2024年5月17日发(作者：五妙珍)

ProteinAggregation:Pathways,InductionFactors

andAnalysis

HANNS-CHRISTIANMAHLER,

1

WOLFGANGFRIESS,

2

ULLAGRAUSCHOPF,

1

SYLVIAKIESE

1

1

2

FormulationR&DBiologics,PharmaceuticalandAnalyticalR&D,nn-LaRocheLtd.,Basel,Switzerland

DepartmentofPharmacy,PharmaceuticalTechnologyandBiopharmaceutics,

Ludwig-Maximilians-UniversitaetMuenchen,Munich,Germany

Received27March2008;revised1August2008;accepted5August2008

Publishedonline29September2008inWileyInterScience().DOI10.1002/jps.21566

ABSTRACT:Controlandanalysisofproteinaggregationisanincreasingchallenge

henatureofproteinin-

teractions,proteinaggregationmayoccuratvariouspointsthroughoutthelifetime

ofaproteinandmaybeofdifferentquantityandqualitysuchassize,shape,morphology.

Itisthereforeimportanttounderstandtheinteractions,causesandanalysesofsuch

aggrega

reviewgivesashortoutlineofcurrentlydiscussedpathwaysandinductionmethods

forproteinaggregationanddescribescurrentlyemployedsetofanalyticaltechniques

andemergingtechnologiesforaggregatedetection,characterizationandquantification.

Amajorchallengefortheanalysisofproteinaggregatesisthatnosingleanalytical

methodexiststocovertheentiresizerangeortypeofaggregateswhichmay

alyticalmethodnotonlyshowsitsspecificadvantagesbutalsohas

itsofdetectionandthepossibilityofcreatingartifactsthrough

samplepreparationbyinducingordestroyingaggregatesneedtobeconsidered

ore,itmayalsobeadvisabletocarefullycompare

analyticalresultsoforthogonalmethodsforsimilarsizerangestoevaluatemethod

performance.

ß2008Wiley-Liss,AmericanPharmacistsAssociationJPharmSci

98:2909–2934,2009

Keywords:proteinaggregation;proteinanalysis;proteinformulation;molecular

weightdetermination;particlesizing;sizeexclusionchromatography;analyticalultra-

centrifugation;lightscattering;SDS–PAGE;orthogonalmethods

INTRODUCTION

ThebreakthroughofrecombinantDNAtechnol-

ogyinthemid1970shasallowedthedevelopment

H.-ontributedequallytothisstudy.

Correspondenceto:Hanns-ChristianMahler(Telephone:

41-61-68-83174;Fax:41-61-68-88689;

E-mail:@)

JournalofPharmaceuticalSciences,Vol.98,2909–2934(2009)

ß2008Wiley-Liss,AmericanPharmacistsAssociation

ofmanyrecombinanttherapeuticproteinsand

thushasresultedinmanyprotein-basedproducts

toreachthemarket.

1,2

Thecontrolandanalysisof

proteinaggregationduringproductionofabio-

therapeuticdrugisanincreasingchallengeto

manypharmaceuticalresearchanddevelopment

ationispotentially

encounteredduringvariousstepsofthemanu-

facturingprocessofbiopharmaceuticals,which

includefermentation,purification,formulation

rmaceuticalsfor

2909

JOURNALOFPHARMACEUTICALSCIENCES,VOL.98,NO.9,SEPTEMBER2009

2910

MAHLERETAL.

clinicaltrialsrequirefullcharacterizationinclud-

ingaccuratequantificationofproteinaggregates

tomeetthedrugproductspecifin

aggregatespotentiallycauseadverseeffects,such

asanimmuneresponse,

3,4

whichmaycause

neutralizationoftheendogenousproteinwith

essentialbiologicalfunctionsleadingtoalife-

threateningsituationforthepatientandaggre-

gatesmayalsopotentiallyimpactthedrug’s

efficacy.

5

Thescientificfactbasetoclearlylink

specifictypesandsizesofaggregatestoimmune

responsesishowevercurrentlystillunderinves-

tialincreaseinimmuneres-

ponsescausedbyaggregateshasbeenreported

previously,

3

whereasincontrastnoenhanced

immunogenicitywasshownforexampleinthe

caseofaggregatedrFVIII.

