Validation of Analytical Methods used for the Characterization, Physicochemical and Functional Analysis and of Biopharmaceuticals. 1 Analytical Method Validation: 1..1 Philosophy: Method validation is described as: the process of demonstrating that analytical procedures are suitable for their intended use. Based on the intended use of the test method, the level of confidence required could be different. For example, it would be different depending on: At what stage of the product life cycle the test method is used? (e.g. product development stage or commercial manufacture). How the test method is applied to the manufacturing process and what purpose it serves? (E.g. process control, product release, raw material evaluation, comparability assessment, characterization, etc.) During the development of a biopharmaceutical, a wide variety of complex analytical techniques such as SDS-PAGE, Western blot, Slot blot, RP-HPLC, SEC-HPLC, ELISA etc. are used to assess the physicochemical and functional characterization of in-process, Drug Substance and Drug Product. As development proceeds from Pre-Clinical to Clinical to Commercial, many of those methods evolve into routine quality control assays that will require validation for their intended use. TABLE 1. - TEST METHOD QUALIFICATION ACTIVITY DURING DIFFERENT STAGES OF PRODUCT LIFE-CYCLE Pre-clinical Specificity is necessary for the new method to be scientifically defendable and suitable for the intended purpose* System Suitability parameters to be identified Intended purpose should be identified for each test method (Intended purpose could be either safety or characterization* or release Predetermined Specifications need not be fully defined (especially if the process is undergoing changes) Clinical Trial batch Qualification - Scientifically defendable and suitable for the intended purpose* Specifications needs to be established Assay related to product safety should be validated / verified Technology Transfer (From R&D to QC) Validation of in-house lot release assays (noncompendial) Verification (if compendial) Created by Dr. Nishodh Saxena Page 1 of 11 Post-approval changes Validation (if changes in the test method or manufacturing process) Verification (if compendial) * Methods used only for Characterization or Comparability Studies need not be fully validated. At the minimum the assay must be scientifically sound and generally suitable for the intended purpose and operationally robust. They are not intended for QC applications.
1.2 The extent of qualification / validation / verification required can be greater or lesser than recommended in Table below as per ICH guideline, based on Risk Analysis, complexity of the test, criticality, stage of product life cycle and the intended purpose (e.g. safety, comparability, characterization, release). Analytical Performance Parameter Identification Impurity Testing Quantitative Limit tests Assay Accuracy - + - + Precision Repeatability Intermediate. Precision. - - + + 1 - - + + 1 Specificity 2 + + + + Limit of Detection (LOD) - - 3 + - Limit of Quantitation (LOQ) - + - - Linearity - + - + Range - + - + - Not normally evaluated; + Normally evaluated 1 In cases where reproducibility has been performed, intermediate precision is not needed 2 Lack of specificity in case of one analytical procedure could be compensated by other supporting analytical procedure(s) 3 May be needed in some cases The following tests shall be validated as per ICH guidelines: a. Identification tests. b. Quantitative tests for impurities content. c. Limit tests for the control of impurities. d. Quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product. Created by Dr. Nishodh Saxena Page 2 of 11
a. Identification tests These tests are intended to ensure the identity of an analyte in a sample. This is normally achieved by comparison of a property of the sample (e.g., spectrum, chromatographic behavior, chemical reactivity, etc.) to that of a reference standard. b. Limit Test for impurities Testing for impurities can be either a quantitative test or a limit test for the impurity in a sample. Either test is intended to accurately reflect the purity characteristics of the sample. Different validation characteristics are required for a quantitative test than for a limit test. c. Quantitative Test for quantification of major components or active ingredients Assay procedures are intended to measure the content or potency of the analyte present in a given sample. d. Assay to determine efficacy of drug substance or drug product. 1.3 Risk Analysis shall be carried out to assess the Risk Rate associated with a Test Method to detect changes in the Quality / Safety / Efficacy (or a combination thereof) of the product being analyzed. Risk Rate = Probability x Severity x Detectability. Notional values shall be assigned as shown below: Probability that the method is likely detect any failure in the material tested 1 = Low (rarely if ever occurs e.g. when the test is sensitive) 2 = Medium (Method frequently requires refinement 3 = High (likely to occur when the test is not reliable or sensitive) Severity In terms of the probable impact on Customer (patient) as a consequence of the ability of the test to evaluate safety and efficacy of the product. 1 = Low (e.g. when used as one of the many identity tests) 2 = Medium 3 = High (e.g. sole test to judge potency or safety) Detectability 1 = Low (when failure of a test is easily noticeable from other supporting tests) 2 = Medium 3 = High (insufficient System Suitability or lack of other supporting tests). Thus the minimum Risk rate = 1x1x1 = 1 and the maximum Risk rate = 3 x 3 x 3 = 27 Created by Dr. Nishodh Saxena Page 3 of 11
