VAL-015 Method Validation Procedure

1. General Requirements

1.1. Validation of an analytical or microbiological method is the process by which it is established by Laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications.  Methods must be re-validated if conditions are changed significantly.

1.2. New and revised analytical and microbiological methods are supported by sufficient Laboratory data to document the validity of these procedures.  The Technical Department is responsible for ensuring that analytical and microbiological testing methods comply with the validated procedures and have been approved by the relevant authorities, before being released into either Laboratory.

1.3. Ideally a method should be based on established techniques requiring commonly available equipment with the greatest selectivity.

1.4. The validation evaluation includes the assessment of the clarity and completeness of the description of the method, the determination of the need for the method and documentation that the methods have been appropriately validated.  It is essential to establish that analytical procedures produce data that are sufficiently accurate and precise for their intended purpose.

1.5. Revised procedures should compare the limitations of the current assay and the advantages offered by the proposed method.

1.6. All analytical procedures must be validated prior to generating data that is intended for regulatory submissions or that are used to test finished products, raw materials or packaging components.

1.7. Cleaning processes are validated to the degree appropriate to their intended use.

1.8. Analytical and Microbiological Method Validation should:

– Identify the need for the assay.

– Describe the capability of the specific method proposed.

– Provide a rationale for use of the new/modified procedure.

– Biological testing, for example sterility, endotoxin testing and the microbiological examination of non-sterile products are validated in line with Pharmacopoeial or other standard requirements.

1.9. The proposal should contain a complete description of the method in sufficient detail so a technician may replicate it with ease.

The method should include:

– All important operational parameters.

– Specific instructions e.g. preparation of reagents, media, storage of reference standards.

– Methodology for the performance of systems suitability tests.

– Descriptions of blanks used.

– Safety precautions.

– Explicit formulas for the calculation of test results.

1.10. Thorough and complete documentation of the method validation should be provided.
The documentation should include summaries of experimental data and calculations substantiating each of the applicable analytical performance parameters.

1.11. Performance characteristics are expressed in terms of analytical parameters:
accuracy, precision, specificity, limit of detection, limit of quantitation, robustness, linearity and range and apply to both Biological and Chemical assays.

1.12. It is unrealistic to apply the same acceptance criteria to a Biological assay and microbial evaluations as is applied to Chemical testing since by their very nature biological systems contain inherent variability.  It is not possible to define and control all the key factors that affect the assays.  Microbiological validation must be supported by good assay design, the elimination of systematic bias in the assay and proper analysis and validity testing.  The source of the test method should be noted in the validation documentation.

1.13. Microbiological test methods should address the following limitations during validation:

– The incubation temperature and time supports the growth of the appropriate organisms.

– The medium used supports the growth of the appropriate organisms.

– The disinfectants may inhibit growth.

– That sampling procedures, sampling handling and transport may affect test results.

2. Assay Categories

Assay procedures vary from highly exacting analytical determinations to subjective evaluations of attributes.  Therefore different test methods require different validation schemes.

2.1. Category I

Analytical methods for quantitation of major excipients and/or active ingredients, and preservatives in finished goods.

2.2. Category II

Analytical methods for determination of impurities or degradation compounds in finished goods.  These methods include quantitative assays and limit tests, titrimetric and bacterial endotoxin tests.

2.3. Category III

Analytical methods for determination of performance characteristics, e.g. sterility testing, dissolution and drug release for pharmaceutical products.

3. Data Elements Required for Assay Validation

* May be required depending on the nature of the specific test.

4. Analytical Performance Parameters

4.1. Accuracy

4.1.1. Definition

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value.  Accuracy may often be expressed as percent recovery by the assay of known, added amounts of analyte.  Accuracy is a measure of the exactness of the analytical method that is true for all practical purposes.

4.1.2. Determination

The accuracy of an analytical method may be determined by applying that method to samples or mixtures of excipients to which known amounts of analyte have been added both above and below the normal levels expected in the samples.  The accuracy is then calculated from the test results as the percentage of analyte recovered by the assay.

4.2. Precision

4.2.1. Definition

The precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogeneous sample.  The precision of an analytical method is usually expressed as the standard deviation or relative standard deviation (coefficient of variation).  Precision may be a measure of either the degree of reproducibility or of repeatability of the analytical method under normal operating conditions.  In this context, reproducibility refers to the use of the analytical procedure in different laboratories.  Intermediate precision expresses within-Laboratory variation, as on different days, or with different analysts to the use of the analytical procedure within a Laboratory over a short period of time using the same analyst with the same equipment.  For most purposes, repeatability is the criterion of concern in analytical procedures.

4.2.2. Determination

The precision of an analytical method is determined by assaying a sufficient number of aliquots of a homogeneous sample to be able to calculate statistically valid estimates of standard deviation or relative standard deviation (coefficient of variation).  Assays in this context are independent analyses of samples that have been carried through the complete analytical procedure from sample preparation to final test result.

