006-Analytical Method Development

8/13/2019 006-Analytical Method Development

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Birgit Schmauser | June 20103 |

Analytical method development Analytical method development

Originator, FirstOriginator, First --time Generic andtime Generic and MultisourceMultisource GenericGeneric

VerifyVerify identity, potency,identity, potency,purity of API and FPP bypurity of API and FPP bypharmacopoeialpharmacopoeial methodsmethodsand inand in --house methodshouse methods

DeriveDerive identity,identity,potency, purity of APIpotency, purity of APIand FPP by in houseand FPP by in housemethodsmethods

EstablishEstablish identity,identity,potency, purity ofpotency, purity of

API and FPP by API and FPP byinin--house methodshouse methods

AnalyticalAnalyticalmethodsmethods

PharmacopoeiasPharmacopoeiasInformation fromInformation fromregulatory agenciesregulatory agencies(publicly available) &(publicly available) &literature dataliterature data

Originator Originator ´́ssspecificationsspecifications

FPP qualityFPP qualitystandardsstandards

PharmacopoeiasPharmacopoeiasInformation fromInformation fromregulatory agenciesregulatory agencies(publicly available) &(publicly available) &

literature dataliterature data

Originator Originator ´́ssspecificationsspecifications

API qualityAPI qualitystandardsstandards

MultisourceMultisource GenericGenericFirstFirst --time Generictime GenericOriginator Originator





HPLCHPLC --method to assay potency and puritymethod to assay potency and purity – – risk assessmentrisk assessment

„„ ImplementImplement ““ for routine usefor routine useAdapt toAdapt to /modify for routine use /modify for routine useAdaptAdapt to routine useto routine use

UseUse pharmacopoeialpharmacopoeialreference materialsreference materials

ExtractExtract (&(& reproducereproduce ) reference) referencematerialsmaterials

EstablishEstablish referencereferencematerialsmaterials

VerifyVerify impurities fromimpurities fromPharmacopoeiaPharmacopoeiaCharacteriseCharacterise „„ inin --househouse ““impurities/impurities/ degradantsdegradants(Response factors)(Response factors)

DeriveDerive impurities/impurities/ degradantsdegradantsfrom Originator from Originator CharacterizeCharacterize „„ inin --househouse ““impurities/impurities/ degradantsdegradantsCalculate response factorsCalculate response factors

CharacteriseCharacterise allallimpurities/impurities/ degradantsdegradantsCalculate Response factorsCalculate Response factors(qualification by clinical(qualification by clinicaluse)use)

IdentifyIdentifyimpurities/impurities/ degradantsdegradants

SelectivelySelectively screenscreen /detect /detectany impurity orany impurity or degradantdegradantEstablishEstablish potencypotency

MultisourceMultisource GenericGenericFirstFirst --time Generictime GenericOriginator Originator





Interchangeability (IC) ofInterchangeability (IC) of multisourcemultisource genericgeneric FPPsFPPs((Essential similarityEssential similarity with Innovator FPP)with Innovator FPP)

PharmaceuticalPharmaceutical ++ BioequivalenceBioequivalenceEquivalenceEquivalence

IC =IC = PEPE ++ BEBE





Pharmaceutical equivalencePharmaceutical equivalence – – FPPsFPPs meet themeet the samesame oror comparablecomparable standardsstandards byby use ofuse of

equivalent analytical methodsequivalent analytical methods•• Same API (Same API ( chemicalchemical andand physicalphysical equivalence)equivalence)

•• Same dosage form and route of administrationSame dosage form and route of administration•• Same strengthSame strength•• Comparable labelingComparable labeling

– – Equivalence in pharmaceutical developmentEquivalence in pharmaceutical development

– – Equivalence in stabilityEquivalence in stability

– – Equivalence in manufacture (WHOEquivalence in manufacture (WHO --GMP)GMP)





Prequalification requirementsPrequalification requirements – – Validation of analytical methods is aValidation of analytical methods is a prerequisiteprerequisite forfor

prequalification of product dossiersprequalification of product dossiers•• NonNon--compendialcompendial APIs andAPIs and FPPsFPPs are tested with methodsare tested with methods developeddeveloped

by the manufacturer by the manufacturer •• ForFor compendialcompendial APIs andAPIs and FPPsFPPs thethe „„applicabilityapplicability““ of of pharmacopoeialpharmacopoeial

methodsmethods toto particular particular productsproducts must bemust be demonstrateddemonstrated (verification)(verification) – – Analytical methods must be developed and validatedAnalytical methods must be developed and validated

according to TRS 823, Annex 5,according to TRS 823, Annex 5, Validation of analyticalValidation of analytical

procedures used in the examination of pharmaceutical procedures used in the examination of pharmaceuticalmaterialsmaterials ; ICH Q2 (R1); ICH Q2 (R1)

