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BIOPHYSICALCHARACTERIZATIONOF

PROTEINSINDEVELOPING BIOPHARMACEUTICALS

SECONDEDITION

BiomolecularDiscovery,RelayTherapeutics, Cambridge,MA,UnitedStates

STEVEN A.BERKOWITZ

Consultant,Sudbury,MA,UnitedStates

Elsevier

Radarweg29,POBox211,1000AEAmsterdam,Netherlands

TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom 50HampshireStreet,5thFloor,Cambridge,MA02139,UnitedStates

Copyright©2020ElsevierB.V.Allrightsreserved.

Nopartofthispublicationmaybereproducedortransmittedinanyformorbyanymeans,electronicor mechanical,includingphotocopying,recording,oranyinformationstorageandretrievalsystem,without permissioninwritingfromthepublisher.Detailsonhowtoseekpermission,furtherinformationaboutthe Publisher’spermissionspoliciesandourarrangementswithorganizationssuchastheCopyrightClearanceCenter andtheCopyrightLicensingAgency,canbefoundatourwebsite: www.elsevier.com/permissions

ThisbookandtheindividualcontributionscontainedinitareprotectedundercopyrightbythePublisher(other thanasmaybenotedherein).

Notices

Knowledgeandbestpracticeinthis fieldareconstantlychanging.Asnewresearchandexperiencebroadenour understanding,changesinresearchmethods,professionalpractices,ormedicaltreatmentmaybecomenecessary.

Practitionersandresearchersmustalwaysrelyontheirownexperienceandknowledgeinevaluatingandusingany information,methods,compounds,orexperimentsdescribedherein.Inusingsuchinformationormethodsthey shouldbemindfuloftheirownsafetyandthesafetyofothers,includingpartiesforwhomtheyhaveaprofessional responsibility.

Tothefullestextentofthelaw,neitherthePublishernortheauthors,contributors,oreditors,assumeanyliability foranyinjuryand/ordamagetopersonsorpropertyasamatterofproductsliability,negligenceorotherwise,or fromanyuseoroperationofanymethods,products,instructions,orideascontainedinthematerialherein.

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Contributors

YvesAubin CentreforBiologics,Regulatory ResearchDivision,Evaluation,Biologicsand GeneticTherapiesDirectorate,HealthProducts andFoodBranch,HealthCanada,Ottawa,ON, Canada

StevenA.Berkowitz Consultant,Sudbury,MA, UnitedStates

GeorgeM.Bou-Assaf AnalyticalDevelopment, Biogen,Cambridge,MA,UnitedStates

MarkL.Brader DrugProductAnalytical Development,Moderna,Cambridge,MA, UnitedStates

RichardK.Burdick BurdickStatisticalConsulting,LLC,ColoradoSprings,CO,United States

JohnF.Carpenter DepartmentofPharmaceuticalSciences,CenterforPharmaceuticalBiotechnology,UniversityofColoradoAnschutz MedicalCampus,Aurora,CO,UnitedStates

StephenJ.Demarest EliLillyBiotechnology Center,SanDiego,CA,UnitedStates

ErtanEryilmaz Takeda,Cambridge,Massachusetts,UnitedStates

VernaFrasca FieldApplicationsManager,MalvernPanalytical,Northampton,MA,United States

DarronL.Freedberg StructuralBiologySection, LaboratoryofBacterialPolysaccharides,Silver Spring,MD,UnitedStates

JohnP.Gabrielson ElionLabs,ADivisionof KBIBiopharma,Inc.,Louisville,CO,United States

AndreaHawe CoriolisPharma,Munich, Germany

DamianJ.Houde BiomolecularDiscovery, RelayTherapeutics,Cambridge,MA,United States

DavidA.Keire FoodandDrugAdministration, St.Louis,MO,UnitedStates

FrancisKinderman Amgen,ThousandOaks, California,UnitedStates

LeeMakowski DepartmentofBioengineering andDepartmentofChemistryandChemical Biology,NortheasternUniversity,Boston,MA, UnitedStates

JohnP.Marino NationalInstituteofStandards andTechnology,InstituteforBioscienceand BiotechnologyResearch,Rockville,MD,United States

AlanG.Marshall IonCyclotronResonance Program,NationalHighMagneticField Laboratory,FloridaStateUniversity,Tallahassee,FL,UnitedStates;Departmentof Chemistry & Biochemistry,FloridaState University,Tallahassee,FL,UnitedStates

A.J.Miles InstituteofStructuralandMolecular Biology,BirkbeckCollege,UniversityofLondon,London,UnitedKingdom

JohnS.Philo AllianceProteinLaboratories,San Diego,CA,UnitedStates

AngelikaReichel CoriolisPharma,Munich, Germany

DenizB.Temel Bristol-MyersSquibb,Devens, Massachusetts,UnitedStates

B.A.Wallace InstituteofStructuralandMolecularBiology,BirkbeckCollege,Universityof London,London,UnitedKingdom

DanielWeinbuch CoriolisPharma,Munich, Germany

WilliamF.WeissIV BioproductResearchand Development,LillyResearchLaboratories,Eli LillyandCompany,Indianapolis,IN,United States

SarahZölls CoriolisPharma,Munich,Germany

Prefacesforthesecondedition

Criticaltothedevelopmentofany successfultherapeuticdrugisourabilityto identifyandmanufacturethedrugsuch thatitsbene fi cialtherapeuticeffectcanbe safelydeliveredtothepatient.Inthecaseof proteinbiopharmaceuticals,theselarge, heterogeneous(complex)andmarginally stablemoleculesareoftenverysensitiveto theirmicro-environment.Thismakesthe processofdevelopingandmanufacturinga proteintherapeuticextremelychallenging. Throughoutthisentireprocessaproteinbiopharmaceuticalmustmaintainitscomplex anddelicatestructure(orconformation)to realizeitsbeneficialtherapeuticattributes, whileavoidingthepotentialharmfuleffects infailingtoachievethisgoal.

Whenwesetouttowritethe firstedition ofthisbook,ourgoalwastoprovidea generalresourcethatspeci ficallydealtwith themanychallengesassociatedwiththe testingandcharacterizationofthehigher orderstructureandbiophysicalpropertiesof proteinbiopharmaceuticalsfromapractical pointofviewtosupportitssafetyand bene ficialtherapeuticactivity.Asstatedin thebook’ s firstprefacewewantedtokeep thereaderfocusedonobtainingapragmatic understandingandknowledgeoftheutility ofbiophysicaltoolsandhowtheyareusedto meetthesechallengesbyunderstanding whatinformationcanrealisticallybeextractedfromthesetools.Whilewefeltwehad initiallyachievedourgoal,theprogressionof timeinevitablyledtobetterandimproved

scienti ficdevelopmentsandtotherealizationthattherewasroomforimprovements.

Asaresult,inwritingthissecondedition wehaveundertakenthejobofupdatingold information,correctingmistakes,improving clarityandtheintroductionofnewtopics thatwerenotcoveredinthe fi rstedition. Therefore,wegatheredourco-authorsonce again,invitedafewnewones,andtasked ourselveswiththegoaltoachievethese objectives.Insodoing,alloriginalchapters havebeenupdated,correctedand enhanced,whilenewchaptershavebeen added.

