2 validation tutorial jntu pharmacy
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2 validation tutorial jntu pharmacy
- 1. 1 BY Dr. Suman Pattanayak Associate Professor Department of Pharma Analysis & QA. Vijaya Institute of Pharmaceutical Sciences for Women M. Pharm/ I Sem Advance Pharmaceutical Analysis
- 2. OverviewOverview What is Validation?What is Validation? – This section will define validation and will put its meaning inThis section will define validation and will put its meaning in terms pertinent for a chemical engineer.terms pertinent for a chemical engineer. Food and Drug Administration (FDA)Food and Drug Administration (FDA) – This section will explain the role of the FDA in validation and theThis section will explain the role of the FDA in validation and the guidelines it sets forth.guidelines it sets forth. Equipment ValidationEquipment Validation – This section will explain what role unit operations equipmentThis section will explain what role unit operations equipment plays in validation and why that is important.plays in validation and why that is important. Process ValidationProcess Validation – This section will explain the implications of validation in theThis section will explain the implications of validation in the overall manufacturing process.overall manufacturing process. Applications to Facility DesignApplications to Facility Design – This section will discuss considerations to facility design in lightThis section will discuss considerations to facility design in light of validation.of validation.
- 3. What is Validation?What is Validation? According to the Food and Drug Administration (FDA), the goal ofAccording to the Food and Drug Administration (FDA), the goal of validation is to:validation is to: ““establish documented evidence which provides a high degree ofestablish documented evidence which provides a high degree of assurance that a specific process will consistently produce aassurance that a specific process will consistently produce a product meeting its predetermined specifications and qualityproduct meeting its predetermined specifications and quality attributes.”attributes.” [1][1]
- 4. What is Validation?What is Validation? What does this mean?What does this mean? – An quantitative approach is needed to prove quality,An quantitative approach is needed to prove quality, functionality, and performance of afunctionality, and performance of a pharmaceutical/biotechnological manufacturing process.pharmaceutical/biotechnological manufacturing process. – This approach will be applied to individual pieces of equipmentThis approach will be applied to individual pieces of equipment as well as the manufacturing process as a whole.as well as the manufacturing process as a whole. – Guidelines for validation are set by the FDA, but the specifics ofGuidelines for validation are set by the FDA, but the specifics of validation are determined by the pharmaceutical/biotechvalidation are determined by the pharmaceutical/biotech company.company.
- 5. What is Validation?What is Validation? Phases of ValidationPhases of Validation – Validation is broken down into three phases:Validation is broken down into three phases: Installation Qualification (IQ)Installation Qualification (IQ) Operational Qualification (OQ)Operational Qualification (OQ) Performance Qualification (PQ)Performance Qualification (PQ) – These three protocols are used to define tests that willThese three protocols are used to define tests that will demonstrate that the process consistently and repeatedlydemonstrate that the process consistently and repeatedly produces the desired productproduces the desired product..
- 6. What is Validation?What is Validation? Installation Qualification (IQ)Installation Qualification (IQ) – This is the first step in validation.This is the first step in validation. – This protocol insures that the system/equipment and itsThis protocol insures that the system/equipment and its components are installed correctly and to the originalcomponents are installed correctly and to the original manufacturer’s specifications.manufacturer’s specifications. – Calibration of major equipment, accessory equipment, and/orCalibration of major equipment, accessory equipment, and/or utilities should be performed in this step as well.utilities should be performed in this step as well.
- 7. What is Validation?What is Validation? Operational Qualification (OQ)Operational Qualification (OQ) – This step proceeds after the IQ has been performed.This step proceeds after the IQ has been performed. – In the OQ, tests are performed on the critical parameters of theIn the OQ, tests are performed on the critical parameters of the system/process. These are usually the independent and/orsystem/process. These are usually the independent and/or manipulated variables associated with the system/equipment.manipulated variables associated with the system/equipment. – All tests’ data and measurements must be documented in orderAll tests’ data and measurements must be documented in order to set a baseline for the system/equipment.to set a baseline for the system/equipment.