•• To be used within GLP and GMP environmentsTo be used within GLP and GMP environments





To beTo be robust, transferable,robust, transferable,

accurateaccurate andand preciseprecise forforspecification setting, stabilityspecification setting, stabilityassessment and QC release ofassessment and QC release ofprequalifiedprequalified product batchesproduct batches

ToTo optimiseoptimise ,, scalescale --upup andand

transfer transfer aa stablestable andandcontrolledcontrolled manufacturingmanufacturingprocess for theprocess for theprequalification productprequalification product

To proveTo prove

bioequivalencebioequivalence afteraftercritical variationscritical variations totothethe prequalifiedprequalifieddossier dossier

AtAt advancedadvanced phase of pharmaceutical developmentphase of pharmaceutical development

To understand theTo understand the profileprofile of relatedof relatedsubstances and to studysubstances and to study stabilitystability

To startTo start

measuringmeasuring

the impact ofthe impact of

key productkey product andand manufacturingmanufacturingprocess parametersprocess parameters onon consistentconsistentFPP qualityFPP quality

To develop a stable andTo develop a stable andreproducible formulation forreproducible formulation forthe manufacture ofthe manufacture ofbioequivalence, dissolution,bioequivalence, dissolution,stability and pilotstability and pilot --scalescalevalidation batchesvalidation batches

ToTo determinedeterminebioavailabilitybioavailability ininhealthy volunteershealthy volunteers

AtAt initialinitial phase of pharmaceutical developmentphase of pharmaceutical developmentMETHODSMETHODSPHARMACEUTICALPHARMACEUTICALCLINICALCLINICALUse of analytical methods

Use of analytical methods--

genericsgenerics





Method development life cycleMethod development life cyclePlanning

Development and Validation Policy

Objectives/Requirements of Method

Information GatheringResource Gathering

Method developmentInitital Method Development

Pre-Validation EvaluationMethod Optimization

RobustnessSystem Suitability

DevelopmentPlan –Project

Customer EvaluationTesting

Validation Experiments

Method Transfer Experiments Filed Method in Use

PeriodicallyMonitoring/Review

of Methodsin Control Labs





Validation should verify the suitability of an analyticalValidation should verify the suitability of an analyticalmethodmethod for its intended purposefor its intended purpose

Validation should be founded on method developmentValidation should be founded on method development

performed beforehand thatperformed beforehand that suggest the suitability andsuggest the suitability androbustnessrobustness of the methodof the method

Validation may be performed in different waysValidation may be performed in different ways(individual purpose) according to common standards(individual purpose) according to common standards





t R

A b s o r b a n c e





Validation protocolValidation protocol – – Method principle / objectiveMethod principle / objective

– – Listing of responsibilitiesListing of responsibilities

•• Laboratories involved and their role in the validationLaboratories involved and their role in the validation

– – Method categorizationMethod categorization

– – List of reagents (including test lots) and standardsList of reagents (including test lots) and standards

– – Test procedures to evaluate each validation parameter and proposTest procedures to evaluate each validation parameter and propos ededacceptance criteriaacceptance criteria

– – Plan or procedure when acceptance criteria are not metPlan or procedure when acceptance criteria are not met

– – Requirements for the final reportRequirements for the final report

The validation process cannot proceed until the protocol andThe validation process cannot proceed until the protocol andall parties involved approve the acceptance criteriaall parties involved approve the acceptance criteria





Innovator versus GenericsInnovator versus Generics

Validated methods:Validated methods:GMP and GLPGMP and GLPValidated methodsValidated methodsEntering of Generics;Entering of Generics;Pharmaceutical development,Pharmaceutical development,Comparability with Innovator Comparability with Innovator

--Validated methodsValidated methodsPost marketing phase IVPost marketing phase IV

--Method validationMethod validationcompletedcompleted

Clinical trials phase IIIClinical trials phase III

--Method validationMethod validationsummarysummaryClinical trials phase I and IIClinical trials phase I and II