Globally,theformatofthebookhas remainedthesame,consistingofthreesections.SectionI,whichdealswiththe complexityofproteinsandtherelevanceof biophysicalmethodsinthebiopharmaceuticalindustry.Ithasforthemostpartbeen alteredtoremoveerrorsandachieveclarity. SectionII,whichdiscussesthebiophysical toolsandtechniquesmostcommonlyusedin thebiopharmaceuticalindustrytocharacterizeproteintherapeuticmoleculeshas similarlybeenaltered,buthasalsobeen enhancedbytheadditionofanewchapter (Chapter14)dedicatedtotheareaofchromatographyandelectrophoresis.Thetoolsin thischapter,whichwedidnotcoverinthe firsteditionofthebook(withtheexceptionof size-exclusionchromatography),aretypically notthoughtoforclassifiedasbiophysical tools.Nevertheless,animportantobjectivein addingthischapteristobringmoreattention

totheirunrealizedlinkageaseffectivebiophysicalcharacterizationtoolswithoutgettingtoodeepintothedetailsoftheirinner workings(whichareextensivelycoveredin manyexcellentbooksandreviewarticlesthat aresolelydedicatedtothesetwoenormously importanttechniques).

Overall,however,SectionIIIofthebook hasexperiencedthemostsigni ficantchange andexpansionviatheadditionoffournew chaptersthatcoverthefollowing:

• Chapter15,whichdealswiththebiophysical characterizationofcomplexbiopharmaceuticals;

• Chapter16,whichdealswiththerigorof statisticalanalysis;

• Chapter17,whichdealswithbiopharmaceuticaldevelopability;

• Chapter18,whichdealswithtechnicaldecisionmaking.

Finally,wewouldliketopointoutthat inwritingthissecondeditionwehave madeaparticulareffort,whereverpossible, tobetterlinkandcross-referenceinformationineachchaptertobringmorecohesion tothebookasapposetojustproviding thereaderwithacollectionofisolated chapters.Wethinkhiscohesionisinparticularmadeapparentbythefouradditional chaptersinSectionIII(describedabove).

Intheend,weandourcoauthorshopewe havefurtherenhancedtheinitialobjectiveof the firsteditionofthebook,ofenlightening thereadertothechallenges,toolsandinner workingsofthetaskassociatedwiththe biophysicalcharacterizationofproteinbiopharmaceuticals.Anintegralpartoftoday’ s modernandchallengingworldofdevelopinglifesavingdrugs.

Listofabbreviationsandsymbols

(T)RPS (Tunable)resistivepulsesensing

3D Threedimensional

A22 orB2

Secondviralcoefficient

AAV Adeno-associatedvirus

AC Alternatingcurrent

AC-SINS Affinity-captureself-interactionnanoparticlespectroscopy

AFFF-MALLS

Asymmetric field flowfractionationwithmulti-anglelaserlightscattering

ACN Acetonitrile

ACS Ammoniumcamphorsulfonate

ADCorADCs AnalogtodigitalconverterorAntibodydrugconjugate(s)

ADCC Antibodydependentcell-mediatedcytotoxicity

AF4 Asymmetric flow field flowfractionation

AFM Atomicforcemicroscopy

API Activepharmaceuticalingredient

APR Aggregationproneregions

AQL Acceptablequalitylevel

ASTM Americansocietyfortestingandmaterials

ATP Analyticaltargetprofile

ATR Attenuatedtotalreflectance

AUC Analyticalultracentrifugation

BiAborbsAb Bispecificantibody

BLA Biologicallicenseapplication

BMI Backgroundedmembraneimaging

BSA Bovineserumalbumin

CA Capsidprotein

CAD Collision-activateddissociation

CCD Charge-coupleddevice

CD Circulardichroism

CDER Centerfordrugevaluationandresearch

CDR Complementarity-determiningregion

CEX Cation-exchangechromatography

cGMP Currentgoodmanufacturingpractices

CH Immunoglobulingammaheavychainconstantdomain

CH1orCH1

Immunoglobulingammaheavychainconstantdomain1

CH2orCH2 Immunoglobulingammaheavychainconstantdomain2

CH3orCH3 Immunoglobulingammaheavychainconstantdomain3

CHO Chinesehamsterovary

CIC Cross-interactionchromatography

CID Collisioninduceddissociation xv

cIEF

Capillaryisoelectricfocusing

CIU Collision-inducedunfolding

CL Immunoglobulingammalightchainconstantdomain

CMC ManufacturingandControl

COSY Correlationspectroscopy

cP Centipose

CPL Circularlypolarizedlight

CQAorCQAs Criticalqualityattribute(s)

CSA Camphorsulfonicacid

CZE Capillary(free)zoneelectrophoresis

D Deuteriumortranslationaldiffusioncoefficientorelectricdipole

DAC Deutscherarzneimittel-codex

DC Directcurrent

DI Developabilityindex

DLS Dynamiclightscattering

DoE Designofexperiment

DNA Deoxyribonucleicacid

DOSY Diffusionorderedspectroscopy

DP Drugproduct

dPLIMSTEX

DilutionPLIMSTEX

DRI Differentialrefractiveindexdetector

DS Drugsubstance

DSA 4,4-dimethyl-4-silapentane-1-ammoniumtrifluoroacetate

DSC Differentialscanningcalorimetry

DSF Differentialscanning fluorimetry

DSLS Differentialstaticlightscattering

DSS 4,4-dimethyl-4-silpentane-1-sulfonicacid

DTT Dithiothreitol

ECD Electroncapturedissociationorequivalentcirculardiameter

ECHOS Easycomparabilityofhigherorderstructure

EDTA Ethylenediaminetetra-aceticacid

EM Electromagneticradiationorelectronmicroscopy

EMEA EuropeanMedicinesAgency

ESD Equivalentspherediameter

ESI Electrosprayionization

ESZ Electricalsensingzone

ET Electrontomography

ETD Electrontransferdissociation

EX1

H/Dexchangemechanisminwhichtherateconstantforproteinfolding/unfolding ismuchslowerthantherateconstantforH/Dexchange

EX2 H/Dexchangemechanisminwhichtherateconstantforproteinfolding/unfolding ismuchfasterthantherateconstantforH/Dexchange

Fab Immunoglobulingammafragmentantigenbinding

Fc Immunoglobulingammafragmentcrystallizable(constantregion)

