ADC安全性质控：从快速检测到合规保障的三维管控体系 ADC Safety Quality Control-From Rapid Detection to Regulatory Compliance

ADCSafetyQualityControl——

FromRapidDetectiontoRegulatoryCompliance

Ying-JieLiuPh.D

SeniorProductManager

yingjie.liu@

ACROBiosystemsaimsatbeingacornerstoneoftheglobalbiopharmaceuticalandhealthindustriesbyprovidingproductsandbusinessmodelsinnovation.

1

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ACROCMCSystem

Non-clinicalstudies

Clinicalstudies

Newdrugapplicationandreview

Approvalandlaunch

Early

research

CMCProcessDevelopment

Safety

USP<1223>EP2.6.7USP<86>EP2.6.32

Mycoplasma,Endotoxin,andSterility

IdentityandPotency

ICHQ2(R2);ICHQ6BEP2.7.29;EP5.2.12

Flow-based:cellreleasing,cellphenotyping,andfluorescent

labeling

Process-relatedresidualstesting

Nucleicacid:CHO/HEK293/HEK293T/E.coli/plasmid/Vero/Pichiapastoris/E1A/E1A&SV40LTA

Cellculture:BSA/cytokine/growthfactor/antibiotics/ECM/trypsin

Affinityligand:ProteinA/L/VH3

IVT:dsRNA/T7RNApolymerase/pyrophosphatase/Vacciniacappingenzyme/RNasininhibitor/DNaseactivity/RNaseactivity

Purity

ICHQ2(R2)

ICHQ6B

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WorkflowofAntibodyProduction

Hostcellresidual

•CHOHCD

Processresidues

•ProteinA/L/VH3affinityligand

Safety

•Mycoplasma

•Endotoxin

•Sterile(bacterial,fungal)

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RegulatoryRequirementsforMicrobialTesting

Testingguidanceformicrobiologicaltestingofbiopharmaceuticalproductsincludesthefollowing：

•EP2.6.1:Sterility

•EP2.6.7:Mycoplasmas

•USP<71>:SterilityTests

•USP<63>:MycoplasmaTests

•ICHQ5D:Originandcharacterizationofcellmatricesusedintheproductionofbiotechnology/biologicalproducts

•CBER/FDA,Pointstoconsiderinthecharacterizationofcelllinesusedtoproducebiologicals

•CBER/FDA,Pointstoconsiderinthemanufacturingandtestingofmonoclonalantibodyproductsforhumanuse

•FDA,GuidanceforIndustry:CharacterizationandQualificationofCellSubstratesandOtherBiologicalMaterialsUsedintheProductionofViralVaccinesforInfectiousDiseaseIndications

TheInvisibleThreat-Mycoplasma

•Themaintenanceofcontamination-freeculturesisfundamentaltocell-basedresearch.

•Amongthemostpervasivecontaminationsineukaryoticandstemcellculturesismycoplasma.

•Membersofthisgenusofbacteriaaretypicallysub-microninsizeandlackingacellwall.Thismakesitdifficultorimpossibletodetectbymicroscopyandresistanttomanyantibiotictreatments.

•Althoughmycoplasmacontaminationdoesnotusuallycausecelldeath,itcancausean

alteredmetabolism,slowedgrowthrates,andchromosomalaberrationsofcells

Contaminatedcelllinescanbesocompromisedthattheybehaveasacompletelydifferentline.

Fetalbovineserum,orbytransmissionfromother

contaminatedcellcultures.

Mycoplasmacanalsoquickly

spreadtocontaminateotherareasofthelabthroughaerosolsand

particulatesgeneratedduringculturehandling.

Bycoughing,sneezing-orsimplytalking-humans

generateaerosolsthatcarryvariousMycoplasma.

.