--++Preclinical trialsPreclinical trials--++R & D on APIR & D on API

GenericsGenericsInnovator Innovator





Validation CharacteristicsValidation Characteristics

++++++++RobustnessRobustness++--++--RangeRange

++--++--LinearityLinearity

----++--QuantitationQuantitation LimitLimit

--++----Detection LimitDetection Limit

++++++++SpecificitySpecificity

++--

++--PrecisionPrecision

++--++--AccuracyAccuracylimitlimitquantitativequantitative

AssayAssayImpuritiesImpuritiesIdentificationIdentification





PrecisionPrecision – – Expresses the closeness of agreement between a series ofExpresses the closeness of agreement between a series of

measurements obtained frommeasurements obtained from multiple samplingmultiple sampling of the sameof the samehomogenous samplehomogenous sample

•• SystemSystem precisionprecision

•• MethodMethodprecisionprecision – – Is usually expressed as the standard deviation (S), variance (SIs usually expressed as the standard deviation (S), variance (S 22))

or coefficient of variation (RSD) of a series of measurementsor coefficient of variation (RSD) of a series of measurements

– – Precision may be considered at three levelsPrecision may be considered at three levels

•• RepeatabilityRepeatability (intra(intra--assay precision)assay precision)•• Intermediate PrecisionIntermediate Precision (variability within a laboratory)(variability within a laboratory)•• ReproducibilityReproducibility (precision between laboratories)(precision between laboratories)





Probability function [Probability function [ P(xP(x )])], normal distribution, and, normal distribution, andconfidence interval [CI]confidence interval [CI]

– – Probability (P)Probability (P) , that measurements from a normal distribution fall within [, that measurements from a normal distribution fall within [ µµ--xxnn ,, µµ++ xxnn ]]forfor xxnn = n= n is described by theis described by the ““ erf erf --functionfunction ”” ((µµ == mean)mean) ::

An interval ofAn interval of ±± 33covers 99.73% of valuescovers 99.73% of values N

u m

b e r o

f t i m e s e a c

h v a

l u e o c c u r s

N u m

b e r o

f t i m e s e a c

h v a

l u e o c c u r s

0.99999940.9999994

0.99993660.9999366

0.99730020.9973002

0.95449970.9544997 0.68268950.6826895

PPxx nn

Values





Confidence interval [CI], normal distribution, and probabilityConfidence interval [CI], normal distribution, and probabilityfunction [function [ P(xP(x )])]

– – ProbabilityProbability --PP Confidence interval [CI]Confidence interval [CI]centered around the mean [centered around the mean [ µµ]]in units of sigma [in units of sigma [ ] described by] described by““inverseinverse erf erf --functionfunction ””::

– – AA CI of 95%CI of 95% includes valuesincludes values±± 1.951.95 around the meanaround the mean

3.290533.29053

2.575832.57583

1.959961.95996

1.644851.64485

1.281551.28155

xx ppPP





RepeatabilityRepeatability

– – SixSix replicatereplicate samplesample preparationpreparation stepssteps fromfrom aa homogenouslyhomogenously preparedprepared tablettabletmixturemixture (nominal(nominal valuevalue of API 150 mg)of API 150 mg)

1.32%1.32%1.32%1.32%RSDRSD

1.98 mg/1.98 mg/ 1.32%1.32%23292329SD (SD ( ))148.45 mg/98.96%148.45 mg/98.96%175453175453MeanMean

149.28 mg/99.52%149.28 mg/99.52%17642517642566

151.95 mg/101.30%151.95 mg/101.30%17955717955755

148.08 mg/98.72%148.08 mg/98.72%17501117501144

146.32 mg/97.54%146.32 mg/97.54%17293317293333148.00 mg/98.66%148.00 mg/98.66%17492617492622

147.10 mg/98.06%147.10 mg/98.06%17386517386511

AssayAssayPeakPeak areaareaInjectionInjection

MeanMean ±± 3 SD =3 SD =Confidence interval of 99.73%Confidence interval of 99.73%

98.9698.96 ±± 3x3x 1.321.32 % =% = 9595 %% -- 102.92%102.92%





Intermediate precisionIntermediate precision

– – ExpressesExpresses withinwithin --laboratorieslaboratories variationsvariations (different(different daysdays , different, different analystsanalysts ,,differentdifferent equipmentequipment etc.)etc.)