FcgRIIIa ImmunoglobulingammaFcreceptorRIIIa

FcRn NeonatalFcreceptor

FDA FoodandDrugAdministration

FFF Field flowfractionation

FID Freeinductiondecay

FIX

BloodclottingfactorIX

FL Fluorescence

FT-ICR Fouriertransformioncyclotronresonance

FTIRorFT-IR Fouriertransforminfraredspectroscopy

fuc Fucose

FUV-CD

FVIII

Farultravioletcirculardichroism

BloodclottingfactorVIII gal Galactose

GlcNAc N-acetylglucosamine

GLP

GMP

H/DX-MSorHDX-MS

HSA

HCH

HCl

HCLF

HDC

HDX

Goodlaboratorypractices

Goodmanufacturingpractices

Hydrogen/deuteriumexchangemassspectrometry

Humanserumalbumin

Humangrowthhormone

Hydrochloricacid

Highconcentrationliquidformulation

Hydrodynamicchromatography

Hydrogen/deuteriumexchange

HF5 Hollow fiber flow field fl owfractionation

HGH

hI

HIC

HILIC

HMQC

Humangrowthhormone

Hydrophobicityindex

Hydrophobicinteractionchromatography

Hydrophilicinteractionchromatography

Heteronuclearmultiplequantumcoherencespectroscopy

HMW Highmolecularweight

HOS

HPLC

HRR-DSC

HSQC

Higher-orderstructure

Highperformanceliquidchromatography

Highrampratedifferentialscanningcalorimetry

Heteronuclearsinglequantumcoherencespectroscopy

HT Hightension

HX

Hydrogenexchange

ICH Internationalconferenceonharmonizationoftechnicalrequirementsforregistrationofpharmaceuticalsforhumanuse

icIEF

Imagingcapillaryisoelectricfocusing

IDP Intrinsicallydisorderedprotein

IDR Intrinsicallydisorderedregion

IEC

Ion-exchangechromatography

IEF Capillaryisoelectricfocusing

IF Intrinsic fluorescence

IFNIFNb orIFNb1a

IgG1

Interferon-b-1a

Immunoglobulingamma1orimmunoglobulinG1

ILP Integerlinearprogramming

IM Ionmobility

ITC Isothermaltitrationcalorimetry

IUP Intrinsicallyunstructuredprotein

IUR Intrinsicallyunstructuredregion

IV Intravenousinjection

kD DiffusionInteractionParameter

LC/MS

Liquidchromatography/massspectrometry

LMW Lowmolecularweight

LNPs lipidnanoparticles

LO Lightobscuration

LOQ Limitofquantitation

LS Lightscattering

mAb Monoclonalantibody

MALDI Matrix-assistedlaserdesorption/ionization

MALLSorMALS Multianglelaserlightscattering

man Mannose

MD Moleculardynamics

MEM Maximumentropymethod

MEMS Micro-Electro-MechanicalSystems

MFI Micro- flowimaging

MHz Megahertz

MREor[M.R.E]

Meanresidueellipticity

mRNA Messengerribonucleicacid

MS Massspectrometry

MS/MS Massspectrometry/massspectrometryortandemmassspectrometry

MW Molecularweight

NEM N-ethylmalemide

NIBS Noninvasivebackscatteringtechnique

nIEF Nativeisolelectricfocusing

NIST NationalInstituteofScienceandTechnology

NMR Nuclearmagneticresonanceornuclearmagneticresonancespectroscopy

NNLS Nonnegativeleastsquares

NOE NuclearOverhauserEffect

NOESY

NuclearOverhauserEffectspectroscopy

NTA Nanoparticletrackinganalysis

OCD Orientedcirculardichroism

OD Opticaldensity

OQ Operationqualification

OS-GAGs Oversulfatedglycosaminoglyclans

PBS Phosphatebufferedsaline

PCA Principalcomponentanalysis

PDA Photodiode-array

PDB Proteindatabank

PDI Polydispersityindex

PEG Polyethyleneglycol

PEM Photoelasticmodulator

PFG Pulsed fieldgradient

PFGE Pulsed fieldgradientecho

Phe Phenylalanine

pI Isoelectricpoint

PK Pharmacokinetic

PL Pathlength

PLIMSTEX

Protein ligandinteractionsbymassspectrometry,titration,andH/Dexchange

PMT Photomultipliertube

PPC ProcedurePerformanceCriterion

PPI

Protein-proteininteractions

PPQ

ProcedurePerformanceQualification

PQ Performancequalification

Pr

PTMorPTMs

Probability

Posttranslationalmodification(s)

QA QualityAnalysis

QbD Qualitybydesign

QToF Quadrupoletimeof flight

QTPP Qualitytargetproductpro file

RDCs

Residualdipolarcouplings

RF Radio-frequency

rhGM-CSF

Recombinanthumangranulocytecolonystimulatingfactor

rhuEPO Recombinanthumanerythropoietin

RI Refractiveindex

rmAb Recombinantmonoclonalantibody

RMM Resonantmassmeasurement

RP-HPLC,RPLCorrpLC Reversed-phasehighperformanceliquidchromatographyorreversed-phaseliquid chromatography

RPS Resistivepulsesensing

RS Referencestandard

RT Roomtemperatureorretentiontime

Sor s Standarddeviation

S/N Signaltonoise

SANS Smallangleneutronscattering

SAP SpatialAggregationPropensity

SAXS SmallangleX-rayscattering

SCorSubQ Subcutaneousinjection

SDS-PAGE

SE-AUC

Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis

Sedimentationequilibriumanalyticalultracentrifugation

SEC Size-exclusionchromatography

SE-HPLCorHP-SEC Size-exclusionhighperformanceliquidchromatographyorhigh-performancesizeexclusionchromatography

SEM Scanningelectronmicroscopy

SFC Supercritical fluidchromatography

SIC Self-interactionchromatography

SIMCA Softindependentmodelingofclassanalogy

SIMSTEX Self-associationinteractionsbymassspectrometry,self-titration,andH/DX

SLS Staticlightscattering

SMP Submicronparticles

SMR Suspendedmicrochannelresonator

SPE Solid-phaseextraction

SRCD Synchrotronradiationcirculardichroism

STEM Scanningtransmissionelectronmicroscopy

SUPREX Stabilityofunpuri fiedproteinsfromratesofH/Dexchange

SV-AUC Sedimentationvelocityanalyticalultracentrifugation

SVD Singularvaluedecomposition

SVP Subvisibleparticles

T1 Longitudinalrelaxationtimeconstant

T2 Transverserelaxationtimeconstant

TCEP-HCl

Tris(2-carboxyethyl)phosphinehydrochloride

TDA

TEM

TIC

TM-DSCorMT-DSC

TMU

TPP

Taylordispersionanalysis

Transmissionelectronmicroscopy

Totalioncurrent

Temperature-modulateddifferentialscanningcalorimetry

Targetmeasurementuncertainty

Targetproductpro file

Try Tyrosine

TSP

Trimethylsilylpropionate

Tyr Tryptophan

UPLCorUHPLC

USP

Ultrahighperformanceliquidchromatographyorultra-performanceliquid chromatography

UnitedStatesPharmacopeia

UV Ultravioletlight

UV VIS Ultraviolet visiblespectroscopy

VH

Immunoglobulingammaheavychainvariabledomain

VIS Visiblelight

VL

VLPorVLPs

Immunoglobulingammalightchainvariabledomain

Virus-likeparticle(s)

WAXS WideangleX-rayscattering

WCX

WSD

XIC

Weak-cationexchangechromatography

Weightedspectraldifference

Extractedionchromatogram

Z Netcharge

Thecomplexityofproteinstructure andthechallengesitposesin developingbiopharmaceuticals

aConsultant,Sudbury,MA,UnitedStates; bBiomolecularDiscovery,RelayTherapeutics, Cambridge,MA,UnitedStates

1.1Thebasicsofproteinhigherorderstructure(HOS)

