Dar es Salaam, August, 21-25, 2006Dr. Birgit Schmauser, BfArM, Bonn Verification of applicability of the validated/compendial API analytical method for

Dar es Salaam, August, 21-25, 2006Dar es Salaam, August, 21-25, 2006 Dr. Birgit Schmauser, BfArM, BonnDr. Birgit Schmauser, BfArM, Bonn

Verification of applicability Verification of applicability of the validated/compendialof the validated/compendial

API analytical methodAPI analytical methodfor the final formulationfor the final formulationAssay, dissolution test and Assay, dissolution test and

degradantsdegradants


GuidelinesGuidelines

• ICH Q2AICH Q2A– Validation of Analytical Methods: Definitions and Validation of Analytical Methods: Definitions and

Terminology (CPMP/ICH/381/95)Terminology (CPMP/ICH/381/95)

• ICH Q2BICH Q2B– Validation of Analytical Procedures: Methodology Validation of Analytical Procedures: Methodology

(CPMP/ICH/281/95)(CPMP/ICH/281/95)

• ICH Q6AICH Q6A– Specifications: Test Procedures and Acceptance Specifications: Test Procedures and Acceptance

Criteria for New Drug Substances and New Drug Criteria for New Drug Substances and New Drug Products: Chemical SubstancesProducts: Chemical Substances(CPMP/ICH/367/96 corr)(CPMP/ICH/367/96 corr)


APIAPI

• AssayAssay– Validation with respect to:Validation with respect to:

• Specificity, linearity/range, accuracy, precision, Specificity, linearity/range, accuracy, precision, robustnessrobustness

• ImpuritiesImpurities– Validation with respect to:Validation with respect to:

• Specificity, linearity/range, accuracy, precision, Specificity, linearity/range, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ)limit of detection (LOD), limit of quantitation (LOQ), , robustnessrobustness


FPPFPP

• Formulation of the drug productFormulation of the drug product– Presence of Presence of further APIsfurther APIs– Presence of Presence of excipientsexcipients (individual formulation) (individual formulation)– Presence of Presence of knownknown impurities/degradants of impurities/degradants of

all APIs and potential all APIs and potential newnew degradants or degradants or incompatibility productsincompatibility products


RequirementsRequirements

• Capability of the analytical method(s):Capability of the analytical method(s):– Assay of Assay of each APIeach API in the presence of the in the presence of the

other APIs and all impurities/degradantsother APIs and all impurities/degradants– Assay of Assay of each degradanteach degradant in the presence of in the presence of

all APIs and all other degradants/impuritiesall APIs and all other degradants/impurities– Influence of Influence of formulation componentsformulation components should should

be excluded/controlledbe excluded/controlled


Revalidation IRevalidation I

• Revalidation of analytical methods with respect to:Revalidation of analytical methods with respect to:

– SpecificitySpecificity• presence of new API(s) and impurities/degradants/formulation presence of new API(s) and impurities/degradants/formulation

componentscomponents– RangeRange

• test concentrations of API(s) versus FPPtest concentrations of API(s) versus FPP– AccuracyAccuracy

• influence of formulation componentsinfluence of formulation components– PrecisionPrecision

• influence of formulation and sample preparationinfluence of formulation and sample preparation– LOD/LOQLOD/LOQ

• test concentrations of API(s) versus FPP)test concentrations of API(s) versus FPP)– RobustnessRobustness

• change of column material, column parameters, solvents)change of column material, column parameters, solvents)


Revalidation IIRevalidation II

• Revalidation reflected by ICH Q2A:Revalidation reflected by ICH Q2A:– Revalidation may be necessary in the Revalidation may be necessary in the

following circumstances:following circumstances:• Changes in the synthesis of the drug substanceChanges in the synthesis of the drug substance• Changes in the composition of the finished Changes in the composition of the finished

productproduct• Changes in the analytical procedureChanges in the analytical procedure