- 8. What is Validation?What is Validation? Performance Qualification (PQ)Performance Qualification (PQ) – This is the third and final phase of validation.This is the third and final phase of validation. – This phase tests the ability of the process to perform over longThis phase tests the ability of the process to perform over long periods of time within tolerance deemed acceptable.periods of time within tolerance deemed acceptable. – PQ is performed on the manufacturing process as a whole.PQ is performed on the manufacturing process as a whole. Individual components of the system are not tested individually.Individual components of the system are not tested individually.
- 9. What is Validation?What is Validation? An example validation protocol can be seen here:An example validation protocol can be seen here: sample validation protocolsample validation protocol..
- 10. FDAFDA The FDA is a federal science-based law enforcement agencyThe FDA is a federal science-based law enforcement agency mandated to protect public health.mandated to protect public health. The validation process is regulated by the guidelines andThe validation process is regulated by the guidelines and restrictions set forth by the FDA. However, the actual validationrestrictions set forth by the FDA. However, the actual validation protocol, documentation, and execution is the responsibility of theprotocol, documentation, and execution is the responsibility of the manufacturer. More specifically, this is the responsibility of themanufacturer. More specifically, this is the responsibility of the engineer.engineer.
- 11. FDAFDA Code of Federal Regulations (CFR)Code of Federal Regulations (CFR) – This is the body of regulations, created by the US government,This is the body of regulations, created by the US government, that sets forth the guidelines pertaining to food and drugs.that sets forth the guidelines pertaining to food and drugs. – 21 CFR Part 21021 CFR Part 210 concerns current good manufacturing practiceconcerns current good manufacturing practice in manufacturing, processing, packing, or holding of drugs.in manufacturing, processing, packing, or holding of drugs. – 21 CFR Part 211 concerns current good manufacturing practice21 CFR Part 211 concerns current good manufacturing practice for finished pharmaceuticalsfor finished pharmaceuticals
- 12. FDAFDA – 21 CFR Part 600 pertains to the safe production of biological21 CFR Part 600 pertains to the safe production of biological derived products.derived products. – 21 CFR Part 610 pertains to the safe distribution of biologically21 CFR Part 610 pertains to the safe distribution of biologically derived products.derived products.
- 13. Equipment ValidationEquipment Validation As mentioned earlier, each piece of must be validated in order toAs mentioned earlier, each piece of must be validated in order to legally operate within the facility.legally operate within the facility. The goal is to produce consistent results with minimal variationThe goal is to produce consistent results with minimal variation without compromising the integrity of the product and the personswithout compromising the integrity of the product and the persons operating the equipment.operating the equipment. A plan of validation should be drafted and executed by engineers inA plan of validation should be drafted and executed by engineers in order to satisfy guidelines. The validation plan generally consists oforder to satisfy guidelines. The validation plan generally consists of IQ and OQ sections.IQ and OQ sections.
- 14. Equipment ValidationEquipment Validation Any major equipment changes after the initial validation will result inAny major equipment changes after the initial validation will result in the need for subsequent revalidation.the need for subsequent revalidation. In the end, equipment validation will create specification ranges andIn the end, equipment validation will create specification ranges and tolerances that will be applied to the normal operation of equipment.tolerances that will be applied to the normal operation of equipment.
- 15. Process ValidationProcess Validation The manufacturing process, in addition to the individual equipment,The manufacturing process, in addition to the individual equipment, must be validated.must be validated. The goal is to create a robust manufacturing process thatThe goal is to create a robust manufacturing process that consistently produces a drug product with minimal variation thatconsistently produces a drug product with minimal variation that adheres to quality criteria of purity, identity, and potency.adheres to quality criteria of purity, identity, and potency. A validation plan for the manufacturing process should be draftedA validation plan for the manufacturing process should be drafted and executed by engineers in order to satisfy guidelines. Theand executed by engineers in order to satisfy guidelines. The validation plan usually involves just a PQ section.validation plan usually involves just a PQ section.