Proteinsareanimportantclassoflargebiologicalmoleculesthatareclassifiedmoregenerallyasmacromoleculesorpolymers.However,giventheirbiologicalorigin,theseunique moleculesareoftenreferredtoasbiomacromoleculesorbiopolymers.Theyaretrulycomplex,particularlywhencomparedtosynthetic(man-made)polymersandevenothertypes ofbiopolymers,e.g.,DNA.Oneofthemainreasonsforthiscomplexityarisesfromtheirbasic buildingblocks,whichinsyntheticpolymerchemistryarereferredtoasmonomerunits.In thecaseofmostsyntheticpolymers,thechemicalcompositionconsiststypicallyofonlyone typeofmonomer(althoughsomesyntheticpolymerscalledcopolymersorblock-copolymers arecomposedoftwoorpossiblymoredifferentmonomerunits).Proteinsmadeinnaturevia aprocesscalledtranslationutilizingthegeneticcodearecomposedofnotone,two,oreven threedifferentmonomerunits,butratherarecomposedofasmanyas20different “naturally” occurringmonomerunitscalled aminoacids.These20aminoacids(orproteinogenic aminoacids,whichdoesnotincludetheotherknow,butrareproteinogenicaminoacidsselenocystineorpyrrolysine)arereferredtoasthestandardaminoacids.Althoughnotallproteinscontainall20aminoacids,mostdo.Thepresenceofsuchalargediversityinchemical composition,invirtuallyeveryprotein,isakeyelementfortheirstructuralcomplexity,which inturngivesrisetotheirdiversefunctionality.Indeed,thischemicalcomplexity,coupled withthelargenumberofaminoacidunitsor residues (N)presentinproteins(thatcannumber inthethousands),andtheuniquenessoftheaminoacidslinearsequentialarrangement (whichinproteinchemistryiscalledthe primary (1 )structure,see Fig.1.1A),enablesa

FIG.1.1 (A)Thelinearsequentialorderingofaminoacids(representedbytherectangularblackdashedboxes)in aproteinisreferredtoasitsprimarystructure.Theextremeleftaminoacidcorrespondstotheamino-terminus,while theextremerightaminoacidcorrespondstothecarboxyl-terminusendoftheproteinchain.Thegrayshadedarea correspondstothepeptidebondsthatlinkalltheaminoacidunitsinaprotein,yieldingthepolypeptidebackbone(or chain)indicatedbythered(grayinprintversion)dottedrectangle.(B)Anillustrationoftheplanarstructureoftwo adjacentamideplanes(eachresultingfromthedoublebondcharacter,duetoresonance,ofthepeptidebondshown asblackdashes),correspondingtothelightblue(lightgrayinprintversion)shadedareasin(A),wherethebottom amideplaneisformedfromthepeptidebondbetweenthecarboxylgroupofaminoacid1(containingR1)andthe aminogroupofaminoacid2(containingR2)andthetopamideplaneisformedfromthepeptidebondformed betweenthecarboxylgroupofaminoacid2andtheaminogroupofaminoacid3(containingR3).Duetostericissues, angularrotationaroundCaN(expressedby F,phi)andCCa (expressedby J,psi)bondsarelimited.(C)Arepresentationofacommonsecondarystructure,the a-helix.Thesmallrectangleoutlinedinblackdashescorrespondsto asmallsectionofthehelicalarrangementsoftheamideplanes,shownin(B).

staggeringnumberofdifferentpossibleproteins,20N,tobemade.Giventheenormousarray ofdifferentproteinsthatcanbemade,thecellhasexploitedthisdiversityinproteinstructure tocreateproteinstoperformnearlyeveryfunctionalandstructuralroleneededforits existence.

Inproteins,theaminoacidunitsarelinkedtogetherthroughauniquechemicalbond calledthe peptidebond,whichisalsoreferredtoastheamidelink,see Fig.1.1A.Thecollection ofthesepeptidebondsinagivenproteinformacommonelementfoundinallproteinscalled the polypeptidebackbone or chain,see Fig.1.1A.Auniquefeatureofthepeptidebondisthe planarstructurethatitformsbetweenthecarbonyloxygen,carbonandthe a-carbons(Ca oralphacarbon)ofoneaminoacidandtheamidenitrogen,hydrogenand a-carbonsofan adjacentaminoacid.Theresultingplanarfeatureoftheselinkedatomsarisesasaresultof thepartialdoublebondcharacterthatexistsbetweenthecarbonylcarbon(C)andtheamide nitrogen(N)atomsduetothepresencesofresonancestructures,see Fig.1.1B.Thisplanar structureanditsattributesplayanimportantroleinaprotein’sstructure,asitspresences confinesthepolypeptidebackbonetoonlycertaincon figurations,viastericeffects,whichrestrictstheangularrangeofbondrotationaroundtheCa N (expressedby F,phi)andC-Ca (expressedby J,psi)bonds.Theserestrictionshavebeensummarizedina2-dimensional graphicalplotcalledtheRamachandranplot,developedbyRamachandranandothersin 1963 [1].Suchaplotgraphicallyshowshowcertainstructuralfeaturesofproteinscanonly existwithinalimitedrangeofanglescharacterizedby J and F,e.g., a-helix,see Fig.1.1C. Theserestrictionsplayanimportantroleinthedevelopmentofprotein’sspatialstructure or higherorderstructure (HOS).

1.1.1ThelevelsofproteinHOS

Indevelopingproteinbiopharmaceuticalsandinstudyingproteinsingeneral,themost importantconceptis “structure”.Intheprevioussection,webrieflydiscussedthemostbasic componentofaprotein’sstructure,itslinearsequenceofaminoacids,orprimarystructure. However,thefocusofthisbookisconcernedwithaprotein’ s three-dimensional (3D)orspatial structure ,alsoreferredtoasits conformation orHOS.Ultimately,whenconsideringthestructuresofproteins,itistheHOSinconcertwithitsprimarystructure(whichalsoincludesall theprimarychemicalbondmodificationsthatoccurtoitsaminoacidunits,see Section1.1.4) thatenablesaproteintoproperlyfunctionor,aswewillalsodiscussinlattersections, malfunction.

IntermsofproteinHOS,therearethreedifferentlevelsthathavebeendefined.Thesethree levelsinclude: secondary (2 ), tertiary (3 ),and quaternary (4 )structure,see Fig.1.2.The first twostructurallevelsareconcernedwithasinglepolypeptidechain,whilethelatterisassociatedwithproteinstructuresthatinvolvetheinteractionoftwoormorepolypeptidechains. Aprotein’s2 structurereferstothelocalfoldingpatternsofaprotein’spolypeptidechain,in whichthe a-helix(see Fig.1.2A),the b-sheet,turns,andrandomcoilsarethemostprominent resultingstructuralelementsthatareformed.Theselocalfoldedelementscanfurtherparticipateinhigherlevelsoffoldingthatinvolveanarrayofsecondarystructuralelementsthat giverisetothe final3Dstructureofaproteinreferredtoas3 structureofaprotein;see Fig.1.2B.Thesummationof2 ,3 and(ifpresent)4 structure,alongwithitsentire1 structure,iswhatgivesaproteinitsuniquestructure,chemicalandphysicalpropertiesand thereforeitsuniquefunction.Indeed,itisthisrelationshipbetweenstructureandfunction thatisthegenesisoftheprotein “structure-function” concept,whichstatesthataprotein’ s structuredeterminesitsfunction.