– (e.g. robustness)(e.g. robustness)


SpecificitySpecificity

• IdentificationIdentification– Discrimination between compounds of closely Discrimination between compounds of closely

related structuresrelated structures• positive results (from samples containing the positive results (from samples containing the

analyte)analyte)• negative results (from samples that do not contain negative results (from samples that do not contain

the analyte)the analyte)• components structurally similar to the analyte do components structurally similar to the analyte do

not give positive resultsnot give positive results


Specificity IISpecificity II

• Assay and impuritiesAssay and impurities– Chromatographic proceduresChromatographic procedures

• Representative chromatograms with Representative chromatograms with appropriate appropriate labellinglabelling of individual components of individual components

• Investigation at an Investigation at an appropriate levelappropriate level


Specificity IIISpecificity III

• Chromatogram with Chromatogram with retention times and retention times and chemical structures chemical structures of:of:

• (1) arteannuin B(1) arteannuin B• (2) artemisitene(2) artemisitene• (3) artemisinin(3) artemisinin• (4) artemisinic acid(4) artemisinic acid• (5) artemether (IS)(5) artemether (IS)

• Analytical standard Analytical standard containing 1.2µg/ml containing 1.2µg/ml of each analyte and of each analyte and 0.4 µg/ml IS0.4 µg/ml IS

From: F.C.W. Van Nieuverburgh et al., J Chromatogr. A 1118 (2006) 180-187From: F.C.W. Van Nieuverburgh et al., J Chromatogr. A 1118 (2006) 180-187


Specificity IVSpecificity IV

• Assay and impurities/degradantsAssay and impurities/degradants– Discrimination of analytes where Discrimination of analytes where

impurities/degradants are availableimpurities/degradants are available• AssayAssay

– Demonstration of discrimination of the analyte in the Demonstration of discrimination of the analyte in the presence of all impurities/degradants and/or excipientspresence of all impurities/degradants and/or excipients

» f. ex. assay result unaffected by presence of spiked f. ex. assay result unaffected by presence of spiked impurities/degradants:impurities/degradants:

- Injection of pure API- Injection of pure API

- Injection of API plus - Injection of API plus impurities/degradantsimpurities/degradants


Specificity VSpecificity V


impurities/degradants are availableimpurities/degradants are available• Impurities/DegradantsImpurities/Degradants

– Demonstration of separation of impurities/degradants Demonstration of separation of impurities/degradants individually and/or from excipientsindividually and/or from excipients

» f. ex. spiking of API with appropriate levels of f. ex. spiking of API with appropriate levels of impurities/degradants and/or excipients:impurities/degradants and/or excipients:

Chromatographic profiles of Chromatographic profiles of API API withwith and and without without impurities/degradants/excipientsimpurities/degradants/excipients


Specificity VISpecificity VI


impurities/degradants are impurities/degradants are notnot available available• Comparison of the test procedure to a second Comparison of the test procedure to a second

well-characterized (independent) procedurewell-characterized (independent) procedure– SamplesSamples

» Test samples containing impurities/degradantsTest samples containing impurities/degradants» Test samples stored under relevant stress conditions Test samples stored under relevant stress conditions

(potential degradants arising during shelf life)(potential degradants arising during shelf life)


Specificity VIISpecificity VII


impurities/degradants are impurities/degradants are notnot available available• AssayAssay

– Comparison of Comparison of test resultstest results by the two independent by the two independent proceduresprocedures

• Impurities/DegradantsImpurities/Degradants– Comparison of Comparison of impurity profilesimpurity profiles

• Peak purity assessmentPeak purity assessment– Demonstration that the analyte peak is attributable to Demonstration that the analyte peak is attributable to

only one componentonly one component


Specificity VIIISpecificity VIII

• Peak purityPeak purity• Overlapping Overlapping

peaks in HPLC peaks in HPLC (simulation)(simulation)

