1. Supplementary Training Modules on Good Manufacturing Practices
Validations –part 5
Workshop on
GMP and Quality Assurance of TB products
Kuala Lumpur
Malaysia, 21 – 25 February 2005
Maija Hietava
M.Sci.Pharm
Quality Assurance and Safety: Medicines, Medicines Policy and Standards, Health Technology and Pharmaceuticals Cluster
Tel: Fax:
World Health Organization

2. Analytical validation
Introduction
Why is analytical monitoring necessary?
What is the purpose of analytical validation?
Introduction:
Analytical monitoring of a pharmaceutical product, or of specific ingredients within the product, is necessary to ensure its safety and efficacy throughout all phases of its shelf-life, including storage, distribution, and use. This monitoring should be conducted in accordance with specifications validated during product development.
The principal purpose of analytical validation is to ensure that a selected analytical procedure will give reproducible and reliable results that are adequate for the intended purpose. It is necessary to define properly both the conditions in which the procedure is to be used and the purpose for which it is intended. These principles apply to all procedures described in a pharmacopoeia and to non-pharmacopoeia procedures used by a manufacturing company. These guidelines apply to procedures used to examine chemical and physico­chemical attributes, but many are equally applicable to microbiological and biological procedures.

3. Validation Objectives To introduce the concepts of :
Protocol development
Instrument qualification
Analytical procedure
Extent of validation
Method transfer
The objectives of Part 4, Module 1 are to introduce the concepts of analytical validation:
Protocol development
Instrument qualification
Analytical procedure, with some illustrated examples of some specific characteristics, such as linearity and precision
Extent of validation
Method transfer and
Chemical and physical, biological and microbiological test validation.

4. Validation Validation of analytical procedures requires:
Qualified and calibrated instruments
Documented methods
Reliable reference standards
Qualified analysts
Sample integrity
Validation of analytical procedures requires:
Qualified and calibrated instruments; the qualification is identical to that previously discussed for production equipment.
Documented methods: the test must be documented for the laboratory itself and it should not just be a photocopy of a method, such as pharmacopoeial methods, which sometimes do not spell out exact details.
Reliable reference standards: these should preferably be primary reference materials sourced from the pharmacopoeia commission or a National Control Laboratory, or secondary standards that have been qualified against the primary reference material and have been fully characterized.
Qualified analysts: experience and qualifications as detailed in a previous module within the WHO Basic Training Modules on GMP, on Personnel (Module 8). There should be training and proficiency testing records cross-referenced in the validation report.
Sample integrity: the sample must be beyond reproach – see also the slide for microbiological testing validation.

5. Validation Validation protocol for analytical method
Statement of purpose and scope
Responsibilities
Documented test method
List of materials and equipment
Procedure for the experiments for each parameter
Statistical analysis
Acceptance criteria for each performance parameter
Validation protocol for analytical method:
The validation protocol for an analytical method is very similar to that required for
any process validation. It should include:
Statement of purpose and scope
Responsibilities for approval, execution and review
The documented test method which should have a unique reference number
List of materials and equipment: the instruments, columns, reagents, test kits and so on
Procedure - details of the experiments for each performance parameter
Statistical analysis. These are usually standard deviation, linear regression, and Analysis of Variants or ANOVA.
Acceptance criteria for each performance parameter which should be settled before the experimental phase of the work commences.

6. Validation Qualification of the instrument
Supplier, model and supplier´s manual
Modifications
Installation and operational qualification
Calibration programs
Maintenance schedules
Instrument qualification:
Critical instruments in the QC laboratory have similar qualification requirements to equipment used in the factory. There should be available:
Make, model and maker’s manual.
Any modifications that have been made to the instrument since it was purchased. This is very important because the servicing of the instrument could result in upgrades, e.g. to software, that are not revealed by the service person to the QC supervisor.
Installation and operational qualification.
Calibration programs.
Maintenance schedules.

7. Validation Characteristics of analytical procedures (1) Accuracy
Precision
Repeatability
Reproducibility
Characteristics of analytical procedures:
Accuracy
The accuracy of the procedure is the closeness of the results obtained by the procedure to the true value. Accuracy may be determined by applying the procedure to samples of the material to be examined that have been prepared with quantitative accuracy. Wherever possible, these samples should contain all the components of the material, including the analyte. Possibly three methods: 1. Spike into placebo matrix: active component is added to (“spiked”) in known amounts usually ranging from 25% to 150% of dose strength; 2. Standard addition technique; and 3. Comparison of two methods for equivalence.
Precision
The precision of the procedure is the degree of agreement among individual test results. It is measured by the scatter of individual results from the mean and it is usually expressed as the standard deviation or as the coefficient of variation . 
(The relationship between Accuracy and Precision is shown on the next slide.)
Repeatability (within-laboratory Variation)
This is the precision of the procedure when repeated by the same analyst under the same set of conditions (same reagents, equipment, settings, and laboratory) and within a short interval of time.  
Reproducibility
This is the precision of the procedure when it is carried out under different conditions—usually in different laboratories—on separate, putatively identical samples taken from the same homogeneous batch of material.  