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Full-ProcessSolutionforMycoplasma

DNA

Extraction

ResultAnalysis

DNA

Amplification

PrepareSamples

ExtractionofDNAbybeads

Instrument

•OPE-32SAutomatedNuclideAcid

ExtractionSystem

SamplePreparationkits

•OPA-E101MycoplasmaDNASample

PreparationKit(Magneticbeads)

qPCRReaction

PC

NTC

NEC

Extracts

Quantitationkits

•OPA-S102MycoplasmaRapidDetectionKit(qPCR)

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Validation

EP

2.6.7.MYCOPLASMAS

TheThird-partyverification

GUIDELINEFORMYCOPLASMANATVALIDATION

•Specificity

•Detectionlimit

Positivecut-offpoint:theminimumnumberoftargetsequence

copiespervolumeofsamplethatcanbedetectedin95percentoftestruns

•Robustness

GUIDELINEFORCOMPARABILITYSTUDY

NATmaybeusedinsteadofofficialmethods(indicatorcellculturemethodand/orculturemethod)

specificity(mycoplasmapaneldetected,putativefalsepositiveresults)shouldalsobeconsidered

acomparisonoftherespectivedetectionlimitsofthealternativemethodandofficialmethods

culturemethod-10CFU/mL

cellculturemethod-100CFU/mL

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Sensitivity

24testswereperformedoneachofthe10mycoplasmastandards(10CFU/mL)

AllwerepositiveandcompliantwithorsuperiortotherequirementsofEP2.6.7(10CFU/mL)

ID

MycoplasmaSpecies

Compendiarequirements(CFU/mL)

Results

Limit(CFU/mL)

1

Mycoplasmaorale

10

24/24

1

2

Mycoplasmapneumoniae

10

24/24

0.1

3

Mycoplasmahyorhinis

10

24/24

1

4

Mycoplasmasalivarium

10

24/24

1

5

Mycoplasmafermentans

10

24/24

1

6

Mycoplasmasynoviae

10

24/24

1

7

Mycoplasmagallisepticum

10

24/24

1

8

Mycoplasmaarginini

10

24/24

1

9

Acholeplasmalaidlawii

10

24/24

0.1

10

Spiroplasmacitri

10

24/24

0.1

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Validation-Sensitivity

Scheme:

Eachmycoplasmamustbetestedinatleastthreeseparate10-foldserialdilutions,andparalleltubesmustbepreparedforeachdilutionconcentrationineachassaytoachieveatotalof24resultsforeachdilution

concentration.

Request:

PC

NTC

NEC

Extracts

Thepositivecut-offisdefinedastheconcentrationofmycoplasmathatresultsinatleast95%ofthetestruntestsbeingpositive,i.e.,atleast23validpositivetestresults.

Example：Mycoplasmahyorhinis

CFU/mL

CtValue

Mean

Result

10

Run1

27.12

27.25

26.93

26.98

27.10

27.07

26.80

26.96

27.02

24/24

Run2

27.42

27.50

27.96

27.92

27.71

27.58

28.05

28.01

27.77

Run3

27.13

27.03

27.14

27.22

26.94

26.95

26.90

26.89

27.03

ThermoFisherABI7500

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Coverage

NumberofMollicutesdetectedwithaDatabasematch

Genus

No.

Mycoplasma

>200

Acholeplasma

14

Spiroplasma

25

Ureaplasma

7

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Specificity

Twocommoncelllinesandfourunrelatedstrainswereextractedandtested.

Allwerenegativewhichindicatesthatthekit'smycoplasmadetectionisnotaffectedbyunrelatedcells/strains.

Unrelatedstrain/Cellline

CtValue

Result

Streptococcuspneumoniae

Undetermined

Undetermined

0/2

Lactobacillusacidophilus

Undetermined

Undetermined

0/2

Staphylococcusepidermidis

Undetermined

Undetermined

0/2

Clostridiumsporogenes

Undetermined

Undetermined

0/2

Clostridiumacetobutylicum

Undetermined

Undetermined

0/2

CHO（106cells/mL）

Undetermined

Undetermined

0/2

HEK293（106cells/mL）

Undetermined

Undetermined

0/2

Tcell（107cells/mL）

Undetermined

Undetermined

0/2

Pichiapastoris

Undetermined

Undetermined

0/2

E.coli

Undetermined

Undetermined

0/2

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ApplicabilityandRobustness

Thekitwasalsosuitableforthefollowingconditions,andalltestresultswerenegative,indicatedthattheseconditionsdonotinterferewiththedetectionofmycoplasma.