- 16. Process ValidationProcess Validation Just as equipment validation, major changes after the initialJust as equipment validation, major changes after the initial validation will result in the need for subsequent revalidation.validation will result in the need for subsequent revalidation. In the end, process validation will ensure aIn the end, process validation will ensure a robust product that isrobust product that is highly reproducible over timehighly reproducible over time..
- 17. Applications to Facility DesignApplications to Facility Design Facilities should be designed in order to facilitate any changes thatFacilities should be designed in order to facilitate any changes that may arise after initial validation.may arise after initial validation. In the case of retrofitting, current facilities services (WFI, CIP, SIP,In the case of retrofitting, current facilities services (WFI, CIP, SIP, HVAC, etc.), equipment, and instrumentation should be assessedHVAC, etc.), equipment, and instrumentation should be assessed for revalidation. This assessment will be based on age of thefor revalidation. This assessment will be based on age of the individual system and the needs of the new process.individual system and the needs of the new process.
- 18. ValidationValidation ObjectivesObjectives To introduce the concepts of :To introduce the concepts of : Protocol developmentProtocol development Instrument qualificationInstrument qualification Analytical procedureAnalytical procedure Extent of validationExtent of validation Method transferMethod transfer Chemical and physical, biological, and microbiological testChemical and physical, biological, and microbiological test validationvalidation
- 19. Validation of analytical procedures requires: Qualified and calibrated instruments Documented methods Reliable reference standards Qualified analysts Sample integrity Validation
- 20. 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
- 21. Qualification of the instrument Make, model and maker’s manual Modifications Installation and operational qualification Calibration programs Maintenance schedules Validation
- 22. Characteristics of analyticalCharacteristics of analytical procedures (1)procedures (1) AccuracyAccuracy PrecisionPrecision RepeatabilityRepeatability ReproducibilityReproducibility ValidationValidation
- 23. Inaccurate & imprecise Inaccurate but precise Accurate but imprecise ValidationValidation Relationship between accuracy and precision Accurate AND Precise
- 24. Characteristics of analytical procedures (2)Characteristics of analytical procedures (2) RuggednessRuggedness RobustnessRobustness Variability caused by:Variability caused by: – Day-to-day variationsDay-to-day variations – Analyst-to-analystAnalyst-to-analyst – Laboratory-to-laboratoryLaboratory-to-laboratory – Instrument-to-instrumentInstrument-to-instrument – Chromatographic column-to-columnChromatographic column-to-column – Reagent kit-to-kitReagent kit-to-kit – Instability of analytical reagentsInstability of analytical reagents ValidationValidation
- 25. Characteristics of analytical procedures (3)Characteristics of analytical procedures (3) Linearity and rangeLinearity and range SpecificitySpecificity SensitivitySensitivity Limit of detectionLimit of detection Limit of quantitationLimit of quantitation ValidationValidation
- 26. Linearity of an analyte in a material 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.01 0.015 0.02 0.025 0.03 0.035 0.04 Reference material mg/ml Calculatedanalyteinmg/mL Table of values (x,y) x y # Reference material mg/ml Calculated mg/ml 1 0.0100 0.0101 2 0.0150 0.0145 3 0.0200 0.0210 4 0.0250 0.0260 5 0.0300 0.0294 6 0.0400 0.0410 ValidationValidation
- 27. Linearity StatisticsLinearity Statistics InterceptIntercept -0.0002-0.0002 Limit of Linearity and RangeLimit of Linearity and Range 0.005 – 0.0400.005 – 0.040 mg/mLmg/mL SlopeSlope 1.02371.0237 Correlation coefficientCorrelation coefficient – PearsonPearson 0.99780.9978 – Olkin and PrattOlkin and Pratt 0.99850.9985 Relative procedure standardRelative procedure standard deviationdeviation 3.4%3.4% ValidationValidation