8. Relationship between accuracy and precision
Validation
Relationship between accuracy and precision
The relationship between accuracy and precision can be represented by arrows being shot at a target.
The first small target at the top shows the arrows have landed indiscriminately. This is neither accurate nor precise.
The second target on the left shows the arrows have grouped together nicely but are not on the bullseye. This is precise but inaccurate. This is sometimes called analytical bias and sometimes a correction factor can be applied.
The third, small target shows the arrows AVERAGE is on the bullseye, but the precision is unacceptable.
The fourth, large target shows the arrows are all clustered on or in the bullseye; this shows accuracy and precision.
Accurate AND Precise

9. Validation Characteristics of analytical procedures (2) Ruggedness
Robustness
Variability caused by:
Day-to-day variations
Analyst-to-analyst
Laboratory-to-laboratory
Instrument-to-instrument
Chromatographic column-to-column
Reagent kit-to-kit
Instability of analytical reagents
Characteristics of analaytical procedures: (Contd)
Ruggedness and Robustness
Robustness, and ruggedness, of an analytical procedures is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters, and thus provides an indication of the reliability of the method during normal usage, under various conditions.
Ruggedness is due to factors external to the method; robustness is due to factors internal to the method.
Things that may cause variability include:
Day-to-day variations in e.g. temperature, relative humidity, etc.
Analyst-to-analyst
Laboratory-to-laboratory
Instrument-to-instrument
Chromatographic column-to-column
Reagent kit-to-kit or lot-to-lot variation
Time from sample preparation to assay
Instability of analytical reagents

10. Validation Characteristics of analytical procedures (3)
Linearity and range
Specificity
Sensitivity
Limit of detection
Limit of quantitation
Characteristics of analytical procedures: (Contd.)
Linearity and range
The linearity of an analytical procedure is its ability to produce results that are directly proportional to the concentration of analyte in the samples. The range of the procedure is an expression of the lowest and highest levels of analyte that have been demonstrated to be determinable with acceptable precision, accuracy, and linearity.
Specificity
The specificity or selectivity of a procedure is its ability to measure the analyte in a manner that is free from interference from other components in the sample being examined (for example, impurities arising from manufacture or degradation or ingredients other than the analyte).
Sensitivity
The specificity or sensitivity is the capacity of the test procedure to record small deviations in concentration. It is the slope of the calibration curve. A more general use of the term to encompass limit of detection and or limit of quantitation should be avoided.
Limit of detection
The limit of detection is the lowest level of analyte that can be detected, but not necessarily determined in a quantitative fashion.
Limit of quantitation
The limit of quantitation is the lowest concentration of analyte in a sample that may be determined with acceptable accuracy and precision when the required procedure is applied.

11. Validation LOQ, LOD and SNR Limit of Quantitation Limit of Detection
Signal to Noise Ratio
Peak B LOQ
Peak A LOD
There are no specific criteria set for the Limit of Quantitation (LOQ) and Limit of Detection (LOD) but guidance is available from specifications and pharmacopeias.
The noise is measured by running the instrument at maximum gain with no test being processed. The ripple generated is noise due to the instrument’s electronics, etc. The peak is measured relative to this noise and the ratio is calculated. This is known as the Signal to Noise Ratio (SNR).
Generally:
LOD SNR should be greater than 2:1. Peak A is acceptable for LOD but not for quantitation;
The LOD can be calculated if the standard deviation (SD) of the response (which is standard deviation of the blank) and the slope is determined:
LOD = 3.3 x SD
slope
Similarly LOQ = 10 x SD
The LOQ SNR should generally be above 10:1. Peak B is suitable for quantitation;
Precision as a percentage relative standard deviation should be % at the limit for LOQ.
noise
Baseline

12. Validation Different classes of analytical tests
Class A: To establish identity
Class B: To detect and quantitate impurities
Class C: To determine quantitatively the concentration
Class D: To assess the characteristics
What analytical characteristics are applicable in particular cases?
Not all of the characteristics previously discussed will need to be considered in all validation cases; those applicable should be identified on a case-by-case basis. As a guide, however, the following generalizations may assist.
Methods used for the examination of pharmaceutical materials may be
broadly classified as follows:
• Class A: Tests designed to establish identity, whether of bulk drug substances or of a particular ingredient in a finished dosage form.
• Class B: Methods designed to detect and quantitate impurities in a bulk drug substance or finished dosage form. The detection of impurities, without quantitation, is referred to as a limit test.
• Class C: Methods used to determine quantitatively the concentration of a bulk drug substance or of a major ingredient in a finished dosage form; referred to as assay.
• Class D: Methods used to assess the characteristics of finished dosage forms, such as dissolution profiles and content uniformity.