Verificationitems

Content

qPCRDevicecomparability

ABI7500/7500FastReal-TimePCRSystemABIQuantStudio®5

Bio-RadCFX96Real-TimePCRSystemRocheLightCycler480II

HighcelldensityBackground

CHO(106,107cells/mL)

HEK293(106,107cells/mL)

Samplematrix

OPM-AM103medium

CHOmedia+CHOCell(107cells/mL)GibcoCDCHOMedium（1×)

GibcoFreeStyleCHOT17

DMEM

FBS

DMEM+10%FBS

DMEM+10%FBS+10μg/mLPuro

DMEM+10%FBS+50μg/mLZeo

DMEM+10%FBS+500μg/mLG41810%DMSO

50mg/mLHSA

Applicationscenarios-controlPoint

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Cells(CGT),Cellbank,Rawmaterials,Cellculture,Buffer

Lot-releasetesting

In-processtesting

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Competitor

Brand

ACRO

Thermo

Satorius

(Minervabiolabs)

Roche

Product

MycoplasmaDNADetectionKit

(qPCR)

MycoSEQPlusMycoplasmaDetectionKit

Microsart®ATMPMycoplasma

MycoTOOLMycoplasmaReal-TimePCRKit

Cat.No./Packagesize

OPA-S102

A55124

SMB95-1003/1004

6495605001

50tests

100tests

25tests/100tests

160tests16samples

TypeofPCR

TaqMan®basedqPCRAssay

TaqMan®basedqPCRAssay

TaqMan®basedqPCRAssay

TaqMan®basedqPCRAssay

TargetDNAProbe

520nm(FAMchannel)

520nm(FAMchannel)

520nm(FAMchannel)

520nm(FAMchannel)

InternalControlDNA

Probe

554nm(VICchannel)

546nm(NEDchannel)

612nm(ROXchannel)

554nm(VICTMchannel)(recoverycontrol)

Fluorescencedyes

FAMTMandVICm

FAMmandNEDm

FAMmandROXm

FAMmandVICm

Time

2hours45minutes

4-5hours

4hours

4-5hours

Internal/RecoveryControlinonewell

Yes

Yes

Yes

No

UNGinkit

Yes

No

No

Yes

ExtractionAutomation

Yes

Yes

No

Yes

EP2.6.7compliance

Yes

Yes

Yes

Yes

MycoplasmaRapidDetectionKit(qPCR)

Suitableforallkindsofcomplexmatrixsamples:highcelldensity(10^7

cells/mL),10%DMSO,T/NKcellmedium,etc

ManufacturedinGMP-likefacilityandalignmentwiththeISO13485standardValidatedaccordingto

Strong

EP.2.6.7/USP<1071>,EP5.1.6andUSP<1223>

Benefit

applicability

Quality&Regulatory

Strong

Specificity

Fast

HighBroad

SensitivityCoverage

<3htorun(Preparationincluded)

Multipleprimersandprobesdevelopedforthe16srRNAofmycoplasmaspeciesdonotcross-reactwithClostridium,Lactobacillus,or

Streptococcus,whichare

closelyrelatedtomycoplasma

Coversover250Mollicutes(Mycoplasma,AcholeplasmaandSpiroplasma)species,

includingallmycoplasma

specieslistedin

Pharmacopoeias(EP,USP,JP)

Fullycompliantwithorsuperiortoregulatory

guidanceof10CFU/mL,upto1CFU/mL

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Endotoxin

EndotoxinsarethemaincomponentoftheoutermembraneofGram-negativebacteriaandofvitalimportancetotheirsurvival.Endotoxinscontributetothestructuralintegrityofbacteriaandactasaprotectiveamphipathicbarrier,shieldingbacteriafromchemicalattacks.

