Bevacizumab.docx

Bevacizumab.docx

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Bevacizumab

Bevacizumab

ProposedForCommentVersion0.2

BEVACIZUMABLIGHTCHAIN

DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

BEVACIZUMABHEAVYCHAIN

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

C6638H10160N1720O2108S44            149kDa

ImmunoglobulinG1（human-mousemonoclonalrhuMAb-VEGFγ-chainanti-humanvascularendothelialgrowthfactor）,disulfidewithhuman-mousemonoclonalrhuMAb-VEGFlightchain,dimer[216974-75-3].

Bevacizumabisa recombinanthumanizedmonoclonalIgG1antibodythatbindstoandinhibitsthebiologicactivityofhumanvascularendothelialgrowthfactor（VEGF）.Bevacizumabcontainshumanframeworkregionsandthecomplementarity-determiningregionsofamurineantibodythatbindstoVEGF.Bevacizumabisproducedinasuitablemammaliancellexpressionsystem.Bevacizumabisaclearcolorlessliquid.

Performance-BasedMonograph

（Containstests,procedures,andacceptancecriteriaforthematerialundertest.Italsoincludesthecriteria-basedprocedurestodemonstratethatanAcceptableProcedureisequivalenttotheReferenceProcedures.） 

DEFINITION

 BevacizumabcontainstherecombinantDNA-derivedhumanizedmonoclonalIgG1antibodyhavingmeasuredpotencyofNLT80.0%andNMT125.0%ofthestatedpotencyinalowbioburdensolution.

IDENTIFICATION

•A.BindingAssay

Standardsolution:

USPBevacizumabRSinanappropriatediluent

Samplesolution:

BevacizumabdilutedinanappropriatediluentsimilartothatoftheStandardsolution.

SystemperformancerequirementsandAnalysis:

ProceedasdirectedintheAssayforBindingPotency.

Acceptancecriteria

Measuredpotency:

ThedoseresponseoftheSamplesolutionshouldbesignificantwithdefinedlowerasymptote andupperasymptoteandsimilartothatoftheStandardsolution.Thesignal（ratioofhighestresponseandlowestresponseofthedoseresponsecurve）shouldbe≥5.0.

•B.PeptideMapping

Useachromatographicprocedure.（See BiotechnologyDerivedArticles—PeptideMapping<1055>.）

AnalyzethematerialtobetestedbyasuitabletechniquecapableofresolvingpeptidesgeneratedfromaTrypsindigest. ThedigestisconductedunderreducingconditionsandprovidesNLT90%digestion.Theanalyticalmethodused provides aminimumof90%coverageofthesequence.

Standardsolution:

DigestanddiluteaquantityofUSPBevacizumabRSinanappropriatediluent.

Samplesolution:

Digestanddilute aquantityofBevacizumabinanappropriatediluenttoobtainanominalconcentrationofbevacizumabsimilartothatoftheStandardsolution. 

Analyticalsystem:

UseaprocedurevalidatedasdescribedinMCgeneralchapterAssessingValidationParametersforReferenceandAcceptableProcedures<10>.

Systemperformancerequirements

Specificity:

IntheprofileobtainedfromtheStandardsolution,CDRregions1,2,and3shouldbeidentifiedusingasuitableprocedure.

Analysis

Samples:

StandardsolutionandSamplesolution

ThepeptideprofilesobtainedfromtheStandardsolutionarevisuallycomparedtotheSamplesolution.

Acceptancecriteria:

TheprofileobtainedfromtheSamplesolutioncorrespondstothatoftheStandardsolution.TherelativeretentiontimesofthepeakscorrespondingtotheCDRregions（withrespecttoareferencepeak）intheSamplesolutionshoulddifferfromthoseintheStandardsolutionbyNMT±0.03.

•C.IsoelectricFocusing

AnalyzeBevacizumabusinganisoelectrophoreticfocusingprocedureusingabroadrangeampholyte（equivalentto pIrangeof3.0–10.0）.（SeeBiotechnology-DerivedArticles—CapillaryElectrophoresis<1053>.）

Standardsolution:

USPBevacizumabRSinanappropriatediluent

Samplesolution:

DiluteaquantityofBevacizumabinanappropriatediluenttoobtainanominalconcentrationsimilartothatoftheStandardsolution.

Analyticalsystem:

Useavalidatedprocedure.RefertoMCgeneralchapterAssessingValidationParametersforReferenceandAcceptableProcedures<10>.（Althoughgeneralchapter<10>isdirectedtochromatographicmethods,conceptsintheguidelinearegeneral.）

Systemperformancerequirements

Specificity:

ThepIobtainedfromtheStandardsolution isbetween 7.4and8.0.

Analysis

Samples:

StandardsolutionandSamplesolution

ComparethepIfromtheSamplesolutionandtheStandardsolution.

Acceptancecriteria:

ThepIofthemainpeakobtainedfromtheSamplesolutiondiffersbyNMT±0.2pIunitsfromthepIofthecorrespondingpeakobtainedfromthe Standardsolution.