2. Validation Performance Parameters 2.1 Accuracy Accuracy shall be determined after precision, linearity and specificity have been established. Accuracy shall be required to be determined for: (A) Quantitative Test for Impurities (B) Assay of analyte For Quantitative method for Impurity, a known quantity of impurity shall be spiked to a relatively pure analyte (Drug substance or Drug product) to mimic the matrix (milieu). Impurity shall be spiked in triplicate at three levels over a range that covers the expected impurity content of the sample. A minimum of 9 determinations over 3 different concentration levels covering the specific range (e.g. 3 concentrations/3 replicates) shall be used for spike studies. If a reference material is not available and there are no other methods appropriate for comparison, then accuracy shall be investigated by spiking, either into a representative matrix blank or into a sample containing a low level of analyte (positive sample). To determine accuracy of test method for Assay (Content Test), accuracy shall be established across a specified range of the assay by spiking analyte (e.g. Reference standard) to a placebo (Blank matrix) and comparing the assayed value with the true value. Spiked samples shall be prepared in triplicate at three levels over a range of 50-150% of the target concentration. Accuracy should be reported as percent recovery by the assay of known added amount of analyte in the sample as the difference between mean and the accepted true value together with the confidence intervals. The recovery (%) of the added analyte is calculated as follows: % Recovery = Added amount minus the Original amount (Average value) X 100 Added amount of analyte 2.2 Precision Precision shall be required to be determined for: (A) Quantitative Test for Impurities (B) Assay of analyte Assays included in the Precision determination shall include individual analysis of samples starting from sample preparation (dilution) to the final test result. Created by Dr. Nishodh Saxena Page 4 of 11
Precision of an analytical method shall be determined by assaying a sufficient number of aliquots of a homogenous sample to be able to calculate statistically valid estimates of standard deviation or relative standard deviation (coefficient of variation). It is recommended to use minimum of nine determinations covering the specified range for the procedure (three concentrations and three replicates of each concentration) or using a minimum of six determinations at 100% of the test concentration. Precision shall be measured at two different levels - Repeatability and Intermediate Precision (A) Repeatability (also termed as intra-assay precision, shall be determined under the same operating conditions over a short interval of time). (B) Intermediate precision (also termed as ruggedness, shall be determined under different days, different analysts, different equipments within a laboratory) (A) Repeatability of a method shall be assessed by: 1. For instrument precision or injection repeatability, a minimum of 10 injections of one sample solution is made to test the performance of the chromatographic instrument. 2. For intra-assay precision, data are obtained by repeatedly analyzing, in one laboratory within a short span of time (usually one day), aliquots of a homogenous sample, each of which has been independently prepared according to the method procedure. 3. For these precision studies, the sample preparation procedure, the number of replicate samples to be prepared, and the number of injections required for each sample in the final method procedure shall be defined in the method validation protocol. 4. Acceptance Criteria for instrument precision / injection repeatability, RSD will be 1%. And the intra-assay precision shall be 2%. For an impurity method, at the limit of Quantitation (LOQ), the instrument precision shall be 5% and the intra-assay precision will be 10%. (B) Intermediate precision for a method: The extent to which intermediate precision should be established shall depend on the factors under which the test procedure is used. The objective is to determine which of these factors contribute significantly to variability. Typical factors to be studied include days, analysts, equipment, etc. It is not necessary to study these effects individually and the use of an experimental design (matrix) is encouraged. Created by Dr. Nishodh Saxena Page 5 of 11