6

Therearemonographsandacceptancecriteria/

limitsinthepharmacopoeiasforvisibleand

,insolubleproteinsaggre-

gates)—forexample,UnitedStatesPharmaco-

poeia(USP)<788>,

7

EuropeanPharmacopoeia

(.)2.9.19

8

2.9.20

9

—for

r,limitsforsoluble

aggregateshavetobesetcase-by-caseasthereare

nopredefinedlimitslaiddowningeneralfor

biopharmaceuticalswithinregulatorydocuments.

Inordertocontrolproteinaggregationtoenable

safeandsuccessfulproducts,itisimportantto

understandtheoriginofproteinaggregates,and

theanalyticaltechniquesforcharacterizingtheir

fullsizerange.

Thisreviewarticleaimstocollateanddiscuss

availableliteratureonthemajorcausesofaggre-

gationandtheanalyticalmethods/techniquesto

characterizeproteinaggregates.

PATHWAYSANDINDUCTIONFACTORS

DefinitionandMechanismofProteinAggregation

Theterm‘‘proteinaggregation’’hasbeengiven

manydefinitionsandterminologieswithinthe

literature.

10,11

Theauthorsdefine‘‘proteinaggre-

gates’’asasummaryofproteinspeciesofhigher

molecularweightsuchas‘‘oligomers’’or‘‘multi-

mers’’insteadofthedesireddefi,a

monomer).Aggregatesarethusauniversalterm

forallkindsofnotfurtherdefinedmultimeric

speciesthatareformedbycovalentbondsor

noncovalentinteractions.

Differentmechanismsthatmayleadtoforma-

tionofvarioustypesofaggregatesarecurrently

snosingleprotein

aggregationpathwaybutavarietyofpathways,

whichmaydifferbetweenproteins

12

andmay

inmay

undergovariousaggregationpathwaysdepending

ontheenvironmentalconditions,includingdif-

,theinitial

stateofaproteinthatisproneforsubsequent

econstitutedby

thenativestructure,

13

byadegraded

14

ormodified

structure,

15

byapartiallyunfoldedstructure

15,16

orbythefullyunfoldedstate.

12

Theaggregationprocessingeneralmayleadto

solubleand/orinsolubleaggregateswhichmay

precipitate.

13,17–19

Themorphologyoftheseinso-

lubleaggregatesmaybeintheformofamorphous

orfibrillarmaterialwhichisdependentonthe

alentaggre-

gatesareformedsolelyviaweakforcessuchas

VanderWaalsinteractions,hydrogenbonding,

hydrophobicandelectrostaticinteractions

20

whereascovalentaggregatesmayforexample

formviadisulfidebondlinkagesthroughfreethiol

groups

11,21,22

orbynondisulfidecross-linking

pathwayssuchasdityrosineformation.

23

Aggre-

gationmaybereversible

24

orirreversiblewhere

theirreversibleaggregatescouldbepermanently

eliminatedbypreparativeseparationprocesses

suchasfiltrationtechniques.

25

Theformationof

reversibleaggregatesisoftenconsideredtobe

causedbytheself-assemblyofproteinmolecules,

whichcouldbeinducedbychangesinpHorionic

strengthoftheproteinsolution.

26–30

Onemodelthathasbeenappliedtodescribe

irreversibleproteinaggregationistheLumry-

Eyringtwostatemodel.

31

Accordingtothismodel

thenativeproteinundergoesfirstareversible

conformationalchangetoanaggregation-prone

state,whichsubsequentlyassemblesirreversibly

modelprotein

aggregationistherebycontrolledbyconforma-

tionalandcolloidalmechanisms.

18,25

Inmanycases,aggregationwasdescribedto

followanucleation–propagationpolymerization

mechanism,wherebythenucleuscanbeformed

byanalteredmonomericstructureorbya

multimericspecies.

32

Newreportsalsosuggest

theappearanceofheterogenousnucleationwhich

isinducedbymicro-andnanoparticlesofforeign

matter,whichforexamplecouldbeshedfromthe

equipmentduringprocessing.

33,34

Muchinsightin

proteinaggregationpathwaysisobtainedfrom

researchinthefieldofamyloidfiberformation

35

andsicklecellhemoglobin.