1.121.12RSDRSD --allall176551176551MeanMean --allall ::0.51%0.51%0.28%0.28%1.32%1.32%RSDRSD

91891849549523292329SD (SD ( ))178504178504175695175695175453175453MeanMean179688179688174959174959176425176425661794661794661753321753321795571795575517801117801117634417634417501117501144177342177342176004176004172933172933331785561785561758781758781749261749262217796517796517565617565617386517386511

PeakPeak areaareaanalystanalyst 33

PeakPeak areaareaanalystanalyst 22

PeakPeak areaareaanalystanalyst 11InjectionInjection

Analyst 1: 98.96%Analyst 1: 98.96% ±± 3 x 1.32%3 x 1.32%95.0095.00 – – 102.92%102.92%



AverageAverage of 3of 3 analystsanalysts ±± 3SD3SD ::96.2496.24 – – 102.96%102.96%

MeanMean ±± 3 SD: (1772523 SD: (177252 100%)100%)





ReproducibilityReproducibility – – ExpressesExpresses thethe precisionprecision betweenbetween laboratorieslaboratories

•• CollaborativeCollaborative studiesstudies ,, usuallyusually appliedapplied toto standardisationstandardisation ofof methodologymethodology

– – Transfer of technologyTransfer of technology

– – CompendialCompendial methodsmethods





AccuracyAccuracy

– – ExpressesExpresses thethe closenesscloseness ofof agreementagreement betweenbetween thethe valuevalue whichwhich isisacceptedaccepted either either as aas a conventionalconventional truetrue valuevalue or or anan acceptedacceptedreferencereference valuevalue andand thethe valuevalue foundfound

•• SometimesSometimes referredreferred to asto as „„TRUENESSTRUENESS““

truetruemeanmean





To find outTo find out whether whether aa methodmethod isis accurateaccurate ::

DrugDrug substancesubstance ((assayassay )) – – Application Application ofof thethe methodmethod to anto an analyteanalyte ofof knownknown puritypurity ((e.ge.g .. referencereference

substancesubstance )) – – ComparisonComparison ofof thethe resultsresults ofof oneone methodmethod withwith thosethose of a secondof a second wellwell --

characterisedcharacterised methodmethod ((accuracyaccuracy knownknown ))

DrugDrug productproduct ((assayassay )) – – Application Application ofof thethe methodmethod toto syntheticsynthetic mixturesmixtures ofof thethe drugdrug productproduct componentcomponent

toto whichwhich knownknown quantitiesquantities ofof thethe analyteanalyte havehave beenbeen addedadded•• DrugDrugproductproductmaymay exceptionallyexceptionallybebe usedused asas matrixmatrix

DrugDrug substancesubstance /Drug /Drug productproduct ((ImpuritiesImpurities )) – – Application Application ofof thethe methodmethod toto samplessamples spikedspiked withwith knownknown amountsamounts ofof impuritiesimpurities





AccuracyAccuracy :: ApplicationApplication ofof thethe methodmethod toto syntheticsynthetic mixturesmixtures ofof thethedrugdrug productproduct componentscomponentstoto whichwhich knownknown quantitiesquantitiesofof thethe analyteanalytehavehave beenbeen addedadded

RecoveryRecovery reducedreducedbyby ~10~10 – – 15%15%

SourceSource :: Analytical Analytical Method Method Validation and Instrument PerformanceValidation and Instrument PerformanceVerificationVerification,, EditedEditedbyby ChungChung ChowChowChan,HermanChan,HermanLamLam,,Y.C. Lee andY.C. Lee and XueXue--MingMingZhang, ISBN 0Zhang, ISBN 0--471471--2595325953--5, Wiley &5, Wiley &SonsSons





When to expect Accuracy problemsWhen to expect Accuracy problems – – InsufficientInsufficient selectivityselectivity ofof thethe methodmethod

•• ImpurityImpuritypeakspeaks areare notnot resolvedresolved andand accountaccount for for assayassay valuevalue

– – RecoveryRecovery isis < 100%< 100%

•• IrreversibleIrreversibleadsorptionadsorption ofof analyteanalyte toto surfacessurfaces ofof thethe systemsystem

– – IncorrectIncorrect assayassay valuevalue ofof aa referencereference standardstandard•• DueDue toto decompositiondecompositionofof referencereference standardstandard

– – IncorrectIncorrect assayassay valuevalue duedue toto changechange inin matrixmatrix•• Analytical laboratory still uses the preceding matrix as standar Analytical laboratory still uses the preceding matrix as standar dd





SpecificitySpecificity – – IsIs thethe abilityability toto assessassess unequivocallyunequivocally thethe analyteanalyte inin thethe presencepresence ofofcomponentscomponents whichwhich maymay bebe expectedexpected toto bebe presentpresent ((impuritiesimpurities ,, degradantsdegradants ,,matrixmatrix ……))