ASSAY

•BindingPotency

DeterminethebindingactivityofBevacizumabinasolidphasebindingassaybyusingasuitableantibodyandcoatedrhVEGF165.BindingofBevacizumabisdetectedbyspecificsecondaryantibodyinanenzyme-linkedimmunosorbentassay.PerformacomparisonofadilutionseriesoftheSamplesolutionwithadilutionseriesoftheStandardsolution.

Standardsolution:

USPBevacizumabRSinanappropriatediluent

Samplesolution:

BevacizumabinanappropriatediluenttoobtainanominalconcentrationsimilartothatoftheStandardsolution.

Analyticalsystem:

UseaprocedurevalidatedasdescribedinBiotechnology-DerivedArticles—BiologicalAssayValidation<1033>.

Systemperformancerequirements

Specificity:

TheStandardsolutionprovidesa doseresponseandpassesthesystemsuitabilitycriteria.

Precision

Repeatability:

NMT10%RSD

Intermediateprecision:

NMT10%RSD

Linearity:

Plotthemeasuredpotencyversusexpectedpotency.TheR2isNLT0.95.[Note—Theslopeshouldbe0.80–1.20.]

Accuracy

Relativebias:

NMT12%

Spikerecovery:

90%–110%

Range:

Shouldencompass80.0%–125.0%,andsatisfytheabovecriteriaforlinearity,precision,andaccuracy.

Analysis

Samples:

StandardsolutionandSamplesolution

ThepotencyoftheSamplesolutioniscalculatedrelativetotheStandardsolutionusingasuitableparallellinemethodorparallellogistic（fullcurve）method.（ProceedasdirectedinAnalysisofBiologicalAssays<1034>.）

Calculatethe95%confidencelimitsforeachindependentdeterminationofthemeasuredpotency.

Acceptancecriteria

95%Confidencelimitsforindependentdetermination:

74%–136%

Meanmeasuredpotency:

80%–125%oftheStatedpotencyobtainedasageometricmeanofaminimumofthreeindependentdeterminationsofmeasuredpotency.

95%Confidencelimitsofthemeanmeasuredpotency:

85%–118%.[Note—MeasureasmanyindependentreplicateSamplesolutionsasnecessarytoachievethe95%confidence limits.]

•VEGFNeutralizationPotency

DeterminethepotencyusingasuitableVEGFreceptorexpressingcells（similartoHUVEC）inananti-proliferationassaywithasuitablereadout.PerformacomparisonofadilutionseriesoftheSamplesolutionwithadilutionseriesoftheStandardsolution.

Standardsolution:

USPBevacizumabRSinanappropriatediluent

Samplesolution:

BevacizumabinanappropriatediluenttoobtainanominalconcentrationsimilartothatoftheStandardsolution.

Analyticalsystem:

UseaprocedurevalidatedasdescribedinBiotechnology-DerivedArticles—BiologicalAssayValidation<1033>.

Systemperformancerequirements

Specificity:

TheStandardsolutionprovidesa doseresponseandpassesthesystemsuitabilitycriteria.

Precision

Repeatability:

NMT15%GCV

Intermediateprecision:

NMT15%GCV

Linearity:

Plotthemeasuredpotencyversusexpectedpotency.TheR2isNLT0.95.[Note—Theslopeshouldbe0.80–1.20.]

Accuracy

Relativebias:

NMT15%

Spikerecovery:

85%–115%

Range:

Shouldencompass80.0%–125.0%,andsatisfytheabovecriteriaforlinearity,precision,andaccuracy.

Analysis

Samples:

StandardsolutionandSamplesolution

ThepotencyoftheSamplesolutioniscalculatedrelativetotheStandardsolutionusingasuitableparallellinemethodorparallellogistic（fullcurve）method.（ProceedasdirectedinAnalysisofBiologicalAssays<1034>.）

Calculate95%confidencelimitsforeachindependentdeterminationofthemeasuredpotency.

Acceptancecriteria

95%Confidencelimitsforindependentdetermination:

74.0%–136.0%

Meanmeasuredpotency:

80.0%–125.0%oftheStatedpotencyobtainedasageometricmeanofaminimumofthreeindependentdeterminationsofmeasuredpotency.

95%Confidencelimitsofthemeanmeasuredpotency:

85.0%–118.0%.[Note—MeasureasmanyindependentreplicateSamplesolutionsasnecessarytoachievethe95%confidence limits.]

IMPURITIES

•LimitofNon-GlycosylatedHeavyChain（NGHC）Impurities

AnalyzeBevacizumabusinganelectrophoreticmethodcapableofgivingseparationintherange10–225kDafollowedbyUVdetectionbynormalizationprocedureunderreducingconditions.（SeeBiotechnology-DerivedArticles—CapillaryElectrophoresis<1053>.）

Standardsolution:

USPBevacizumabRSinanappropriatediluent

Samplesolution:

BevacizumabinanappropriatediluenttoobtainanominalconcentrationsimilartothatoftheStandardsolution

NGHCsolution:

CombineaportionoftheStandardsolutionwithPNGaseFenzymeundersuitableconditionstoachieveatleast95%deglycosylation.

Resolutionsolution:

2.0%NGHCsolutionspikedinStandardsolution.

Analyticalsystem:

Useavalidatedprocedure. RefertoMCgeneralchapterAssessingValidationParametersforRefere