FIG.1.2 Illustrationofthethreelevelsofaprotein’sHOS.(A)Representativesecondarystructuralelement,as illustratedbyaribbonrepresentativestructureofan a-helix.(B)Acartoonrepresentationofthefoldingofallthe secondarystructuralelementsinapolypeptidechain,whichgivesrisetothepolypeptide’stertiarystructure.(C)A cartoonrepresentationofthequaternarystructureofaprotein,whichariseswhenthe finalproteinstructureinvolves theassociationofmorethanonepolypeptidechaintoformthe finalfoldedproteinstructure(alsosee Fig.1.3).

Althoughthefoldingandinteractionsofthesecondarystructuralelementscangiveriseto anenormousarrayofdifferentproteintertiarystructures,eachwithuniquepropertiesand functions,it’snotuncommonto findthatthe3 structureofaproteinoftenconsistsofone ormorecommonlyfoldedpatternscalled motifs, super-secondarystructures,or complexfolds [2 4].Thesecommonlyfoldedstructurescontainseveralfoldedsecondaryelements involvingonlyaportionoftheentirepolypeptidechainofaprotein,whichcanblursome ofthedistinctionbetweenaprotein’s2 and3 structure.Hence,onemightlookatmotifs, super-secondarystructures,orcomplexfoldsas “local3 structure”,whilereferringtothe 3 structureoftheentireproteinmoleculeasits “global3 structure”

Anotherstructuralelementthatfurthersubclassifiesthestructurallevelofaprotein betweenwhatwecallaprotein’s2 and3 structureistheconceptof domain [5,6].Domains aretypicallyamuchlargercollectionoffoldedstructuralelementsthanmotifs,supersecondarystructures,orcomplexfolds.Intermsoftheglobalstructureofaprotein,domainscorrespondtooneormoreindependentcompactregionofaprotein’spolypeptidechain,as

FIG.1.3 DifferentrepresentationsoftheHOSofamonomericIgG1antibody.Thetwoheavychainsarecolorcodedinblue(lightgrayinprintversion)andgray,whilethetwolightchainsarebothcolor-codedinred(dark grayinprintversion).(A)AribbonmodelofanIgG1antibody(PDB:1HZH).Theblackcirclecorrespondstothe variabledomainononeoftheIgG1lightchain(VL).(B)AsimplifiedcartoonofthemonomericIgG1antibody indicatingthevarioussectionsofindividualdomainspresent.Theblacklineslinkingthevariousinterchaindomains correspondtoareaswherecovalentlinkagesexist(disulfidebonds)betweendifferentpolypeptidechainsintheIgG1 molecule.TheblackcirclecorrespondstothesameVL domainintheIgG1moleculeasshownin(A).(C)AspacefillingstructuralmodelofthemonomericIgG1antibody.Theblackcircledregionagaincorrespondstothesame VL domainintheIgG1antibodyasshownin(A).(D)AlineardepictionofamonomericIgG1structureshowingall thevariouscovalentlinkages(disulfidebonds)presentintheIgG1antibody.Thosedisulfidebondspresentwithin thesamepolypeptidechainarereferredtoas intrachain disulfidebonds,whilethosedisulfidebondsthatlinktwo differentpolypeptidechainsarereferredtoas interchain disulfidebonds.

indicatedbytheblackcirclesshownin Fig.1.3A C.Proteinscontainingtwoormoredomainsarefrequentlyreferredtoasmultidomainproteins.Intheseproteins,thedomains arechemicallylinkedbyshortsectionsofthepolypeptidechainthataretypicallyhighly flexible,calleda “linker”,butneverthelessexistasstableandindependentfoldedunits.Incertain cases,commondomainstructurescanalsobefoundinproteinsmuchlikethatobservedwith motifs,super-secondarystructures,orcomplexfolds.

Whatisinterestingaboutthesefoldedelementsisthatthereisacertainamountofchange inthe1 structurethatcanbetoleratedwhilestillarrivingat,effectively,thesamefolded structure.Thisobservationexplainsthecommonpresenceofsimilarsecondary,supersecondary,andevendomainstructuresseenindifferentproteinswithdifferentsequences. Hence,theformationofthesebasicfoldingelementscandisplaysomelevelofdiscrepancy intermsoftherequiredorallowableaminoacidsequencevariationsandstillgiveriseto

thesamefunctioningprotein.Thisfeatureplaysanimportantroleinbiologicalevolution,in generatingHOSbuildingblocks,andincontrollingandregulatinggroupsofproteinsthat performverysimilarfunctionsindifferentbiochemicalpathways [7 9].Nevertheless,itis importanttomentionthatinproteins,thereexistmanysequenceregionswhereevenaslight change,i.e.,oneaminoacidchangeoraminorchemicalmodification(e.g.,oxidation,deamidation),cansignificantlyalteraprotein’sstructureandthereforeitsfunction [10,11].

Formanyproteins,however,theuniquefoldedstateofitspolypeptidechainisnotthelast stepinattaininga finaloverall3Dstructure.Manyproteinsarecomposedofmorethanone polypeptidechains,whichmaybeidenticalornonidentical,givingtheseproteinsanadded levelofstructuralcomplexity,4 structure;see Fig.1.2C.

Whenreferringtoaprotein’s4 structure,alackofclarityorconfusioncanunfortunately arise.Anexampleisillustratedin Fig.1.3.Inthis figure,amonomericintactIgG1antibodyis shown.However,thisproteincouldbereferredtoasaproteindimer(madeoftwoidentical proteinunits)oraproteintetramermadeoffourseparatepolypeptidechains,whichinthis casearechemicallycrosslinkedviacovalentbondscalleddisulfidebonds(whichisthemost commonprimarybondusedinnaturetocross-linkpartsofpolypeptides).Suchachoiceof descriptivewordsunfortunatelycanleadtosomeconfusion.Asaresult,somecareshouldbe takenwhendescribingthebasicstructureofaprotein.Inthecaseofthe4 structureofIgG1 molecule,asshownin Fig.1.3,theuseofatetramerinthecontextofits4 structurewouldbe correct.However,inthecontextofacompletefunctioningunit(initslowestcompleteform) themoleculeisamonomer.