36

Intheareaof

DOI10.1002/jpsJOURNALOFPHARMACEUTICALSCIENCES,VOL.98,NO.9,SEPTEMBER2009

2024年5月17日发(作者：五妙珍)

ProteinAggregation:Pathways,InductionFactors

andAnalysis

HANNS-CHRISTIANMAHLER,

1

WOLFGANGFRIESS,

2

ULLAGRAUSCHOPF,

1

SYLVIAKIESE

1

1

2

FormulationR&DBiologics,PharmaceuticalandAnalyticalR&D,nn-LaRocheLtd.,Basel,Switzerland

DepartmentofPharmacy,PharmaceuticalTechnologyandBiopharmaceutics,

Ludwig-Maximilians-UniversitaetMuenchen,Munich,Germany

Received27March2008;revised1August2008;accepted5August2008

Publishedonline29September2008inWileyInterScience().DOI10.1002/jps.21566

ABSTRACT:Controlandanalysisofproteinaggregationisanincreasingchallenge

henatureofproteinin-

teractions,proteinaggregationmayoccuratvariouspointsthroughoutthelifetime

ofaproteinandmaybeofdifferentquantityandqualitysuchassize,shape,morphology.

Itisthereforeimportanttounderstandtheinteractions,causesandanalysesofsuch

aggrega

reviewgivesashortoutlineofcurrentlydiscussedpathwaysandinductionmethods

forproteinaggregationanddescribescurrentlyemployedsetofanalyticaltechniques

andemergingtechnologiesforaggregatedetection,characterizationandquantification.

Amajorchallengefortheanalysisofproteinaggregatesisthatnosingleanalytical

methodexiststocovertheentiresizerangeortypeofaggregateswhichmay

alyticalmethodnotonlyshowsitsspecificadvantagesbutalsohas

itsofdetectionandthepossibilityofcreatingartifactsthrough

samplepreparationbyinducingordestroyingaggregatesneedtobeconsidered

ore,itmayalsobeadvisabletocarefullycompare

analyticalresultsoforthogonalmethodsforsimilarsizerangestoevaluatemethod

performance.

ß2008Wiley-Liss,AmericanPharmacistsAssociationJPharmSci

98:2909–2934,2009

Keywords:proteinaggregation;proteinanalysis;proteinformulation;molecular

weightdetermination;particlesizing;sizeexclusionchromatography;analyticalultra-

centrifugation;lightscattering;SDS–PAGE;orthogonalmethods

INTRODUCTION

ThebreakthroughofrecombinantDNAtechnol-

ogyinthemid1970shasallowedthedevelopment

H.-ontributedequallytothisstudy.

Correspondenceto:Hanns-ChristianMahler(Telephone:

41-61-68-83174;Fax:41-61-68-88689;

E-mail:@)

JournalofPharmaceuticalSciences,Vol.98,2909–2934(2009)

ß2008Wiley-Liss,AmericanPharmacistsAssociation

ofmanyrecombinanttherapeuticproteinsand

thushasresultedinmanyprotein-basedproducts

toreachthemarket.

1,2

Thecontrolandanalysisof

proteinaggregationduringproductionofabio-

therapeuticdrugisanincreasingchallengeto

manypharmaceuticalresearchanddevelopment

ationispotentially

encounteredduringvariousstepsofthemanu-

facturingprocessofbiopharmaceuticals,which

includefermentation,purification,formulation

rmaceuticalsfor

2909

JOURNALOFPHARMACEUTICALSCIENCES,VOL.98,NO.9,SEPTEMBER2009

2910

MAHLERETAL.

clinicaltrialsrequirefullcharacterizationinclud-

ingaccuratequantificationofproteinaggregates

tomeetthedrugproductspecifin

aggregatespotentiallycauseadverseeffects,such

asanimmuneresponse,

3,4

whichmaycause

neutralizationoftheendogenousproteinwith

essentialbiologicalfunctionsleadingtoalife-

threateningsituationforthepatientandaggre-

gatesmayalsopotentiallyimpactthedrug’s

efficacy.

5

Thescientificfactbasetoclearlylink

specifictypesandsizesofaggregatestoimmune

responsesishowevercurrentlystillunderinves-

tialincreaseinimmuneres-

ponsescausedbyaggregateshasbeenreported

previously,

3

whereasincontrastnoenhanced

immunogenicitywasshownforexampleinthe

caseofaggregatedrFVIII.