From: Prof. Siegfried Ebel, University of Wuerzburg, in: Stavros Kromidas, ValidierungFrom: Prof. Siegfried Ebel, University of Wuerzburg, in: Stavros Kromidas, Validierungin der Analytik, Wiley-VCHin der Analytik, Wiley-VCH

AA BB

CC DD


Specificity IXSpecificity IX• Peak purityPeak purity

Fraction 5 was analysedFraction 5 was analysedby MALDIby MALDI

Fatty acids were reacted withFatty acids were reacted withethylene oxide and separatedethylene oxide and separatedby HPLC (Fractions 1-6)by HPLC (Fractions 1-6)

From: Dr. Michael Schmitt, Henkel KGaA, Düsseldorf, in: Stavros Kromidas, ValidierungFrom: Dr. Michael Schmitt, Henkel KGaA, Düsseldorf, in: Stavros Kromidas, Validierungin der Analytik, Wiley-VCHin der Analytik, Wiley-VCH


Specificity with FDCsSpecificity with FDCs

• FDC (e.g. artesunate and amodiaquine)FDC (e.g. artesunate and amodiaquine)– One analytical method for both APIsOne analytical method for both APIs

• Capability of one method to quantify both APIs and Capability of one method to quantify both APIs and to separate/discriminate one API and its to separate/discriminate one API and its impurities/degradants and potential incompatibility impurities/degradants and potential incompatibility products from the other API and its products from the other API and its impurities/degradants/incompatibility productsimpurities/degradants/incompatibility products

– Some reference material for impurities/degradants will be Some reference material for impurities/degradants will be available (spiking experiments applicable)available (spiking experiments applicable)

– Other degradants are not available as reference material Other degradants are not available as reference material (stress testing necessary to generate in situ degradants)(stress testing necessary to generate in situ degradants)


RangeRange

• Minimum specified rangesMinimum specified ranges– AssayAssay

• 80 – 120% of the 80 – 120% of the test concentrationtest concentration– Content uniformityContent uniformity

» 70 – 130% of the 70 – 130% of the test concentrationtest concentration– DissolutionDissolution

» Q-20% - 120%Q-20% - 120%

– Impurities/DegradantsImpurities/Degradants• Reporting level to 120% of specification limitReporting level to 120% of specification limit

• Revalidation is necessary, if the ranges covered Revalidation is necessary, if the ranges covered during validation of the API-methods are during validation of the API-methods are different from those of the FPP-methodsdifferent from those of the FPP-methods(different test concentrations)(different test concentrations)


AccuracyAccuracy

• AssayAssay– Application of the analytical procedure to Application of the analytical procedure to synthetic synthetic

mixturesmixtures of the product components (placebo of the product components (placebo mixture) to which known quantities of the analyte mixture) to which known quantities of the analyte have been addedhave been added

– In case certain product components are In case certain product components are unavailable:unavailable:

• Application of the analytical procedure to the Application of the analytical procedure to the productproduct to which known quantities of the analyte have been to which known quantities of the analyte have been addedadded

• Comparison of results obtained by a Comparison of results obtained by a secondsecond (independent) (independent) procedureprocedure with defined accuracy with defined accuracy


Accuracy IIAccuracy II

• Impurities/DegradantsImpurities/Degradants– Assessment of samples Assessment of samples spikedspiked with known with known

amounts of impurities/degradantsamounts of impurities/degradants– In case certain impurities/degradation In case certain impurities/degradation

products are unavailableproducts are unavailable• Comparison of results obtained by a second Comparison of results obtained by a second

(independent) (independent) procedureprocedure with defined accuracy with defined accuracy


PrecisionPrecision• Assay and impurities/degradantsAssay and impurities/degradants

– RepeatabilityRepeatability• 9 determinations (3 x 3) covering the specified range9 determinations (3 x 3) covering the specified range

oror• 6 determinations at 100% of the test concentration6 determinations at 100% of the test concentration