- 28. LOQ, LOD and SNRLOQ, LOD and SNR Limit of QuantitationLimit of Quantitation Limit of DetectionLimit of Detection Signal to Noise RatioSignal to Noise Ratio noise Peak A LOD Peak B LOQ Baseline ValidationValidation
- 29. Different classes of analytical testsDifferent classes of analytical tests Class A: To establish identityClass A: To establish identity Class B: To detect and quantitate impuritiesClass B: To detect and quantitate impurities Class C: To determine quantitatively theClass C: To determine quantitatively the concentrationconcentration Class D: To assess the characteristicsClass D: To assess the characteristics ValidationValidation
- 30. * A degree of bias may be allowed Characteristic A B quant. B Limit test C D Accuracy X X X* Precision X X X Robustness X X X X X Linearity and range X X X Specificity X X X X X Limit of detection X Limit of quantitation X ValidationValidation
- 31. 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 ValidationValidation
- 32. Analytical method transferAnalytical method transfer Method transfer protocol and procedureMethod transfer protocol and procedure – precisionprecision – accuracyaccuracy – ruggednessruggedness Written and approved specific test methodWritten and approved specific test method Proficiency checkProficiency check Formal acceptance by new laboratoryFormal acceptance by new laboratory ValidationValidation
- 33. Chemical laboratory validation requirementsChemical laboratory validation requirements (1)(1) BalancesBalances ChromatographyChromatography – HPLC, HPTLC, GC, TLCHPLC, HPTLC, GC, TLC Dissolution or disintegration apparatusDissolution or disintegration apparatus Karl Fischer moisture determinationKarl Fischer moisture determination Melting, softening or freezing point apparatusMelting, softening or freezing point apparatus Ovens, refrigerators, incubatorsOvens, refrigerators, incubators ValidationValidation
- 34. Chemical laboratory validation requirementsChemical laboratory validation requirements (2)(2) pH meterpH meter Polarimeter - optical rotationPolarimeter - optical rotation RefractometerRefractometer Spectrophotometer UV/Vis, IR, FTIR, Raman, AASpectrophotometer UV/Vis, IR, FTIR, Raman, AA TimersTimers ViscometerViscometer Volumetric equipmentVolumetric equipment ValidationValidation
- 35. ValidationValidation Typical validation of HPCL assay (1)Typical validation of HPCL assay (1) System suitability (performance check)System suitability (performance check) – system precisionsystem precision – column efficiencycolumn efficiency – symmetry factorsymmetry factor – capacity factorcapacity factor
- 36. ValidationValidation Typical validation of HPLC assay (2)Typical validation of HPLC assay (2) Method validationMethod validation – specificityspecificity – accuracyaccuracy – precisionprecision – linearitylinearity – robustnessrobustness
- 37. Biological assaysBiological assays Can be difficult to "validate"Can be difficult to "validate" "Validity" on a case by case basis"Validity" on a case by case basis Strictly adhere to the Biological Testing monographs inStrictly adhere to the Biological Testing monographs in pharmacopoeiaspharmacopoeias ValidationValidation
- 38. Microbiological testing requiring validationMicrobiological testing requiring validation Microbial limit testingMicrobial limit testing Microbial countMicrobial count Sterility testingSterility testing Preservative effectiveness testingPreservative effectiveness testing Environmental monitoring programEnvironmental monitoring program Biological testingBiological testing ValidationValidation