IdentityIdentity testingtesting – – ToTo ensureensure thethe identityidentity of anof an analyteanalyte

PurityPurity testingtesting – – ToTo ensureensure accurateaccurate statementstatement onon thethe contentcontent ofof impuritiesimpurities of anof an analyteanalyte

AssayAssay – – ToTo allowallow anan accurateaccurate statementstatement onon thethe contentcontent of anof an analyteanalyte in ain a samplesample





SpecificitySpecificity :: OverlayOverlay chromatogramchromatogram of anof an impurityimpurity solutionsolution withwith aasamplesample solutionsolution






SpecificitySpecificity and stabilityand stabilityStressStress stabilitystability testingtesting toto ensureensure thethe stabilitystability indicatingindicating potential of anpotential of ananalyticalanalytical methodmethod

– – Apply Apply diverse stressdiverse stress factorsfactors toto thethe API API – – Apply Apply diverse stressdiverse stress factorsfactors toto thethe FPPFPP

StressStress conditionsconditions :: e.ge.g. Supplement 2 of. Supplement 2 of GenericGeneric GuidelineGuideline; TRS 929, Annex 5; TRS 929, Annex 5

Assure Assure thatthat thethe API API cancan bebe assessedassessed specificallyspecifically inin thethe presencepresence ofofknownknown andand unknownunknown ((generatedgenerated byby stress)stress) impuritiesimpurities

Assure Assure thatthat knownknown impuritiesimpurities //degradantsdegradants cancan bebe specificallyspecifically assessedassessed

inin thethe presencepresence ofof further further degradantsdegradants

ByBy peakpeak puritypurity assessmentassessment and (and ( overlayoverlay of)of) chromatogramschromatograms





StressStress stabilitystability studiesstudies versusversus forcedforced degradationdegradation studiesstudies

5050 °°C / 80% RH (4C / 80% RH (4 weeksweeks ))--HumidityHumidity

365 nm365 nm or or whitewhite fluorescentfluorescent light /light /solid API (solid API ( 1d1d and 7d)and 7d)

UVUV or or LightLight

6060 °°C (4C (4 weeksweeks ))105105 °°CC / solid API (/ solid API ( 1d1d and 7d)and 7d)HeatHeat

--6060 °°CC / 5 ml/ 5 ml solutionsolution ((3h,3h, 6h6h ……7d)7d)HeatHeat

1 g/ml1 g/ml oxygenoxygen bubbledbubbled throughthrough (8(8 hourshours ))0.10.1 – – 2%2% HH22OO 22 (24(24 hourshours ))

0.2 ml0.2 ml 5%5% or or 35%35% HH22 OO 22 / 5 ml/ 5 ml API API --solutionsolution (RT, to 7d & 60(RT, to 7d & 60 °°C,C, 3h3h ))

HH22OO 22 / /OxygenOxygen

pHpH ±± 10 (2 weeks)10 (2 weeks)0.2 ml0.2 ml 1N1N NaOHNaOH / 5 ml/ 5 ml API API --solutionsolution //3h, 6h3h, 6h , 12h, 24h, 12h, 24h ……7d (RT & 607d (RT & 60 °°C)C)

BaseBase

pHpH ±± 2 (2 weeks)2 (2 weeks)0.2 ml0.2 ml 1N1N HClHCl / 5 ml/ 5 ml API API --solutionsolution //3h, 6h3h, 6h , 12h, 24h, 12h, 24h ……7d (RT & 607d (RT & 60 °°C)C)

AcidAcid

StressStress stabilitystability(5(5 – – 15%15% decompositiondecomposition ))

ForcedForced degradationdegradationStressStressparameter parameter





Limit ofLimit of DetectionDetection (LOD, DL)(LOD, DL) – – TheThe LOD of anLOD of an analyticalanalytical procedureprocedure isis thethe lowestlowest amountamount ofof analyteanalyte

inin samplesample whichwhich cancan bebe detecteddetected butbut notnot necessarilynecessarily quantitatedquantitated asasanan exactexact valuevalue

DeterminationDetermination isis usuallyusually basedbased onon – – Signal toSignal to noisenoise ratioratio (~3:1) ((~3:1) ( baselinebaseline noisenoise ))or or

– – StandardStandard deviationdeviation ofof responseresponse (( ) and) and SlopeSlope (S)(S)