– Intermediate precisionIntermediate precision• Effects of random events on the precision of the procedure, Effects of random events on the precision of the procedure,

e.g.e.g.– DaysDays– AnalystsAnalysts– EquipmentEquipment

• To be performed with aTo be performed with a test solution prepared test solution prepared from the drug productfrom the drug product


Detection LimitDetection Limit

• Determination based on Determination based on – Visual evaluation (non-instrumental and instrumental Visual evaluation (non-instrumental and instrumental

methods)methods)– Signal to Noise (Signal to Noise (baseline noisebaseline noise))– Standard deviation of response (Standard deviation of response () and) and

slope (S)slope (S)• DL=3.3DL=3.3/S/S

– Estimation of SEstimation of S» from the calibration curve of the analytefrom the calibration curve of the analyte

– Estimation of Estimation of » from the standard deviation of the from the standard deviation of the blankblank» from the standard deviation (regression line or y-intercept) from the standard deviation (regression line or y-intercept)

of a calibration curve in the range of the DLof a calibration curve in the range of the DL


Quantitation LimitQuantitation Limit

• Determination based on Determination based on – Visual evaluation (non-instrumental and instrumental Visual evaluation (non-instrumental and instrumental

methods)methods)– Signal to Noise (Signal to Noise (baseline noisebaseline noise))– Standard deviation of response (Standard deviation of response () and) and

slope (S)slope (S)• QL=10QL=10/S/S

– Estimation of SEstimation of S» from the calibration curve of the analytefrom the calibration curve of the analyte

– Estimation of Estimation of » from the standard deviation of the from the standard deviation of the blankblank» from the standard deviation (regression line or y-intercept) from the standard deviation (regression line or y-intercept)

of a calibration curve in the range of the QLof a calibration curve in the range of the QL


Robustness IRobustness I

• Reliability of an analysis with respect to Reliability of an analysis with respect to deliberate variations in method parametersdeliberate variations in method parameters– Susceptibility to variations in analytical Susceptibility to variations in analytical

conditions?conditions?• control of analytical conditionscontrol of analytical conditions

ororprecautionary statementprecautionary statement

• establishment of establishment of system suitability parameterssystem suitability parameters


Robustness IIRobustness II

• Examples of variationsExamples of variations– Stability of analytical solutionsStability of analytical solutions– Extraction timeExtraction time

• In the case of liquid chromatographyIn the case of liquid chromatography– Influence of variations of pH in a mobile phaseInfluence of variations of pH in a mobile phase– Influence of variations in mobile phase compositionInfluence of variations in mobile phase composition– Influence of columns (different lots and/or suppliers)Influence of columns (different lots and/or suppliers)– Influence of temperatureInfluence of temperature– Influence of flow rateInfluence of flow rate

• In the case of gas chromatographyIn the case of gas chromatography– Influence of columns (different lots and/or suppliers)Influence of columns (different lots and/or suppliers)– Influence of temperatureInfluence of temperature– Influence of flow rateInfluence of flow rate


Robustness IIIRobustness III• Influence of pH of elution on separation of amino Influence of pH of elution on separation of amino

acids by RP-HPLCacids by RP-HPLC

From: Waters, in: Stavros Kromidas, Validierung in der Analytik, Wiley-VCHFrom: Waters, in: Stavros Kromidas, Validierung in der Analytik, Wiley-VCH


RobustnessRobustness

• Electropherograms under identical conditions by Electropherograms under identical conditions by different analytical equipment different analytical equipment

From: Dr. Michael Krämer, NOVARTIS, Basel, in: From: Dr. Michael Krämer, NOVARTIS, Basel, in: Stavros Kromidas, Validierung in derStavros Kromidas, Validierung in derAnalytik, Wiley-VCHAnalytik, Wiley-VCH


DissolutionDissolution

• Applicability of the Applicability of the analytical methodanalytical method used for used for assay and impurities/degradantsassay and impurities/degradants– Sample preparationSample preparation– RangeRange