- 39. Validation of microbial test procedures (1)Validation of microbial test procedures (1) Virtually impossible to completely validate testVirtually impossible to completely validate test procedures for every microorganismprocedures for every microorganism Neutralize /inactivate inhibitory substances, or diluteNeutralize /inactivate inhibitory substances, or dilute Periodic media challengePeriodic media challenge Media QCMedia QC Reliable methodsReliable methods ValidationValidation
- 40. Validation of microbial test procedures (2)Validation of microbial test procedures (2) Incubation temperature and timeIncubation temperature and time Media may not grow all microorganismsMedia may not grow all microorganisms Variations in media may affect recoveryVariations in media may affect recovery Inhibitory disinfectants or preservativesInhibitory disinfectants or preservatives SampleSample – proceduresprocedures – handling, storage, transporthandling, storage, transport ValidationValidation
- 41. Microbiological viable count methodMicrobiological viable count method validation (1)validation (1) MethodsMethods – pour plate / spread platepour plate / spread plate – membrane filtrationmembrane filtration – Most Probable NumberMost Probable Number SSample sizeample size Test dilutionTest dilution Inoculation sizeInoculation size ValidationValidation
- 42. Microbiological viable count methodMicrobiological viable count method validation (2)validation (2) Membrane filtration conditionsMembrane filtration conditions Incubation conditionsIncubation conditions Acceptance criteriaAcceptance criteria ValidationValidation
- 43. Sterility testing validation requirementsSterility testing validation requirements Media growth promotion, sterility, pHMedia growth promotion, sterility, pH Product validationProduct validation Stasis testingStasis testing Environmental monitoringEnvironmental monitoring Negative controlsNegative controls Challenge organismsChallenge organisms ValidationValidation
- 44. ResourcesResources [1] www.fda.gov[1] www.fda.gov – 21 CFR Part 210:21 CFR Part 210: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm? CFRPart=210&showFR=1CFRPart=210&showFR=1 – 21 CFR Part 211:21 CFR Part 211: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm? CFRPart=211&showFR=1CFRPart=211&showFR=1 – 21 CFR Part 600:21 CFR Part 600: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm? CFRPart=600&showFR=1CFRPart=600&showFR=1 – 21 CFR Part 610:21 CFR Part 610: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm? CFRPart=610&showFR=1CFRPart=610&showFR=1
- 45. ResourcesResources [2] www.validationworld.com[2] www.validationworld.com – Information concerning IQ: http://www.cqionline.com/knowledge/iq.htmlInformation concerning IQ: http://www.cqionline.com/knowledge/iq.html – Information concerning OQ: http://www.cqionline.com/knowledge/oq.htmlInformation concerning OQ: http://www.cqionline.com/knowledge/oq.html – Information concerning PQ: http://www.cqionline.com/knowledge/pq.htmlInformation concerning PQ: http://www.cqionline.com/knowledge/pq.html [3] Patricia Stewart-Flaherty. Performance Validation. Presentation. 13 Apr.[3] Patricia Stewart-Flaherty. Performance Validation. Presentation. 13 Apr. 2003.2003. [4] Sofer, Gail and Zabriskie, Dane W., ed.[4] Sofer, Gail and Zabriskie, Dane W., ed. Biopharmaceutical ProcessBiopharmaceutical Process ValidationValidation. New York: Marcel Dekker, 2000.. New York: Marcel Dekker, 2000. [4] Avis, Kenneth E., Wagner, Carmen M., Wu, Vincent L., ed.[4] Avis, Kenneth E., Wagner, Carmen M., Wu, Vincent L., ed. Biotechnology: Quality Assurance and ValidationBiotechnology: Quality Assurance and Validation. Buffalo Grove, Illinois:. Buffalo Grove, Illinois: Interpharm Press, Inc., 1999.Interpharm Press, Inc., 1999.
- 46. ResourcesResources [5] Carleton, Fredrick J. and Agalloco James P. ed.[5] Carleton, Fredrick J. and Agalloco James P. ed. Validation ofValidation of Pharmaceutical ProcessesPharmaceutical Processes. New York: Marcel Dekker, Inc., 1999.. New York: Marcel Dekker, Inc., 1999.
Editor's Notes
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- 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.
