two laboratories. Interlab precision. Generally done in labs working with collaboration / pharmacopeia methods Generally not required in industries. Ruggedness (USP) is defined as the degree of reproducibility of results under variety of conditions like different labs, Environmental conditions, materials , operators, instruments and analysts. Robustness – deliberate changes in operations conditions Ruggedness-normal , expected operations conditions (many labs) Reproducibility- normal operations conditions in 2 or 3 labs (clients)
- 18. Accuracy • Expresses the closeness of agreement between the value which is accepted either as a conventional true value (Expected) or an accepted reference value and the value found (Observed value by method) Sometimes referred to as „TRUENESS“/ Sameness Agreement of test results with the true value Expressed in % Recovery
- 19. Accuracy- Approaches Spiking known amount in sample (HCD,SEC) and calculating the spike recovery (3 conc /3 replicates) Spiking of ref.mateterial in placebo. Known amount (purity) of sample is taken and analysed to check method recovery(80-120% acceptable range ) sample Expected Observed % Recovery sample-1 100 98 98 sample-2 80 81 101 sample-3 20 19 95
- 20. Precision and Accuracy Accurate & precise Accurate & imprecise Inaccurate & precise Inaccurate & imprecise
- 21. Linearity Linearity of an analytical procedure is its ability (within a given range) to obtain test results which 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 (r) Y-intercept Slope of regression line Residual sum of squares (R2) Plot of recovey
- 22. Linearity Approaches Different con (minimum 5) of sample Vs responses (PAHPLC,OD 280,HCD) Spiking of known amount of impurity/substances into test sample (SEC ,CEX, Glycan)
- 23. Range 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
- 24. Range-Approaches Data from linearity/accuracy is used to check this. Have to check how method behaves in different ranges like Ex: Complete range of method used in linearity (0.1 to 120 %) Lower range of linearity/accuracy samples (0.1 to 10%) Middle range of linearity/accuracy samples (10 to 100%) Higher range of linearity/accuracy samples (50-120%)
- 25. Limit of Quantitation (LOQ, QL) 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 accurac Determination is usually based on Signal to noise ratio (~10:1) (baseline noise) -- Standard deviation of response (s) and Slope (S) Viaual evaluation Calibration curve SD of blanks
- 26. Limit of Detection (LOD, DL) 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) Standard deviation of response (s) and Slope (S) Viaual evaluation Calibration curve SD of blanks
- 27. LOD and LOQ calculations
- 28. Robustness Robustness Robustness of an analytical procedure should show the reliability of an analysis with respect to deliberate variations in method parameters The evaluation of robustness should be considered during the development phase
- 29. Robustness-Approaches Deliberate changes like reagents/buffers pH, concentration, temperature, injection volume, Incubation time , Samples stability, pippete errors etc depending on type of methods 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
- 30. Verification of Reference Method or pharmacopeia method The suitability of compendial method must be verified that they are suitable for the DS/DP analysis in that laboratory conditions following characteristics should be checked. Specificity Intermediate precision Stability of samples solution (defined if critical)
- 31. Revalidation consideration To Repeat validation of an validated method to ensure continued compliance with established requirements after a defined time or in following situations. Found that Method performance changes with time Where some changes were made for improvement in Method , Process , DP formulation Product etc. Degree of validation depends on type of change/ its criticality
- 33. Other consideration When using a non- pharmacopeia method (justification is needed, comparison with Pha.method,complete validation required ) Stress studies should demonstrate that impurities and degradents from the sample do not interfere with the quantitation of sample.
- 34. References ICH Q2: validation of analytical procedures, Text and methodology USP: validation of compendial method FDA :Analytical procedures and methods validation (Guidance for industry-draft guidance) Emea : recommends to follow ICH Q 2 WHO :supplementary guidelines on GMP :validation Annexure 2 : Analytical method validation Book: validation of analytical method from agilent technologies Book: Method validation in pharmaceutical analysis by j.Ermer and J.H.mill
- 35. Learning starts from questioning ??????????
- 36. Learning is a continuous process For giving this opportunity Have A Nice Weekend