• Applicability of the Applicability of the dissolution methoddissolution method– Appropriateness of Appropriateness of drug releasedrug release acceptance criteriaacceptance criteria

• Solubility criteria of the APIsSolubility criteria of the APIs

– Appropriateness of Appropriateness of test conditionstest conditions andand acceptance acceptance criteriacriteria

• Dissolution affecting bioavailabilityDissolution affecting bioavailability• Changes in formulation or manufacturing variables affecting Changes in formulation or manufacturing variables affecting

dissolutiondissolution


Dissolution IIDissolution II

• Applicability of the analytical method used Applicability of the analytical method used for assay and impurities/degradantsfor assay and impurities/degradants– Potential parameters for revalidationPotential parameters for revalidation

• SampleSample preparation preparation– Stability of analytes in the dissolution medium?Stability of analytes in the dissolution medium?– Preparation of an injectable sample volume according to Preparation of an injectable sample volume according to

the analytical method?the analytical method?– Precision of analysis of the prepared dissolution sample?Precision of analysis of the prepared dissolution sample?

• RangeRange of test concentrations of API / impurities / of test concentrations of API / impurities / degradants according to the validated ranges?degradants according to the validated ranges?

– Test concentration of prepared dissolution sample Test concentration of prepared dissolution sample versus test concentration of FPP sampleversus test concentration of FPP sample


Dissolution IIIDissolution III

• Applicability of the dissolution methodApplicability of the dissolution method– Appropriateness of drug release acceptance criteriaAppropriateness of drug release acceptance criteria

• Solubility of the APIs (ICH Q6A Definitions)Solubility of the APIs (ICH Q6A Definitions)– Rapidly dissolving productsRapidly dissolving products

» Not less than 80% of the label amountNot less than 80% of the label amount of the drug of the drug substance dissolves substance dissolves within 15 minuteswithin 15 minutes in each of the in each of the following media: following media: pH 1.2, pH 4.0, pH 6.8pH 1.2, pH 4.0, pH 6.8

– Highly water soluble drugsHighly water soluble drugs

» Drugs with a Drugs with a dose/solubility volumedose/solubility volume of of less than or equal to less than or equal to 250 ml250 ml over a over a pH range of 1.2 to 6.8pH range of 1.2 to 6.8

– Low solubility drugsLow solubility drugs

» Drugs with a dose/solubility volume of Drugs with a dose/solubility volume of more thanmore than250 ml250 ml


Dissolution IVDissolution IV

• Appropriateness of drug release acceptance Appropriateness of drug release acceptance criteriacriteria– Solubility of the APIsSolubility of the APIs

• ProblemProblem with low solubility drugs: with low solubility drugs:– Solution of the drugs Solution of the drugs may become a time-limiting stepmay become a time-limiting step

» Dissolution also dependent on the strength of the drug Dissolution also dependent on the strength of the drug productproduct

» Dissolution test cannot reflect batch to batch consistencyDissolution test cannot reflect batch to batch consistency

• Possible solution of the problemPossible solution of the problem– Extending the dissolution volumeExtending the dissolution volumeandand– Validation of the dissolution procedure with extended volume Validation of the dissolution procedure with extended volume

(applicability of the pharmacopoeial procedure)(applicability of the pharmacopoeial procedure)


Dissolution VDissolution V

• Sink conditionsSink conditions– Ph. Eur 2.9.3: ..the material already in solution does Ph. Eur 2.9.3: ..the material already in solution does

not exert a significant modifying effect on the rate of not exert a significant modifying effect on the rate of dissolution of the remainder…dissolution of the remainder…

– „„Sink conditions normally occur in a volume of Sink conditions normally occur in a volume of dissolution medium that isdissolution medium that is at least at least 3 to 10 times the saturation medium3 to 10 times the saturation medium

– ConsequentlyConsequently: the amount of API contained in the : the amount of API contained in the dosage form should be soluble in NMT 300 ml of dosage form should be soluble in NMT 300 ml of dissolution mediumdissolution medium


Dissolution VDissolution V

• Applicability of the dissolution methodApplicability of the dissolution method– Appropriateness of test conditions and acceptance Appropriateness of test conditions and acceptance

criteria (criteria (ICH Q6AICH Q6A))• Dissolution significantly affecting bioavailabilityDissolution significantly affecting bioavailability

– Have relevant developmental batches exhibited unacceptable Have relevant developmental batches exhibited unacceptable bioavailability?bioavailability?