•• 3.33.3 /S/S

l l h d d l





Limit ofLimit of

QuantitationQuantitation

(LOQ, QL)(LOQ, QL)

– – TheThe LOQLOQ isis thethe lowestlowest amountamount ofof analyteanalyte in ain a samplesample whichwhich cancan bebequantitativelyquantitatively determineddetermined withwith suitablesuitable precisionprecision andand accuracyaccuracy

•• TheThe quantitationquantitationlimitlimitisis usedused particularlyparticularlyfor for thethe determinationdetermination ofof impuritiesimpuritiesand/and/ or or degradationdegradation productsproducts

DeterminationDetermination isis usuallyusually basedbased onon – – Signal toSignal to noisenoise ratioratio (~10:1) ((~10:1) ( baselinebaseline noisenoise ))or or

– – StandardStandard deviationdeviation ofof responseresponse (( ) and) and SlopeSlope (S)(S)•• 1010 /S/S

l l h d d lA l i l h d d l





NoiseNoise

LODLODSignal toSignal to NoiseNoise = 3:= 3: 11

LOQLOQ

Signal toSignal to NoiseNoise = 10:= 10: 11

LOD, LOQ and Signal toLOD, LOQ and Signal to NoiseNoise Ratio (SNR)Ratio (SNR)

A l i l h d d lA l i l h d d l





LOQLOQ – – QuantitationQuantitation byby SNRSNR isis acceptedaccepted

– – QuantitationQuantitation byby StandardStandard deviationdeviation ofof responseresponse (( ) and) and SlopeSlope (S)(S)(10(10 /S)/S) isis moremore adequateadequate asas itit involvesinvolves thethe responseresponse ofof thethe actualactual

analyteanalyte

– – Best to calculate in the region close to yBest to calculate in the region close to y --interceptintercept

A l i l h d d lA l ti l th d d l t





LOQ andLOQ and impuritiesimpurities – – InIn determinationdetermination ofof impuritiesimpurities inin APIs APIs andand FPPsFPPs thethe LOQLOQ shouldshould bebe

determineddetermined inin thethe presencepresence of APIof API

•• LOQLOQshouldshould bebe NMTNMTreportingreportinglevellevel

•• LOQLOQshouldshould bebe givengiven relative torelative tothethe testtest concentrationconcentrationof APIof API – – SpecificitySpecificity ofof impurityimpurity determinationdetermination shouldshould alwaysalways bebe

demonstrateddemonstrated inin thethe presencepresence of APIof API at APIat API specificationspecification levelslevels

•• SpikingSpikingof testof test concentrationconcentration(API/FPP)(API/FPP)withwithimpuritiesimpuritiesatat levelslevels ofof their their

specificationspecificationrangerange

A l ti l th d d l tA l ti l th d d l t





SpikingSpiking – – API testAPI test concentrationconcentration ((normalisednormalised ))•• 0.1 mg/ml (100%)0.1 mg/ml (100%)

– – ImpurityImpurity spikingspiking concentrationsconcentrations•• 0.001 mg/ml (1%)0.001 mg/ml (1%) – – specificationspecification limitlimit•• 0.0001 mg/ml (0.1%)0.0001 mg/ml (0.1%) – – limitlimit ofof quantitationquantitation ((minimumminimum requirementrequirement ))

API at testAPI at test concentrationsconcentrations

APIAPI belowbelow testtest concentrationsconcentrations

A l ti l th d d l tA l ti l th d d l t





LinearityLinearityof anof an analyticalanalytical procedureprocedure isis itsits abilityability ((withinwithin aa givengiven rangerange ))toto obtainobtain testtest resultsresults whichwhich areare directlydirectly proportional toproportional to thetheconcentrationconcentration ((amountamount ) of) of analyteanalyte inin thethe samplesample

– – If If therethere isis a lineara linear relationshiprelationship testtest resultsresults shouldshould bebe evaluatedevaluatedbyby appropriateappropriate statisticalstatistical methodsmethods

•• CorrelationCorrelation coefficientcoefficient (r)(r)•• Y Y--interceptintercept•• SlopeSlope ofof regressionregression lineline

•• ResidualResidual sumsum ofof squaressquares•• PLOT OF THE DATAPLOT OF THE DATA

Anal tical method de elopmentAnalytical method development





UsualUsual acceptanceacceptance criteriacriteria for for a lineara linear calibrationcalibration curvecurve – – r > 0.999r > 0.999 ;; yy--interceptintercept a < 0 to 5% ofa < 0 to 5% of targettarget concentrationconcentration