6

Therearemonographsandacceptancecriteria/

limitsinthepharmacopoeiasforvisibleand

,insolubleproteinsaggre-

gates)—forexample,UnitedStatesPharmaco-

poeia(USP)<788>,

7

EuropeanPharmacopoeia

(.)2.9.19

8

2.9.20

9

—for

r,limitsforsoluble

aggregateshavetobesetcase-by-caseasthereare

nopredefinedlimitslaiddowningeneralfor

biopharmaceuticalswithinregulatorydocuments.

Inordertocontrolproteinaggregationtoenable

safeandsuccessfulproducts,itisimportantto

understandtheoriginofproteinaggregates,and

theanalyticaltechniquesforcharacterizingtheir

fullsizerange.

Thisreviewarticleaimstocollateanddiscuss

availableliteratureonthemajorcausesofaggre-

gationandtheanalyticalmethods/techniquesto

characterizeproteinaggregates.

PATHWAYSANDINDUCTIONFACTORS

DefinitionandMechanismofProteinAggregation

Theterm‘‘proteinaggregation’’hasbeengiven

manydefinitionsandterminologieswithinthe

literature.

10,11

Theauthorsdefine‘‘proteinaggre-

gates’’asasummaryofproteinspeciesofhigher

molecularweightsuchas‘‘oligomers’’or‘‘multi-

mers’’insteadofthedesireddefi,a

monomer).Aggregatesarethusauniversalterm

forallkindsofnotfurtherdefinedmultimeric

speciesthatareformedbycovalentbondsor

noncovalentinteractions.

Differentmechanismsthatmayleadtoforma-

tionofvarioustypesofaggregatesarecurrently

snosingleprotein

aggregationpathwaybutavarietyofpathways,

whichmaydifferbetweenproteins

12

andmay

inmay

undergovariousaggregationpathwaysdepending

ontheenvironmentalconditions,includingdif-

,theinitial

stateofaproteinthatisproneforsubsequent

econstitutedby

thenativestructure,

13

byadegraded

14

ormodified

structure,

15

byapartiallyunfoldedstructure

15,16

orbythefullyunfoldedstate.

12

Theaggregationprocessingeneralmayleadto

solubleand/orinsolubleaggregateswhichmay

precipitate.

13,17–19

Themorphologyoftheseinso-

lubleaggregatesmaybeintheformofamorphous

orfibrillarmaterialwhichisdependentonthe

alentaggre-

gatesareformedsolelyviaweakforcessuchas

VanderWaalsinteractions,hydrogenbonding,

hydrophobicandelectrostaticinteractions

20

whereascovalentaggregatesmayforexample

formviadisulfidebondlinkagesthroughfreethiol

groups

11,21,22

orbynondisulfidecross-linking

pathwayssuchasdityrosineformation.

23

Aggre-

gationmaybereversible

24

orirreversiblewhere

theirreversibleaggregatescouldbepermanently

eliminatedbypreparativeseparationprocesses

suchasfiltrationtechniques.

25

Theformationof

reversibleaggregatesisoftenconsideredtobe

causedbytheself-assemblyofproteinmolecules,

whichcouldbeinducedbychangesinpHorionic

strengthoftheproteinsolution.

26–30

Onemodelthathasbeenappliedtodescribe

irreversibleproteinaggregationistheLumry-

Eyringtwostatemodel.

31

Accordingtothismodel

thenativeproteinundergoesfirstareversible

conformationalchangetoanaggregation-prone

state,whichsubsequentlyassemblesirreversibly

modelprotein

aggregationistherebycontrolledbyconforma-

tionalandcolloidalmechanisms.

18,25

Inmanycases,aggregationwasdescribedto

followanucleation–propagationpolymerization

mechanism,wherebythenucleuscanbeformed

byanalteredmonomericstructureorbya

multimericspecies.

32

Newreportsalsosuggest

theappearanceofheterogenousnucleationwhich

isinducedbymicro-andnanoparticlesofforeign

matter,whichforexamplecouldbeshedfromthe

equipmentduringprocessing.

33,34

Muchinsightin

proteinaggregationpathwaysisobtainedfrom

researchinthefieldofamyloidfiberformation

35

andsicklecellhemoglobin.

36

Intheareaof

DOI10.1002/jpsJOURNALOFPHARMACEUTICALSCIENCES,VOL.98,NO.9,SEPTEMBER2009