1.1.2StabilizingtheHOSofproteins

Inallthreelevelsofaprotein’sHOS(i.e.,2 ,3 ,and4 ),variouschangesintheconformationofthepolypeptidechain(s)occurasaproteinfoldstoreachits finalnativestructure. Thesechangesaretypicallyaccompaniedbyanincreaseinoverallstructuralorder,which impartsasignificantreductionintheprotein’sentropythatbyitselfishighlyunfavorable, intermsoftheoverallfree-energychange.However,asaproteinfolds,variousweaknoncovalent(secondary)bondsformviaionic,dipoles(hydrogenbonds),nonpolar(hydrophobic effect),andvanderWaalsinteractions.Theseweakbondsinvolvetheinteractionsof aminoacidsidechains,aswellaselementsofthepolypeptidebackbone,particularlythe amidehydrogen.Whileindividuallytheseinteractionsareweak,duringthefoldingprocess theirlargenumberandthecooperativewaytheyformprovidethenecessaryenthalpicand entropicdrivingforces(releaseofstructuredwaterviathehydrophobiceffect)tooverridethe largeunfavorabledecreaseinentropythatoccursasaproteinfoldsintoitsnative(moreordered)conformation.Thestabilizationofthefoldedprotein,however,isonlymarginal. Comparingthelevelofstabilizationagainsttheaveragethermalenergycontentofaprotein molecule(whichisequalto kT,where k ¼ Boltzmannconstantand T ¼ temperature)and thedistributionofthisenergy,intermsoftheamountofthermalenergypermolecule,avariety oftheseweaksecondarybondscanbebrokenasafunctionoftime.Suchspatialandtemporal rupturingoftheseweaksecondarybondsenablesaproteintodisplaydynamicstructuralpropertiesinitsconformation(sometimesreferredtoas proteinbreathing).Thisdynamicproperty canplayanimportantroleinaprotein’sfunction [12 15] andstability [16,17].Thisdynamic property,however,canalsoconstituteaweaknessforproteinbiopharmaceuticals,giventhe

widerangeofstressfulenvironmentsanaveragebiopharmaceuticalmustendureduringits biosynthesis,purification,formulation,packaging/storage,patienthandling,anditsadministration.Hence,insearchingforagoodtherapeuticbiopharmaceutical,scientistslookformoleculeswithhighstability,suchthatthedynamicpropertiesoftheproteindonotresultinloss ofactivityoradversestructuralchanges.Proteinsthathavesuchattributesaresaidtohave gooddevelopabilityproperties.

Inadditiontoweaksecondarybonds,stabilizationoftheHOSofaproteincanbeachieved throughprimarybondsformedbetweenfoldedelementswithinaprotein.Asalready mentioned,themostcommonsuchbondisthedisulfidebond,see Fig.1.3D.Althoughthe numberofdisulfidebondsfoundinagivenproteintypicallyamountstoonlyafewsuch bondsperproteinmolecule(andmaynotevenexistwithinsomeproteins),theyoftenplay importantrolesinaprotein’soverallstructure-functionandstability [18].Disul fidebonds canoccurbothwithinasinglepolypeptidechain(wheretheyarereferredtoas intrachain disulfidebonds;see Fig.1.3C)andbetweentwodifferentpolypeptidechainsinthesameprotein(wheretheyarereferredtoas interchaindisulfidebonds;see Fig.1.3C).Disulfidebonds alsooccurbetweentwodifferentproteinmoleculeswheretheyfunctiontostabilizelarge complexmultiproteinsupramolecularstructures [19].Unfortunately,however,theformation ofdisulfidebondscangoastrayleadingtoalteredHOSstructuresoraggregatesviadisulfide scramblingorexchangebetweenotherdisulfidebondsorfreecysteineresiduesinthesame proteinordifferentproteins.Thesemodesofproteindegradation [20 27] areanotherreason whythebiopharmaceuticalscientistneedtoconstantlyscrutinizethestructureofthebiopharmaceuticalduringitsdevelopment.

1.1.3DynamicspropertiesofaProtein’sHOS

TheHOSofvirtuallyallproteinsisprimarilyheldtogetherbyalargearrayofrelatively weakbonds.Inthecontextofaprotein’sthermalenergycontent,thesebondscanbreak enablingvariouslevelsof fluctuationswithinaprotein’sHOSthatcanspananenormous timerange,from10 15 stotensofsecondsandevenlonger [12,28].Again,the fluctuations inaprotein’sconformationessentiallyoccurbecauseoftheopeningorbreakingofvarious weaksecondarybonds.Theextentofthese fluctuationsintermsofamplitudeandlocation isverydependentonmanyfactors,e.g.,environmentalconditions,thestrengthofeachsecondarybond,thedistributionofthesebondswithintheprotein,aswellasthedistributionof thermalenergywithintheprotein.Variationsinthese(andother)factorswilldeterminethe locationofwhichsecondarybondswillbreakinaprotein’sHOSandtherefore,thenatureof theconformationalchange(s)andthepopulationofproteinmoleculesinaspeci ficconformationasafunctionoftime.Whilethesechangesareforthemostpartcontainedtoaregion wherethesecondarybond(s)break,changesmightalsoextendtootherareasoftheprotein, viaallostericeffects.Duetotherandomnatureofthethermalenergy fluctuationswithina protein,arangeofdifferentconformationsandpopulationsofdifferentconformationalstates willexistatanyonetime.Forthemostpart,theextentofchangeinaprotein’sHOSaretypicallynotthatlargeandareoftenreversibleallowingthealteredproteinstructuretoreturnto itsmorestableconformations.

Consequently,insolutionproteinsexistasan ensemble ofdifferentconformations,rather thanasasingle fixeduniqueconformation.Thisensembleislimitedandcontrolledbythe

1.Thecomplexityofproteinstructureandthechallengesitposesindevelopingbiopharmaceuticals

interplayoftheoverallstructureoftheproteinanditsphysicochemicalenvironment.However,underappropriateconditions,involvingsomeformofstressorsubtlechangesinaprotein’schemicalstructure,changesinconformationmaycauseaproteintodisplaydifferent physicochemicalproperties.Inthecaseofaproteinbiopharmaceutical,changesinitsphysicochemicalpropertiescouldalterthedrug’sabilitytobindwithitstherapeutictargetor enableittobindtodifferentmaterialsitencounters,e.g.,variouscontainerclosuresurfaces [29 33].Otherpossibleadverseeventsincludetheformationofaggregatesthatarenonfunctionaland/orevenmoreconcerning,immunogenic [34 36].Itshouldbenotedthatthe formationofaggregatesandtheirassociatedlinktolossofproteinfunctionand/orimmunogenicitycorrespondstooneofthemostcommonformsofproteindegradationthatisclosely monitoredinthebiopharmaceuticalindustry.

1.1.4Finerstructuralalterationofproteins

Onceaproteinissynthesized,orasitisbeingsynthesized,additionalprimarystructural changescanoccurinvivo.Inmostcases,thesechangesareduetoadditionalenzymaticprocessingreactionsinvolvingamultitudeofpotentialchemicalmodificationstovariousamino acids,aswellaschangesinvolvingcleavageorcross-linkingreactions.Thesereactionsmayor maynotplayanimportantroleinthenormalfunction/activityofaprotein,butrathermay representalterationsthatplayouttothedetermentofthecelloreventheorganismduetoan immunogenicresponse.Generally,mostmodificationsareconfinedtotheprotein’ssurface. However,modificationscanalsooccurtotheprotein’sinteriorduetothedynamicproperties ofitsstructure(whichexposestheseburiedinternalareas)orduringitssynthesiswhenthese normallyburiedinternalareashadnothadachancetoproperlyfold.Suchalterationscan leadtochangesinthelocalorglobalHOSoftheprotein.Ingeneral,thesemodifications arereferredtoas posttranslationalmodifications (PTMs).Principally,PTMsoccurinvivoand thenumberofdifferentPTMsthataproteincanexperienceisquitelarge [37].Ineukaryotes, oneofthemorecommon(andbiopharmaceuticallyrelevant)PTMsisglycosylation.This modificationinvolvestheenzymaticadditionofcarbohydrate(alsocalledglycanorsugar) unitstoaproteinatspeci ficasparagine(wheretheyarecalledN-linkedglycan)orserine orthreonine(wheretheyarecalledO-linkedglycan)aminoacid [38].WhilemostPTMsoccur invivo(insidethecell),PTMscanalsooccurinvitro(outsidethecell).TheselatterPTMs, however,typicallyrepresentformsofproteindegradationthatoccurduetodirectphysical orchemicalinteractions(e.g.,oxidation,deamidation,glycation,etc.)andarealsoofgreat concerninthebiopharmaceuticalindustryastheyareoftenlinkedtoinstabilityleadingto loseofdrugactivityandadverseeffects [3,39 44].