2.3 Specificity An investigation of specificity shall be conducted during the validation of: (A) Identification tests, (B) Test for quantification of impurities (C) Assay (content test). Note: It may not be possible to demonstrate that an analytical procedure is specific for a particular analyte (complete discrimination), in which case, a combination of two or more analytical procedures are recommended to achieve the necessary discrimination. (A) Identification Tests (to ensure the identity of the analyte) Suitable identification tests shall be performed to discriminate between the compounds of closely related structures, which are likely to be present. Obtaining positive results from samples with a known reference material and obtaining negative results from samples, which do not contain the analyte or contain material structurally similar to or closely related to the analyte, shall confirm the discrimination. (B) Tests for Quantification of Impurities and (C) Assays (Content) the approach for validation of Specificity are similar for both tests and shall be done by any of the following ways, depending on: (i) In case where the impurities are available or can be generated (by subjecting the samples to relevant stress conditions such as heat, acid/base hydrolysis, oxidized, reduced, UV radiation, etc. to generate material closely related to the analyte). For the assay: It shall be done by spiking pure substances (Drug substance or Drug product) with appropriate levels of impurities and / or excepients and demonstrating that the assay results obtained is unaffected by the presence of these materials (by comparison with the assay result obtained from un-spiked samples). For the impurity tests: the discrimination should be established by spiking drug substance or drug product with appropriate levels of impurities and demonstrating the separation of these impurities individually and / or from other components in the sample matrix. Created by Dr. Nishodh Saxena Page 6 of 11
(ii) In case where impurities are not available: If impurity or degradation product standards are not available, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure, e.g., pharmacopoeia method or other validated analytical test procedure. For chromatographic procedures, specificity can be demonstrated by the resolution of the components in a stress sample (containing all potential components closely related to the analyte) and comparing it with the resolution obtained with normal analyte. The specificity test shall be done once the column type, mobile phase composition, flow rate, and detection modes, are set. 2.4 Limit of Detection. For non-instrumental methods, the LOD shall be determined by the analysis of samples with known concentration of analyte and by establishing the minimum level at which the analyte can be reliably be detected. Several approaches for determining the LOD are possible, depending on whether the procedure is non instrumental or instrumental. Approaches other than those listed below may be acceptable. a) Based on Visual evaluation (as in the case of a SDS-PAGE, Slot-Blot or Western blot) - The LOD is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably seen. b) Based on Signal -to- Noise- This approach can only be applied to analytical procedures those exhibit baseline noise. The determination of signal-to-noise ratio is performed to comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio of 3 (or 2:1) is generally considered acceptable for estimating the detection limit. In case of chromatography, the presentation of the relevant chromatograms is considered acceptable for justification. c) Based on the standard deviation of the blank Analyzing an appropriate number of blank samples and calculating the standard deviation of these responses perform measurement of the magnitude of analytical background response. The LOD may be expressed as: LOD = 3.3σ / S Where σ = The standard deviation of the response Created by Dr. Nishodh Saxena Page 7 of 11
S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways. d) Based on the calibration curve A specific calibration curve should be studied using samples containing an analyte in the range of LOD. The residual standard deviation of a regression line or the standard deviation of y- intercepts of regression lines may be used as the standard deviation. In cases where an estimated value for the detection limit is obtained by calculation or extrapolation, this estimate shall be subsequently validated by independent analysis of a suitable number of samples known to be near or prepared at the detection limit. 2.5 Limit of Quantitation: a) Based on Visual Evaluation The LOQ is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision against a series of reference concentrations. b) Based on Signal-to-noise approach can only be applied to instrumental methods which exhibit baseline noise. It is performed by comparing measured signals from samples with known concentrations of analyte with those of blank samples and by establishing the minimum concentration at which the analyte can be reliably quantified. A typically acceptable signal-tonoise ratio is 10:1. Standard deviation of the Response and the Slope The LOQ may be expressed as: LOQ = 10 σ / S Where σ = the standard deviation of the response and S = the slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways, like c) Based on the standard deviation of the blank -Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses. d) Based on the calibration curve A specific calibration curve should be studied using samples containing an analyte in the range of LOQ. The residual standard deviation of a regression line or the standard deviation of y- intercepts of regression lines may be used as the standard deviation. Created by Dr. Nishodh Saxena Page 8 of 11