13. Validation Characteristic A B quant. B Limit test C D Accuracy X X*
Validation
Characteristic A
B quant. B
Limit test C D
Accuracy X X*
Precision
Robustness
Linearity and range
Specificity
Limit of detection
Limit of quantitation
This table offers guidelines to the characteristics that are relevant in each case. There will clearly be occasions when certain characteristics marked as not being required may be necessary and vice versa.
The purpose for which the testing is being made may have a bearing on the choice of characteristics and the extent to which they are specified.
For example, although Classes B, C, and D are all referred to in Table 1 as requiring consideration of precision, the stringency applied may be different. For estimation of an impurity it may not be necessary to be as precise as for quantitative assessment of a bulk drug substance. By the same token, a degree of bias may be acceptable in determining the accuracy of a test for uniformity of content (Class D) that would not be permissible for a quantitative assessment of the concentration of an ingredient in a finished dosage form (Class C). Similarly, a test designed to establish the identity of a new drug entity, for which no previous data have been lodged, may need to be considerably more searching than tests designed to verify the identity of a long-established drug substance to be included in a pharmacopoeia.
A different emphasis may be required for pharmacopoeial as opposed to registration purposes. For example, robustness is a critical characteristic for pharmacopoeial methodology but may be less significant for a manufacturer’s release specification.
Suggested Reading:
WHO Expert Committee on Specifications for Pharmaceutical Preparations. Thirty-second Report. Geneva, World Health Organization, 1992 (WHO Technical Report Series No. 823). Annex 5: Validation of analytical procedures used in the examination of pharmaceutical materials.
ICH Harmonised Tripartite Guidelines. Text on validation of analytical procedures and validation of analytical procedures: Methodology. October and November 1996 respectively.
* A degree of bias may be allowed

14. Validation Extent of validation
New methods require complete validation
Pharmacopoeial methods require partial validation (or verification)
Significant changes mean partial revalidation
equipment changes
formula changed
changed suppliers of critical reagents
Extent of validation required:
New (from manufacturer/literature) methods require complete validation.
Methods in pharmacopoeias require partial validation, if the method has not been previously validated for that specific drug product. Manufacturers should validate pharmacopoeial methods to ensure they work with their own products - as a minimum accuracy and specificity. The USP monograph states: “Already established general assays and tests - should also be validated to verify their accuracy (and absence of possible interference) when used for a new product or starting materials.”
At least partial revalidation is required whenever significant changes are made which could reasonably be expected to affect the results obtained, e.g. in case of instrument change, product formula change, changed suppliers of critical reagents, method.

15. Validation Analytical method transfer
Method transfer protocol and procedure
precision
accuracy
ruggedness
Written and approved specific test method
Proficiency check
Formal acceptance by new laboratory
Analytical method transfer:
An analytical method may need to be transferred from, say Research and Development laboratory, to the QC laboratory. This should:
require documented evidence of method transfer
require a method transfer protocol and procedure
The protocol should address performance parameters: - precision - accuracy - ruggedness
Documentation must be written and approved for the specific test method before the transfer is verified.
Proficiency: The method must be followed by the new laboratory without input (or coaching - since any training must already have been given) from the originator laboratory. There should be three assays in the new laboratory which are compared to originator’s results.
Formal acceptance is then required by the new laboratory’s management.

16. Validation Typical validation of HPCL assay (1)
System suitability (performance check)
system precision
column efficiency
symmetry factor
capacity factor
The system suitability tests are carried out during the method development phase, prior to method validation. These tests are designed to evaluate the performance of the entire system.
It is done by analysing a “system suitability” sample, which consists of the main components, including impurities. This may also contain excipients, which may interfere with peaks of interest.
The system suitability is evaluated in terms of the following parameters:
- system precision
- column efficiency (usually >2000)
- symmetry factor (acceptance criteria 0.9 to 2.5)
- capacity factor (acceptance criteria NLT 1.5)

17. Validation Typical validation of HPLC assay (2) Method validation
specificity
accuracy
precision
linearity
robustness
Following a system suitability test, the actual analytical method is then validated by checking:
Specificity: by checking that the method is free of interference from excipients, impurities, etc.
Accuracy: by checking that the method gives closeness to true results.
Precision: by checking that the method is precise.
Linearity: by checking that the method will produce results that are directly proportional to the concentration of analyte in the samples.
Robustness: by checking that the method will withstand deliverate changes.