1.2Thesearchforhowproteinsattaintheircorrect HOS:theproteinfoldingproblem

Inthe1950sand1960s,biophysicalresearchledscientiststotherealizationthataprotein’ s HOSiseffectivelydictatedbyitsprimarysequence.ChristianAnfinsenwasthekeyscientist whoformalizedthisidea,andin1972wasawardedtheNobelPrizeinchemistryforhiscontributions [45].Inthescientificliterature,thisideahasbeenfrequentlyreferredtoasthe

I.Proteinsandbiophysicalcharacterizationinthebiopharmaceuticalindustry

“An finsendogma” orthe “thermodynamichypothesis”.Thefoldingpathaproteintakesto achieveitscorrectfunctionalHOSisintrinsicallydictatedbyits1 structure(whichmayalso includePTMs).Howthefoldingprocessadvancessoef ficiently,incombinationwiththeway aproteinissynthesizedinvivo,inthespeci ficphysicochemicalenvironmentwithinthecell, hasfascinatedscientistsformanyyears [46].Thisfascinationstemsfromtherealizationthat proteinsachievetheircorrectHOSwithinamatterofmillisecondstoseconds!

Inthe1960s,CyrusLevinthalprosedthefollowinginterestingandsimpleproblemconcerningproteinfolding.Foraproteinconsistingof100aminoacidsinaninitiallyunfolded state,howlongwouldittakethisproteinto find,throughacompletelyrandomprocess, itsappropriatenativeHOSgivenitsphysicochemicalenvironment [28]?Thisproblemis nicelyrestatedinthewordsofAmitKesselandNirBen-Talintheirbook “Introductionto Proteins:Structure,FunctionandMotion” [47] asfollows:

Assumingthattheproteinfoldingprocessinvolvesthefreesamplingofallpossibleconformationsofthe protein(i.e.,ofeachresidueindependently),andthateachresiduehasatleastthreestates,thenthefoldingofa 100-residueproteinisexceptedtosample3100 ¼ 5 1047 conformations.Nowifweassumethatittakesa protein1picosecondtosampleasingleconformation,thenthetimeittakestosampleallpossibleconformationsinorderto findtherightoneshouldbe3100 10 12 s ¼ 5 1035 s ¼ 1.6 1028 years.Thisperiodof timeisabout1018 timeslongerthantheageoftheuniverse!!

ThissimpleproblemproposedbyLevinthaliscalled “Levinthal’sParadox” andwasasignificantdrivingforceforthegeneratingwhatiscalled “theproteinfoldingproblem”.Clearly, thenatureofproteinfoldingisnowhereassimpleasstartingwiththecompletelysynthesized andunstructured(denaturedorrandomcoil)formofaprotein,whichisthenallowedtoundergoacompletelyrandomsamplingprocessofconformationalspace.Proteinfoldingmust proceedviaaprocessthatisenormouslymoreefficient,buthow!!?Answerstothisproblem appeartoliewithintheideaofa “funnel-shapedfoldingenergylandscape ” [48 52],see Fig.1.4,whichmightpossiblytakeadvantageofthewayproteinsaremadeinvivoalong withaconceptof “divideandconquer”.Inthisprocessaproteinproceedstofoldthrough ahierarchyofsubassemblyunitscalleda “foldon” [53,54].Theseunitscanfoldsomewhat independentofeachotherinparalleltoformrelativelylocalhigherorderstructuresthat caneventuallycollapseintothe finalnativeHOSoftheprotein.

Ingeneral,thefunnelingprocessofproteinfoldingislikelynotassimpleasthatportrayed in Fig.1.4A.Rather,itisexpectedtobemorecomplexandtreacherous,asindicatedin Fig.1.4B.Inthelatterscenario,afoldingproteincouldencounterconformationalstates thatarenotasoptimallyfoldedasitsnativestateandcontainhighactivationenergybarriers thatinhibititssearchto findthemoststableconformation.Hence,theproteininthesestates would finditselftrapped,duetothehighenergyofactivationneededtotransitionthemisfoldedstatebackintoamoreunfoldedstatesoitcan finditsmorestableandnativeform. Althoughthesemisfoldedproteinformsmaybeencounteredatverylowlevelsundernormal conditions,thesituationcouldescalateunderstressedconditions,suchasforcingacellto producealargequantityofoneproteininaveryshortperiod.Forsuchasituation,ahigher frequencyofmisfoldedormetastablefoldedproteinstatescouldbeencounteredleavingthe biopharmaceuticalscientistwithamoredifficultpurificationprocessthatresultsinalower proteindrugyield. 1.2ThesearchforhowproteinsattaintheircorrectHOS:theproteinfoldingproblem

FIG.1.4 Agraphicalviewofthethree-dimensionalfunnel-shapedenergylandscapeforproteinfolding.Thetopof eachfunnelcorrespondstothecompletelyunfoldedprotein.Thebottomofeachfunnelplotcorrespondstothefully foldedproteinmoleculeinitsnativestate,whichundercloserscrutinyactuallyconsistsofalargearrayofslight differentenergeticallyfoldedstates(conformations)thatdifferinmostcasesbyasmallamountoffreeenergythus enablingthenativeproteintoexistinsolutionasanensembleofdifferentconformations.(A)Afoldingprocessfreeof situationswhereitcanbetrappedinincompleteorpartialfoldedstate.(B)Afoldingprocessthatenablespartially foldedproteinstobepotentiallytrappedduetothepresenceofsmallershapedfoldingfunnelswithrelativelylarge energyofactivationthatmustbeovercomeinordertoescapeand findits finalnativestate.