4.3. Specificity

4.3.1. Definition

The specificity of an analytical method is its ability to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the sample matrix.  Specificity may often be expressed as the degree of bias of test results obtained by analysis of samples containing added impurities, degradation products, related chemical compounds, or placebo ingredients when compared to test results from samples without added substances.  The bias may be expressed as the difference in assay results between the two groups of samples.  Specificity is a measure of the degree of interference (or absence thereof) in the analysis of complex sample mixtures.

4.3.2. Determination

The specificity of an analytical method is determined by comparing test results from the analysis of samples containing impurities, degradation products, or placebo ingredients with those obtained from the analysis of samples without impurities, degradation products, or placebo ingredients.  The bias of the assay, if any, is the difference in test results between the two groups of samples.  When impurities or degradation products are unidentified or unavailable, specificity may be demonstrated by analysis by the method in question of samples continuing impurities or degradation products and comparing the results to those from additional purity assays (e.g. chromatographic assay, phase solubility differential scanning calorimetry).  The degree of agreement of test results is a measure of the specificity.

4.4. Limit of Detection

4.4.1. Definition

The limit of detection is a limit test parameter.  It is the lowest concentration of analyte in a sample that can be detected, but not necessary quantitated, under the stated experimental conditions.  Thus, limit tests merely substantiate that the analyte concentration is above or below a certain level.  The limit of detection is usually expressed as the concentration of analyte, (e.g. percentage, parts per billion) in the samples.

4.4.2. Determination

Determination of the limit of detection of an analytical method will vary depending on whether it is an instrumental or a non-instrumental procedure.  For instrumental procedures, different techniques may be used.  Some investigators determine the signal-to-noise ratio by comparing test results from samples with known concentrations of analyte with those of blank samples and establish the minimum level at which the analyte can be reliably detected.  A signal-to-noise ratio of 2:1 or 3:1 is generally accepted.  Other investigators measure the magnitude of analytical background response by analysing a number of blank samples and calculating the standard deviation of this response.  The standard deviation multiplied by a factor, usually 2 or 3 provides an estimate of the limit of detection.  This limit is subsequently validated by the analysis of a suitable number of samples known to be near to or at the limit of detection.

For non-instrumental methods, the limit of detection 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 reliably detected.

4.5. Limit of Quantitation

4.5.1. Definition

Limit of quantitation is a parameter of quantitative assays for low levels of compounds in sample matrices, such as impurities in bulk drug substances and degradation products in finished Pharmaceuticals.  It is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions.  The limit of quantitation is expressed as the concentration of analyte, (e.g. percentage, parts per billion) in the sample.

4.5.2. Determination

Determination of the limit of quantitation of an analytical method may vary depending on whether it is an instrumental or a non-instrumental procedure.  For instrumental procedures, a common approach is to measure the magnitude of analytical background response by analysing a number of blank samples and calculating the standard deviation of this response.  The standard deviation multiplied by a factor, usually 10, provides an estimate of the limit of quantitation.  This limit is subsequently validated by the analysis of a suitable number of samples known to be near or prepared at the limit of quantitation.  For non-instrumental methods, the limit of quantitation 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 detected with acceptable accuracy and precision.

4.6. Linearity and Range

4.6.1. Definition of Linearity

The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range.  Linearity is usually expressed in terms of the variance around the slope of the regression line calculated according to an established mathematical relationship from test results obtained by the analysis of samples with varying concentrations of analyte.

4.6.2. Determination of Range

The range of an analytical method is the interval between the upper and lower levels of analyte (including these levels) that have been demonstrated to be determined with precision, accuracy, and linearity using the method as written.  The range is normally expressed in the same units as test results, (e.g. percent, parts per million) obtained by the analytical method.

4.6.3. Determination of Linearity and Range

The linearity of an analytical method is determined by mathematical treatment of test results obtained by analysis of samples with analyte concentrations across the claimed range of the method.  The treatment is normally a calculation of a regression line by the method of least squares of test results versus analyte concentrations.  In some cases, to obtain proportionality between assays and sample concentrations, the test data may have to be subjected to a mathematical transformation prior to the regression analysis.  The slope of the regression line and its variance provide a mathematical measure of linearity; the Y-intercept is a measure of the potential assay bias.  Plotting the test results graphically as a function of analyte concentration on appropriate graph paper may be an acceptable alternative to the regression line calculation.

The range of the method is validated by verifying that the analytical method provides acceptable precision, accuracy, and linearity when applied to samples containing analyte at the extremes of the range as well as within the range.

4.7. Robustness

4.7.1. Definition

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

4.7.2. Examples of typical variations are:

a. Variations in pH of testing solutions

b. Temperature

c. Chemical composition of the testing solutions

d. Variations in testing consumables and durables

e. Stability of the solutions

f. Extraction time

g. Time taken to do the test.

5. System Suitability Testing

System suitability 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.  System suitability test parameters to be established for a particular procedure depend on the type of procedure being validated.

6. Reference Standards

It is not a universal requirement to use a reference (primary) standard each time a product is tested.  A reference standard is often called a primary standard. However, a house standard/working standard may be used in place of the reference standard if it has been properly qualified against that primary standard, to determine its purity.

For validation of a new chemical entity for development work, it is mandatory that a primary standard is used as the product purity of the active to be in used in the trials is often unknown..

7. Summary of Changes