18. Validation Biological assays Can be difficult to "validate"
"Validity" on a case by case basis
Strictly adhere to the Biological Testing monographs in pharmacopoeias
Biological assays:
Biological Method Validation: Examples are rabbit pyrogen testing. The principles and performance parameters listed in the general monograph in the various pharmacopoeias for method validation can be applied to biological assays systems. However, it can be difficult to "validate" a biological assay using the characteristics previously described. Biological systems contain a larger variability than a chemical or physical test system. It is not realistic to apply the same acceptance criteria to biological systems since it is not possible to totally define all the key factors that affect the assays let alone control them.
Validation is still a critical issue with biological systems. Consequently, there must be good assay design, elimination of systematic bias and “built-in” validity.
"Validity" is assessed on a case-by-case basis: validation design should include the following:
The assay is performed in triplicate;
The assay includes three different dilutions of the standard preparation and three dilutions of sample preparations of activity similar to that of the standard preparation;
The assay layout is randomised; ideally the analyst should be “blinded” to sample and location on the matrix;
If the test sample is presented in serum or formulated with other components, the standard is likewise prepared.
Lastly, the biological testing monographs in pharmacopoeias must be strictly adhered to with no deviations permitted.

19. Microbiological testing requiring validation
Microbial limit testing
Microbial count
Sterility testing
Preservative effectiveness testing
Environmental monitoring program
Biological testing
Microbiological methods also need validation. In each microbiological testing laboratory, the types of methods which must be validated or verified include:
Microbial limit testing - such as limit tests for indicator organisms, nominated in pharmacopoeias (BP/EP and USP specify strains of Escherichia coli, Staphylococcus aurens, Pseudomonas aeruginosa and Salmonella typhimurium).
Microbial count - such as total viable aerobic counts of starting materials and finished products.
Sterility testing
Preservative effectiveness testing
Environmental monitoring program - such as water, air and surface monitoring
Biological testing - such as large plate assays for vitamins or antibiotics.

20. Sterility testing validation requirements
Media growth promotion, sterility, pH
Product validation
Stasis testing
Environmental monitoring
Negative controls
Challenge organisms
Sterility test validation requirements:
The official sterility test must be validated against each product since inhibitory
substances, even when present at very low levels, may stop a contaminant growing within the prescribed time. Sterility testing validation requirements include:
The media must be competent before use. That is, each batch of sterilized media should be tested for growth promotion (fertility test), sterility and pH.
Product validation; by spiking a low level of challenge organisms and demonstrating clear, visible evidence of growth within 3 days for bacteria and 5 days for fungi.
“Stasis testing” at the end of the incubation period to demonstrate that culture media supports growth for the full period. Note that this is not a mandated requirement, but is recommended as part of Good Laboratory Practice (GLP).
Environmental monitoring of sterility test environment must be satisfactory; settle plates and operator checks should be performed.
Negative controls are required to demonstrate that the analyst has the necessary dexterity to carry out the test manipulations properly. Usually the negative control is double sterilized product but its choice should also reflect the manipulations required for the test sample. Negative controls are usually conducted before and after a testing session.
The challenge test organisms are:
Staphylococcus aureus, Bacillus subtilis, Pseudomonas aerugihosa, Aspergillus niger, Clostridium sporogenes, and Candida albicans. For each challenge organism, the inoculum level is CFU. Validation tests using live microorganisms must not be undertaken in the sterility testing area.

21. Validation
Question no 34
What is the validation parameter showing that results of analysis are almost all in line??
Sterility test validation requirements:
The official sterility test must be validated against each product since inhibitory
substances, even when present at very low levels, may stop a contaminant growing within the prescribed time. Sterility testing validation requirements include:
The media must be competent before use. That is, each batch of sterilized media should be tested for growth promotion (fertility test), sterility and pH.
Product validation; by spiking a low level of challenge organisms and demonstrating clear, visible evidence of growth within 3 days for bacteria and 5 days for fungi.
“Stasis testing” at the end of the incubation period to demonstrate that culture media supports growth for the full period. Note that this is not a mandated requirement, but is recommended as part of Good Laboratory Practice (GLP).
Environmental monitoring of sterility test environment must be satisfactory; settle plates and operator checks should be performed.
Negative controls are required to demonstrate that the analyst has the necessary dexterity to carry out the test manipulations properly. Usually the negative control is double sterilized product but its choice should also reflect the manipulations required for the test sample. Negative controls are usually conducted before and after a testing session.
The challenge test organisms are:
Staphylococcus aureus, Bacillus subtilis, Pseudomonas aerugihosa, Aspergillus niger, Clostridium sporogenes, and Candida albicans. For each challenge organism, the inoculum level is CFU. Validation tests using live microorganisms must not be undertaken in the sterility testing area.