•Heatresistance-250℃,30min

•Molecularpolarity-easytogatherandadsorb,unevenpollution

Pyrogen

Endotoxin

•Toxicity-Endotoxinsarepyrogens,provokingastronginnateimmuneresponse,maycausefeveranddiarrhoea.Thepresenceofendotoxinstypicallyleadstohypotension,respiratoryfailureandreducedoxygendelivery.Strongendotoxemiacanleadtosepsisandeventuallydeath.

IntheproductionandqualitycontrolofpharmaceuticalproductsunderGMPconditions,theacceptedviewisthattheabsenceofendotoxinmeanstheabsenceofpyrogen.

UndertheproductionconditionsofGMP,bacterialendotoxinisthekeyofqualitycontrolofpyrogen.

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RegulatoryRequirementsforEndotoxinTesting

Testingguidanceforendotoxintestingofbiopharmaceuticalproductsincludesthefollowing：

•RabbitPyrogenTest(RPT)

•BacterialEndotoxinsTest(BET)

•LimulusAmoebocyteLysate(LAL)Assay

MethodA.Gel-clotmethod:limittest

MethodB.Gel-clotmethod:quantitativetestMethodC.Turbidimetrickineticmethod

MethodD.Chromogenickineticmethod

MethodE.Chromogenicend-pointmethodMethodF.Turbidimetricend-pointmethod

•RecombinantFactorCEndotoxintestingassay

2.6.32.TESTFORBACTERIALENDOTOXINSUSINGRECOMBINANTFACTORC

<86>BacterialEndotoxinsTestUsingRecombinantReagents

EliLilly’sEmgality(galcanezumab),thefirstdrugapprovedbytheU.S.FDAtohavebeenreleasedusingrFc.

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LALMethods-SomeInherentWeaknesses

A.SampleSensitivity

1.Turbidimetric/ChromogenicLimitations

•Unsuitableforturbid/coloredproductsduetoopticalinterference;precipitatesmaymimicpositiveresults.

•Chromogenicassaysriskprecipitateformationduringacidtermination,especiallyinpH-sensitivesamples.

2.Gel-ClotVesselDependence

•Siliconizedglass/plasticinhibitsgelformationorspectrophotometricreadings.

B.Chemical/PhysicalInterferences

1.ChemicalImpairments

•EDTAchelatescations,fluoresceindenaturesproteins,pHoutside6.0-7.5disruptsreactions;dilutionreducessensitivity.

2.PhysicalBarriers

•Highviscosity/endotoxinadsorptionhindersLALinteraction,affectingmostmethodswithoutuniversalfixes.

C.Reagent&ValidationDependence

1.ReagentSourceVariability

•Differentlysatemanufacturerscauseresultinconsistencies,requiringre-validationforsourcechanges.

2.MandatoryProductValidation

•Eachproductneedsendotoxinrecoverytesting,highlightinglimiteduniversalityandhighcomplexity.

D.MethodologicalFlaws

•Modifiedlow-LALmethodsmaysacrificesensitivity;standardizedmethodsstrugglewithcomplexmatrices(e.g.,biologicals,TCM).

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PharmacopoeiasandRegulationsRelatedto

EndotoxinTesting-Comparison

USP

EuropeanPharmacopeia

JapanesePharmacopeia

Unlessspecifiedinanindividualmonographor

GeneralNotices,thetestsinthischapterare

consideredalternativetestsandusersmustmeetthe

requirementsinGeneralNotices6.30.

ThereplacementofanLALbasedmethodprescribed

inamonographbyanrFC-basedmethodis

consideredastheuseofanalternativemethodas

describedinthePh.Eur.GeneralNotices.