- This is a typical plot of raw data to check for linearity and range. The table of raw data on the right hand side has been plotted using linear regression to obtain a line of best fit. The x axis is the reference material that was weighed out and the y axis the calculated values from the instrument readings. These are plotted (the blue line with the points as a yellow box and error bars) and a linear regression analysis is performed. The two yellow lines are the 2-sided 95% confidence limits.
- Determination of linearity: The data from the chart on the previous slide is analysed to obtain: The Intercept value It is important not to force the line through zero as the xy intercept should be known in order to also check slope. Determination of Limit of Linearity and Range The assay should only be used within the range and linearity validated. The working range if the slope is linear should be 75% to 125% of the predicted content of the analyte. Slope “Slope” checks the ability to measure accurately small increments of analyte. This method shows a nearly one-to-one relationship. Correlation coefficient (r2) The correlation coefficient is calculated. Better than 0.99 is required but better than 0.999 should be routinely obtained for example, in case of HPLC assays. Standard deviation The standard deviation should be calculated. This generally should be below 2%. However, some manual methods, such as volumetric methods can be as high as 3%. Although linearity is acceptable, the value of 3.4% is unsatisfactory and could give arbitrarily high or low results resulting in possible release of sub- or super-potent material, or rejection of acceptable material.
- 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 slope The LOQ SNR should generally be above 10:1. Peak B is suitable for quantitation; Precision as a percentage relative standard deviation should be 5 - 10% at the limit for LOQ.
- 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.
- 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 TechnicalReport 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 1994 and November 1996 respectively.
- 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.
- 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.
- Chemical laboratory validation requirements: (Note to trainer: The trainer should discuss the PQ requirements of laboratory instruments, including calibration. When should it be done? And how? The list below should be checked in the laboratory and the requirements listed should be used by the trainer to stimulate discussion.) Balances: IQ and OQ, linearity, range, precision, accuracy. Chromatography: HPLC, HPTLC, GC, TLC: IQ and OQ plus - e.g. pump for solvent delivery – ripple, pressure, leaks; injector sample delivery precision; detector checks (eg variable UV, Refractive Index (RI) or diode array); and computer validation. Dissolution or disintegration apparatus: IQ, OQ and calibration. Computer validation if relevant. Karl Fischer: IQ, OQ. Computer validation if relevant. Melting, softening or freezing point apparatus: IQ, OQ. Check against calibrators. computer validation if relevant. Ovens, refrigerators, incubators, furnaces: IQ, OQ, heating time, cooling, thermal mapping. Data loggers may need computer validation.
- Chemical laboratory validation requirements: (Contd.) (Note to trainer: The list below should be checked in the laboratory and the requirements below should be used by the trainer to stimulate discussion.) pH meter: IQ, OQ, linearity, stability, slope, temperature. Polarimeter: Optical rotation - IQ, OQ and calibration against quartz discs or sucrose, computer validation if required. Refractometer: IQ, OQ. Temperature stability, water bath IQ and OQ if relevant, precision and accuracy. Spectrophotometer: UV/Vis, IR, FTIR, Raman, Atomic Absorption (AA): IQ, OQ and calibration. Computer validation if required. Timers: IQ, OQ and calibration against National Time Standard. Viscometer: IQ, OQ and calibration. Volumetric equipment: Autotitrators, nonaqueous titration equipment: IQ, OQ and linearity, precision, accuracy. Computer validation if required. Volumetric Glassware: pipettes, burettes, volumetric flasks: IQ.
- 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)
- 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.
- 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.
- 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.