RSD (RSD ( wrtwrt calibrationcalibration curvecurve ) < 1.5) < 1.5 --2%2%

r > 0.997 r < 0.997

Analytical method developmentAnalytical method development




RangeRange – – TheThe rangerange of anof an analyticalanalytical procedureprocedure isis thethe intervalinterval

betweenbetween thethe upper upper andand lower lower concentrationconcentration ((amountsamounts ) of) ofanalyteanalyte inin thethe samplesample for for whichwhich itit hashas beenbeen demonstrateddemonstrated

thatthat thethe analyticalanalytical procedureprocedure has ahas a suitablesuitable levellevel ofofprecisionprecision ,, accuracyaccuracy andand linearitylinearity







RangeRange – – AssayAssay•• 80 to 120%80 to 120% of test concentrationof test concentration

– – ContentContent uniformityuniformity•• 70 to 130%70 to 130% of test concentrationof test concentration

– – DissolutionDissolution•• QQ--20% to 120%20% to 120%

– – ImpuritiesImpurities•• ReportingReporting levellevel – – 120%120% ofof impurityimpurity specificationspecification limitlimit

– – AssayAssay && ImpuritiesImpurities•• ReportingReporting levellevel to 120%to 120% ofof assayassay specificationspecification






LinearityLinearity isis limitedlimited to 150% ofto 150% of shelf shelf lifelife specificationspecification ofof impuritiesimpurities

– – TestTest concentrationconcentration cancan bebeusedused toto determinedetermine impuritiesimpurities

– – ToTo determinedetermine drugdrug substancesubstance((assayassay )) thethe testtest concentrationconcentration

mustmust bebe diluteddiluted – – TheThe rangerange isis 00 – – ~ 150% of ~ 150% of

impurityimpurity specificationspecification







RobustnessRobustness – – RobustnessRobustness of anof an analyticalanalytical procedureprocedure shouldshould showshow

thethe reliabilityreliability of anof an analysisanalysis withwith respectrespect toto deliberatedeliberatevariationsvariations inin methodmethod parametersparameters

– – TheThe evaluationevaluation ofof robustnessrobustness shouldshould bebe consideredconsideredduringduring thethe developmentdevelopment phasephase – – If If measurementsmeasurements areare susceptiblesusceptible toto variationsvariations inin

analyticalanalytical conditionsconditions thethe analyticalanalytical conditionsconditions shouldshould

bebe suitablysuitably controlledcontrolled or or aa precautionaryprecautionary statementstatementshouldshould bebe includedincluded inin thethe procedureprocedure






InfluenceInfluence ofof buffer buffer pHpH andand buffer buffer concentrationconcentration in mobilein mobile phasephaseonon retentionretention timestimes of API andof API and impuritiesimpurities

ConclusionConclusion :: TheThe buffer buffer compositioncomposition shouldshould bebe maintainedmaintained in ain a rangerange ofof8585 ±± 0.5%0.5%

11.1811.189.619.614.774.7715.2615.26BufferBuffer concconc . 87%. 87%

6.666.666.166.163.433.437.847.84BufferBuffer concconc . 83%. 83%8.348.347.497.493.943.9410.4610.46buffer buffer pHpH 6.96.9

8.388.387.517.513.943.9410.4510.45buffer buffer pHpH 5.95.9

8.268.267.437.433.863.8610.4610.46 As As isisImpurityImpurity CCImpurityImpurity BBImpurityImpurity A A API API






SystemSystem suitabilitysuitability testingtesting – – BasedBased onon thethe conceptconcept thatthat equipmentequipment ,, electronicselectronics ,,

analyticalanalytical operationsoperations andand samplessamples toto bebe analysedanalysedconstituteconstitute an integralan integral systemsystem thatthat cancan bebe evaluatedevaluated asas

suchsuch – – SuitabilitySuitability parametersparameters areare establishedestablished for for eacheach analyticalanalytical

procedureprocedure individuallyindividually•• DependDepend onon thethe typetype ofof analyticalanalytical procedureprocedure






MethodMethod stabilitystability – – SystemSystem suitabilitysuitability over over timetime•• SampleSample solutionsolution stabilitystability

– – A A solutionsolution ofof stavudinestavudine isis stablestable for for ~ 2 h,~ 2 h, thenthen itit startsstarts toto degradedegradetoto thyminethymine