2.7 Linearity A linear relationship should be evaluated across the range of the analytical procedure. It may be demonstrated directly on the drug substance by dilution of a standard stock solution. It should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. The test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. In some cases to obtain linearity between the response of an analyte and its concentration, the test data may have to be subjected to a mathematical transformation (e.g. immunoassays, bioassays). The Data from the regression line itself may be helpful to provide mathematical estimates of the degree of linearity, correlation coefficient, y-intercept, slope of the regression line, and residual sum of squares. A plot of the data should be included. For assay methods, the linearity study is to be performed by preparing standard solutions (five different concentrations are recommended, from 50 to 150% of the target analyte concentration. Standards should be prepared and analyzed by a minimum of three times. For impurity methods, linearity is to be determined by preparing standard solutions at five concentration levels over a range such as 0.05 2.5 wt%. For an assay method for a drug substance or drug product, linearity criteria is that the correlation coefficient for each of three curves (five concentration levels each) shall be 0.99 for the range of 80 120% of the target concentration. For an impurity method, the correlation coefficient for each of three curves (five concentration levels each) will be 0.98 for the range of 0.1 2.5% of the main component concentration. 2.7 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. The standard deviation, relative standard deviation (coefficient of variation), and confidence interval should be reported for each type of experiment performed. For an assay method, the acceptable range will be defined as the concentration interval over which linearity, accuracy and precision are obtained. Normal range shall be from 80 to 120% of the test concentration. For an impurity method, the acceptable range will be defined as the concentration interval over which linearity and accuracy are obtained as per their acceptance criteria, normally up to 120% of the specification. Created by Dr. Nishodh Saxena Page 9 of 11
For Content uniformity, the range shall cover a minimum of 70 to 130% of the test concentration, unless a wider, more appropriate range, based on the nature of the dosage form is justified. If assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the range from the reporting level of the impurities to 120% of the assay specification. 2.8 Robustness For the purpose of robustness is to show the reliability of an analysis with respect to deliberate variations in method parameters. A risk analysis approach is recommended to be adopted to identify parameters that are likely to vary from their normal set values. Refer to Risk analysis described under Para 1.2. Examples for risk factors, which should be considered, are: Sample preparation Worst case scenario Stability of sample preparations and solutions Incubation times Different analysts Different labs Identical technology, but different types of equipment System Suitability; Calibration and adjustment 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. One consequence of the evaluation of robustness should be that a series of system suitability parameters is established to ensure that the validity of the analytical procedure is maintained whenever used. In the case of liquid chromatography, examples of typical variations which shall be determined are: Suitability of solutions Influence of variations in ph on the mobile phase, Influence of variations in mobile phase composition, Different columns (different lots and / or suppliers), Temperature, Flow rate. Methanol or Trifluoroacetic acid content in mobile phase Created by Dr. Nishodh Saxena Page 10 of 11
The above method parameters may be evaluated one factor at a time or simultaneously as a part of a factorial experiment. Obtaining data on the effects of all the above mentioned parameters may allow a range of acceptable values to be included in the final method procedure. For example, if column performance changes over time, adjusting the mobile-phase strength to compensate for changes in the column may be allowed if such data are included in the validation. 2.9 System suitability System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analysed constitute an integral system that can be evaluated as such to ensure that the validity of the assay is maintained. System suitability tests are an integral part of chromatographic and electrophoresis methods. These tests are used to verify that the resolution and reproducibility of the system are adequate for the analysis to be performed. It is to check the system to ensure system performance is unchanged before or during the analysis. Parameters such as plate count, tailing factors; resolution and reproducibility (% RSD retention time and area for six repetitions) are determined and compared against the specifications set for the method. These parameters are measured during the analysis of system suitability (using a sample that is a mixture of main components and expected by-products). Created by Dr. Nishodh Saxena Page 11 of 11