- Validation of microbial test procedures: According to some people it is conceded that it is virtually impossible to completely validate test procedures for every microorganism that may be objectionable. Methods are, however, generally validated for recovery of specified indicator organisms nominated by pharmacopoeias and which generally are required to be absent from the product. Test method validation demonstrates that any inhibitory substances present in the sample have been neutralized/inactivated or diluted to a sub-inhibitory level. It involved inoculation of culture media with low levels of specified indicator organisms, in the presence and absence of the product/material to be tested. The capability of the media to promote the growth of organisms may be affected by the media preparation process, sterilization and storage procedures. The age of the culture media may affect growth promotion and selective properties of the media. For this reason, the shelf-life of culture media should be validated, and media not used beyond its expiry. All batches of prepared culture media should be subject to QC to ensure media is suitable for its intended purpose. Methods that the manufacturer selects must be able to reliably detect the presence of objectionable organisms such as Pseudomonas species, fungi, Escherichia coli, etc.
- Validation of microbial test procedures: (Contd.) The following parameters and reasons to validate microbiological methods include the facts that: Incubation temperature and time may not support the growth of all organisms. A classic example is demonstrated by water where the natural flora grow better at temperatures below 30oC, and for a longer period than is normally given other samples - up to or greater than 5 days. Media may not support the growth of all organisms. The use of Tryptic Soya Agar for water analysis may underestimate the microflora by up to 3 logs depending on time and temperature compared to say R2A or Plate Count Agar. Variations in media may affect recovery. For example, a recall of a product containing pseudomonas was necessary when media beyond its shelf life was used to screen for the objectionable microorganism. Disinfectants or preservatives may inhibit growth. Water with chlorine (even low levels) should have sodium thiosulphate added to the sample container to immediately neutralize the free chlorine. Sampling procedures, sample handling and transport may affect test results. If the sample is contaminated when the sample is drawn, or very long storage and very hot or cold storage temperatures are used, then the sample will be affected. A cold chain may need to be demonstrated if the laboratory is a long way from where the sample is drawn.
- Microbial viable count method validation: Methods commonly used - Pour plate or spread plate method at optimum dilution. Dilutions allowed to determine optimum 1:10 but generally no more than 1:100. - Membrane filtration method – is prefered for filterable products. -Most Probable Number – MPN, is generally the least accurate method for microbial counts. However, for certain product groups with very low bioburden where membrane filtration is not an option, it may be the most appropriate method. Sample size and dilutions The sample size should be of the order of 10g or 10mL although 1 – 5 g may be used for smaller lots. Test solution and dilutions must be specified. Use within a certain maximum period of time from preparation must be part of the validation. Challenge organisms for validationUse the organisms from a recognized type culture collection recommended by pharmacopoeias. Staphylococcus aureus Bacillus subtilis Escherichia coli Candida albicans Aspergillus niger Challenge inoculum10 –100 cfu total inoculated at 0.1ml to 100mL test solution. Organisms to be separately challenged and not mixed in one test
- Microbial viable count method validation: (Contd.) Membrane filtration (MF) conditionsFilter must be sterile with a hydrophobic edge if substances with antimicrobial agents are present. The mean pore size should be 0.45 micrometer not 0.2 micrometer because 0.2 causes inhibitory effects called mean pore diameter paradox. Solutions are also much slower to filter through a 0. 2 micrometer filter and can cause problems if filtering viscous liquids. The number of washes must be validated especially for solutions with inhibitory substances present. Incubation conditionsDuring test method validation, visible evidence of growth of the bacterial challenge organisms should be evident within 48 hours after incubation at 30°-35°C. Visible evidence of growth of fungal challenge organisms should be evident within 3-5 days after incubation at 20°-25°C. During performance of the actual viable aerobic count test, the incubation conditions for the aerobic bacterial count should generally be 30°-35°C for 5 days and for the fungal count, 20°-25°C for 5 days. Acceptance criteriaRecovery compared to positive control. For satisfactory validation of the total viable aerobic count method the BP/EP require challenge organism recovery counts to differ by not more than a factor of 5 from the control counts; the USP requires at least a 70% recovery rate.
- 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 10-100 CFU. Validation tests using live microorganisms must not be undertaken in the sterility testing area.