•• ImpurityImpurity--spikedspiked samplesample solutionsolution stabilitystability A A solutionsolutioncontainingcontainingstavudinestavudine spikedspiked withwithitsits impurityimpuritythyminethyminedoesdoes notnot allowallowtotoclearlyclearlydistinguishdistinguishbetweenbetween degradationdegradationandand spikespike A A solutionsolutioncontainingcontainingstavudinestavudine of aof a FPPFPP--stabilitystabilitysamplesample solutionsolutiondoesdoes notnot allowallowtoto clearlyclearlydistinguishdistinguishbetweenbetween FPPFPP--stabilitystabilitydegradationdegradationandand samplesample solutionsolution

degradationdegradationShouldShould bebe analysedanalysed immediatelyimmediately






ValidationValidation datadata andand acceptanceacceptance criteriacriteria ::

– – Precision of Precision of impurity determinationimpurity determination

– – Precision of Precision of API determinationAPI determination

– – Method precision of Method precision of released API (dissolution)released API (dissolution)

% RSD 0.0% RSD 0.0MethodMethod precisionprecision

% RSD 0.33% RSD 0.33 – – 2.252.25SystemSystem precisionprecision

% RSD% RSD ≤≤ 2.02.0 Acceptance Acceptance criterioncriterion% RSD 0.08% RSD 0.08 Average Average peakpeak areaarea

% RSD% RSD ≤≤ 10.010.0 Acceptance Acceptance criterioncriterion

% RSD 0.4% RSD 0.4 Average Average peakpeak areaarea






SpecificationSpecification rangerange (USL(USL --LSL)LSL) – – ProcessProcess variabilityvariability ((usuallyusually ±± 2 SD)2 SD) – – AnalyticalAnalytical variabilityvariability ((±± 33 ))

•• ~ NMT 30% of total~ NMT 30% of totalspecificationspecification rangerange

AnalyticalAnalytical variabilityvariability ProcessProcess variabilityvariability – – ReliabilityReliability ofof evaluationevaluation ofof major major processprocess variablesvariables byby analyticalanalytical

proceduresprocedures dependsdepends onon analyticalanalytical variabilityvariability

– – ImpuritiesImpurities

•• LOQLOQandand specificationspecificationlimitlimit((e.ge.g.. qualificationqualificationlimits NMT 0.15%)limits NMT 0.15%) – – ResponseResponse factorsfactors (LOQ(LOQmodifiedmodifiedbyby responseresponse factor factor ))






MethodsMethods for for cleaningcleaning validationvalidation – – Method for assay and related substances used in stability studieMethod for assay and related substances used in stability studie s of API ands of API and

FPPFPP•• SpecificitySpecificity (in(in samples taken from a cleaning assessmentsamples taken from a cleaning assessment))•• LLinearityinearity of responseof response (from(from50% of the cleaning limit to50% of the cleaning limit to110x this concentration0x this concentration; R; R22 ≥≥ 0.9900)0.9900)•• PrecisionPrecision

– – RepeatabilityRepeatability(RSD(RSD ≤≤ 5%)5%) – – iinter nter mediate precision [ruggedness (USP)]mediate precision [ruggedness (USP)] – – ReproducibilityReproducibility

•• LLimitsimits of detection andof detection and quantitationquantitation•• A A ccuracyccuracy or recovery fromor recovery from rinsaterinsate ((≥≥ 80%)80%) , swabs, swabs ((≥≥ 90%)90%) , and process, and process

surfacesurface ((≥≥ 70%)70%)•• RR angeange ((lowest level is at least 2x higher thanlowest level is at least 2x higher than LOQLOQ ))



Summary / ConclusionSummary / Conclusion




yy

AnalyticalAnalytical proceduresprocedures playplay aa criticalcritical rolerole ininpharmaceuticalpharmaceutical equivalenceequivalence andand riskriskassessmentassessment / /managementmanagement – – Establishment ofEstablishment of productproduct --specificspecific acceptanceacceptance criteriacriteria

– – Assessment Assessment ofof stabilitystability ofof APIs APIs andand FPPsFPPs

Validation ofValidation of analyticalanalytical proceduresprocedures shouldshould demonstratedemonstratethatthat theythey areare suitablesuitable for for their their intendedintended useuse

Validation ofValidation of analyticalanalytical proceduresprocedures deservesdeserves specialspecialattentionattention duringduring assessmentassessment ofof dossiersdossiers for for prequalificationprequalification




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006-Analytical Method Development

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