» Development of test conditions and acceptance criteria Development of test conditions and acceptance criteria which can distinguish batches with unacceptable which can distinguish batches with unacceptable bioavailabilitybioavailability

• Changes in formulation or manufacturing variables affecting Changes in formulation or manufacturing variables affecting dissolutiondissolution

– Control of these changes by another procedure and acceptance Control of these changes by another procedure and acceptance criterioncriterionoror

– Development of test conditions and acceptance criteria which Development of test conditions and acceptance criteria which can distinguish these changescan distinguish these changes


Major problems Major problems • SolubilitySolubility of Artemisinins of Artemisinins

– Sink condition cannot be establishedSink condition cannot be established• (+) Addition of solubilizers could help establish a (dis)solution (+) Addition of solubilizers could help establish a (dis)solution

testtest• (-) The test would disconnect dissolution and bioavalability (-) The test would disconnect dissolution and bioavalability

and could only serve as parameter for batch to batch and could only serve as parameter for batch to batch consistencyconsistency

• Disintegration could be considered as additional parameterDisintegration could be considered as additional parameter

• StabilityStability of Artemisinins of Artemisinins– Artesunate decomposes (to DHA) in buffers required Artesunate decomposes (to DHA) in buffers required

for dissolution testing (e.g. pH 1.2, pH 4.5)for dissolution testing (e.g. pH 1.2, pH 4.5)• Dissolution could only be performed at a Dissolution could only be performed at a neutral pHneutral pH (~ 7.0) (~ 7.0)


Deficiencies from PQDeficiencies from PQ

• Validation of precisionValidation of precision– Precision of the drug substance solution lower than Precision of the drug substance solution lower than

precision of the drug product solutionprecision of the drug product solution– Acceptance criteria for precision of the drug Acceptance criteria for precision of the drug

substance solution wider than for precision of the substance solution wider than for precision of the drug product solutiondrug product solution

– Acceptance criteria much wider than real values Acceptance criteria much wider than real values assessedassessed

– Acceptance criteria of assay specifications and Acceptance criteria of assay specifications and precision do not matchprecision do not match

• (3 x RSD outside the specification range)(3 x RSD outside the specification range)


Deficiencies from PQ IIDeficiencies from PQ II

• Assay of API and impurities/degradantsAssay of API and impurities/degradants– No acceptable mass balance found between No acceptable mass balance found between

assay of API and impurities/degradantsassay of API and impurities/degradants– Quantitation limit of impurities too highQuantitation limit of impurities too high

• ICH requirement on threshold for identification and ICH requirement on threshold for identification and qualification of unknown impurities cannot be qualification of unknown impurities cannot be fulfilledfulfilled


Deficiencies from PQ IIIDeficiencies from PQ III

• DissolutionDissolution– Necessary information on development of Necessary information on development of

dissolution test not presenteddissolution test not presented– Dissolution method (pharmacopoeial) not Dissolution method (pharmacopoeial) not

presented along with development of presented along with development of dissolution test and/or validation of dissolution test and/or validation of applicability of analytical methodsapplicability of analytical methods

– Test conditions and acceptance criteria of the Test conditions and acceptance criteria of the dissolution test not justifieddissolution test not justified


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Dar es Salaam, August, 21-25, 2006Dr. Birgit Schmauser, BfArM, Bonn Verification of applicability of the validated/compendial API analytical method for