<G4-4-180>describesproceduresandconsideration

inmeasurementwhenusingrecombinantprotein

reagentsforendotoxinassayasalternativemethods,

inadditiontolysatereagentsandtestmethodsin

BacterialEndotoxinsTest.

AtestforbacterialendotoxinsusingrFCorrCRcanbe

usedinthesamewayasLAL-basedmethods,after

demonstrationofitsfitnessforuseforthespecific

substanceorproduct.

Regulatoryauthoritiesmayrequiresupplementaldata

andusersareencouragedtodiscusswitheach

regulatoryauthority.

AtestforbacterialendotoxinsusingrFCcanbeused

inthesamewayasLAL-basedmethods,after

demonstrationofitsfitnessforuseforthespecific

substanceorproduct.

Ifthesereagentsforendotoxinassayareusedasan

alternativemethod,confirmthataccuracy,precision,

sensitivity,specificity,etc.areequalorbetter

comparedtoBacterialEndotoxinsTest<4.01>using

lysatereagents.

Touserecombinantreagents,supplier’sprimary

validationdatacanbeused.

TherFCcanbeusedinthesamewayasLAL-based

methods,afterdemonstrationoffitnessforusefor

thespecificsubstanceorproduct.

Therecombinantprotein-reagentsforendotoxinassay

arenotidenticalto‘‘anamoebocytelysateprepared

frombloodcorpuscleextractsofhorseshoecrab’’

specifiedinBacterialEndotoxinsTest<4.01>.

IncludesmethodsforrFCandrCR.

IncludesmethodsforrFC.

IncludesmethodsforrFCandrCR.

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EndotoxinDetection-Methods

Comparison

content

ConventionalTachypiensAmebocyteLysate

RecombinantCfactorendotoxinassay

Gelationmethod

Turbiditymethod

Colorimetricmethod

Endpointturbiditymethod

Dynamicturbiditymethod

Matrixcolorimetricmethod

Dynamic

colorimetricmethod

Nature

Qualitative,

Semi-quantitative

Quantify

Quantify

Quantify

Quantify

Quantify

Instrumentandequipment

37℃waterbath

Microplatereader，Turbiditymeter

Temperature

controlled

microplatereader,

Turbiditymeter

Spectrophotometer,Microplatereader

Temperature

controlled

microplatereader

Fluorescencedetection(Wavelength:380/440nm）

Detectionresult

Manualreadingandrecording(Subjective)

Automate,

Electronicdata

Detection

sensitivity

0.03、0.06、0.125、0.25EU/mL

0.01-100EU/mL

0.1-1EU/mL

0.005-50EU/mL

0.005-5EU/mL

Reagentsource

Lysatesofhorseshoecrabblooddeformedcells

Recombinantexpressionofhorseshoe

crabfactorCeliminatesdependenceon

animalderivedreagentsandconformsto

the3Rsubstitutionprinciple.

Reactionprinciple

Cascadereaction(involvingthreeserineproteasomes,withGfactorbypassinterference)

ThesinglefactorCreactionhasmuch

higheraccuracyandprecisionthanthe

horseshoecrabreagentmethod.

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EndotoxinStandardsAreBenchmarkedandTraced

1000000

△RFU-Blank

△RFU-Blank

ACROkitstandard

USPEndotoxinStandard

10000

100

1

0.0010.010.1110

EndotoxinsConc.(EU/mL)

1000000

ACROkitstandard

CHPEndotoxinStandard

10000

100

1

0.0010.010.1110

EndotoxinsConc.(EU/mL)

Usingendotoxin(USP,Cat.No.U1235503)asastandard,therecoveryofACROkitstandardsrangedfrom80%to120%,meetingguidelines.

Usingtheendotoxinstandard(Cat.No.150601)asthestandardcurve,therecoveryrateoftheACROKitstandardwaswithintherangeof50%-200%,whichmettherequirements

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LOQ&Precision&Accuracy

Specificity

FalsepositiveendotoxinresultsinaDCproductcausedby(1->3)-b-D-glucansacquiredfromasterilizingcellulosefilter

Finalproduct-releasetestingincludedanassayforendotoxin,performedbytheLimulusamebocytelysate(LAL)gelclotmethodwithacut-offof0.06endotoxinunits/mL(EU/mL).BGarebiologicallyactive,immunomodulatory,andareknowntoreactwiththefactorG-activatedcoagulationpathwayintheprimitiveimmunesystemofthehorseshoecrab.BGhavebeenusedasvaccineadjuvantsincombinationwithaLeishmaniavaccineandanHIVDNA

vaccine.

Antibodydrugorproteinprocesssamplesarepronetohighlevelsofβ-glucan,resultinginfalsepositivesforendotoxintesting

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Endotoxinresiduesinβ-glucanatconcentrationsof10μg/mLand1ug/mLweredetectedusingrFC.Nonon-specificsignalwasdetected.Incontrast,thedynamicchromogenicmethodforβ-dextrandetectiondetectsendotoxinsandnon-specificsignals.ThisindicatesthatrecombinantfactorCdoesnotreactwithβ-glucan,demonstratinggoodspecificityoftherFCmethod.

24

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MatrixInterferenceData

Matrix

Recovery

DilutionFactor

1MNaCl

140%

16

20mMCaCl2

82%

8

20mMMgCl2

98%

4

1MSodiumacetate,pH5.0

73%

16

50mMSodiumacetate,pH3.5100mMTris,pH10.9

100mMGlycine,pH3.5

74%

20

113%

80

109%

16

1×PBS,pH7.5

102%

40

1×PBS,pH6.0

91%

20

50mMTris,100mMGlycine,225mMArginine,150mMNaCl,0.005%Tween80,pH7.5with11%Trehalose

81%

4

50mMTris,100mMGlycine,25mMArginine,150mMNaCl,pH7.5with0.01%Tween80with11%Trehalose

88%

16

Essential8FlexBasalMedium(Thermofisher,Cat.No.A2858501)

74%

4

mTeSRTMPlus(Stemcell,Cat.No.100-0276)

82%

32

CelTheraGMPTCellExpansionMedium(Acrobiosystems,Cat.No.GMP-CM3101)

93%

4

RPMI1640Medium(Hyclone,Cat.No.SH30809.01)

109%

8

DMEMMedium(Basalmedia,Cat.No.L120KJ)

104%

8

CTSmOpTmizermT-CellExpansionSFM,nophenolred,bottleformat(Gibco,Cat.No.A3705001)

91%

16

12.5mMHistidineBuffer,pH6.5

88%

4

KeytrudaFormulation(1.55mg/mLL-histidine,0.2mg/mLpolysorbate80,70mg/mLsucroseinwater,pH5.2-5.8)

76%

64

HemlibraFormulation(26.1mg/mLL-arginine,3.1mg/mLL-histidine,0.5mg/mLpoloxamer188,adjustedtopH6.0withL-asparticacid)

100%

4

30%DMSO

99%

16

HSA(25mg/mL)

73%

64

Toripalimab(40mg/mL)

83%

40

MultipleElectrolytesInjection(Baxter)

96%

4

100%FBS

109%

10

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Comparability

Differentmethodswereusedtodetectendotoxinresiduesinfivesamples,andthedeviationbetweenthedetectionresultsofrFCmethodandLALmethodiswithin2times.

Sample

Endotoxin

limit

LAL(Endpoint)

LAL(Kinetic)

rFc(RES-A056)

RecombinantHumanInterferonα-1b

10EU/mL

<0.02EU/mL

<0.01EU/mL

<0.16EU/mL

Humaninsulininjection

32EU/mL

<0.02EU/mL

<0.01EU/mL

<0.02EU/mL

CelTheraGMPTCell

ExpansionMedium

(ACROBiosystems,

Cat.No.GMP-

CM3101)

10EU