1.2.1Invivoproductionofproteins:revisitingtheproteinfoldingproblem

Anotheruniqueattributeofproteinsisthecomplexmannerwithwhichtheyaremade invivo.Proteinsynthesisinvolvesacomplexarrayofcellularmachinery,themaincomponentofwhichistheribosome.Invivo,proteinsaresynthesizedfromtheN-terminusto theC-terminusinasequentialmanneratarateof50 300aminoacids/min [55,56].Thespecificorderingandchemicalcouplingoftheaminoacidsforagivenproteinisachievedbya processcalled translation,whichcontrolstheproteinsynthesisprocessdictatedbythegenetic codinginformationstoredinaspecificmessengerRNA(m-RNA).Asthenascentprotein chainissynthesizedandexposedtothecellmatrix,itcanbegintofold.However,itshould benotedthatthe first50 60aminoacidsinthegrowingpolypeptideareinitiallylimitedto someextentintheirabilitytofreelyfold,duetothephysicalrestrictions(sterichindrance)of theenvironmentwithintheribosome [57].Thisideaofconcurrent,invivo,proteinsynthesis andfoldingarereferredtoas cotranslationalproteinfolding [58] andlikelyplaysanimportant roleinthefoldingofnewlysynthesizedpolypeptide.

1.2ThesearchforhowproteinsattaintheircorrectHOS:theproteinfoldingproblem

Theimportanceofcotranslationalproteinfoldingmostlikelyarisesbecauseonlythe growingpolypeptidechainthathasadvancedbeyondtheribosometunnelwillbeableto fullyparticipateinthefoldingprocess.Thisallowsonlyaportionofthegrowingprotein chaintofoldwithouttheinterferencefromotherpartsoftheproteinthathaseithernot beensynthesizedorislocatedintheribosometunnel.Asaresult,thisshouldimprovethe efficiencyofthesequentialfoldingofthelocalhigherorderstructuralelementscharacterized asfoldonunitstoproceedinamoreorderlymanner.SuchfoldonunitsmostlikelycorrespondtolocalHOSelementsthatarepresentinthe finalnativeprotein.Nevertheless,as theselocalhigherorderstructuralelementsareformed,theymustsearchoutandundergo higherlevelsoffoldingastheproteinchaincontinuestogrow.Asaresult,thesevarioushierarchyoffoldedstructuralelementsareprobablynotarrangedorpackedoptimally(asthey areintheprotein’ s finalnativestate)untiltheentireproteinisfullysynthesizedandrelease fromtheribosome.Oncethishappens,whatremainingloosearrangementoffolded(or partiallyfolded)structuralelementsthatstillexistmustcollapseintothe finalnativestructure oftheprotein.This finalconsolationoffoldedorpartiallyfoldedstructuralelementsmost likelyproceedsthroughtheinteractionsofkeyaminoacidsidechainstomakethe finalfunctionalprotein(notwithstandinganyadditionalchangesinHOSresultingfromPTMsandthe formationofquaternarystructures).

Consequently,cotranslationalproteinfoldingconstrainsandtosomeextentguidesthe overallproteinfoldingprocess.Bylimitingthenumberoffoldingpathways(speci fically badfoldingpathways,whichwouldsignificantlyincreasetheamountoftimerequiredto findthecorrectnativeconformation)availabletoaprotein,relativetothesituationwhere foldingonlybeginsoncetheproteinisfullysynthetizedandreleasefromtheribosome,could maketheroleofcotranslationalproteinfoldingtrulyanimportantattributeinthesuccessful foldingofaprotein.

1.2.2Invivoproductionofproteins:avoidingandeliminatingfoldingerrors viatheuseofchaperones

Invivo,therearemechanismsinvolvingotherproteins,called chaperones,thathelpproteinsthatarefoldingavoidthesituationofbeingmisfolded.Thistaskisachievedviathe chaperone’sabilitytoassistafoldingproteintoavoidfoldingtrapsbyparticipatinginthe proteinfoldingprocessthroughprotein proteininteractions [59 63].Inadditiontochaperones,therealsoexistsinvivocellularmachinerywhosefunctionistoidentifythepresencesof misfoldedproteinsandeliminatethemviaproteolytichardwareexistingwithinthecell [64]. However,thesesystemsarenotperfect,andfailuretoremoveorpreventtheseerroneously foldedproteinsfromaccumulatingwithinthecellcanalterthecell,causingadverseeffects thatcouldeventuallyleadtoitsdeath.Inthecaseofproducingaproteinbiopharmaceutical, onceamisfoldedproteinisreleasedintothecellculturemedia,itthenbecomestheproblem fortheprocessscientisttodevelopappropriatepuri ficationstrategiestoremovethemisfoldedproteinfromthe finalproteindrugproduct.Iftheseerroneouslyfoldedproteinsare notremoved,theycouldleadtoadverseeffectswhenthe finaldrugproductisadministered toapatient.Hencebiophysicalanalysisofthebiopharmaceutical’sHOSagainbecomesan importantactivityindevelopingproteinbiopharmaceuticalswithminimallevelsofthese misfoldedformsinthe finaldrugproduct.

1.3Surprisesintheworldofproteinfolding:intrinsicallydisorderedor unstructuredproteins(anapparentchallengetotheprotein Structure Functionparadigm)

Withinthepasttwodecades,ithasbeenrealizedthatmanyproteins,especiallyineukaryotesormulticellularorganism,existwithinthecellwithnodefinedHOS [28].Rather,these proteinsappeartobedisorderedorunstructured,approachingwhatmightbecalleda randomcoilstructure,anomaloustowhatisfrequentlyseenwithsyntheticpolymersor denaturedproteins.However,whentheseproteinsinteractwiththeirtargetmolecule(s) theycommonlyappeartotakeonaleveloforganizedHOS.Hence,thisstructuraldisorder istransientinmanycasesandadisorder-to-ordertransitionoccursduringtheirfunctioning (i.e.,interactingwiththeirbindingtarget).Suchbehaviorcouldplayanimportantrolein allowingtheseproteinstobindtoanarrayofdifferentpartnermoleculesbytakingadvantage oftheplasticityoftheirpolypeptidechain’ s flexibility [28].Thisprocessisliabletobemodulatedbyotherfactorswithinthecell,whichcontrolandregulatethebindingpartnersthey interactwith.Indeed,thelevelofdisorderedproteinsishigherineukaryotesormulticellular organism,incomparisontoprokaryotes,wherehighlevelsofsignalingandregulationis required.Thisuniqueclassofproteinshasbeenreferredtoasintrinsicallydisorderedproteins(IDPs)orintrinsicallyunstructuredproteins(IUPs) [65,66].Theexistenceofthese IDPswouldappeartopresentachallengetotheparadigmofstructure functiondiscussed earlierinthischapter.

WiththerealizationoftheexistenceofIDPs,manyofthelargerandomcoil-likeregionsof proteinsconsistingof20 30ormoreaminoacidsinlengtharenowbeingreferredtoas intrinsicallydisorderedorunstructuredregions(IDRsorIURs) [66].Thesestructuralelementsarecommonlyseenaslinkersbetweenorderedproteinregionssuchasdomainswhere theyarethoughttoalsoplayimportantrolesinprovidingprotein flexibility,allowingproper foldingortofacilitatedomain domaininteractionsordomainbindingtofunctioningbindingtargets.Atpresent,IDPshavenotmadetheirwayintothebiopharmaceuticalindustry, althoughitisprobablyonlyamatteroftimeuntilsuchaproteindrugwillappear.

1.4Proteinsandthebiopharmaceuticalindustry:problemsand challenges

Althoughproteinscanbechemicallysynthesizedexternaltothecell,theirhighcost(which isafunctionofproteinsize),aswellastheiroverallcomplexityleadstopooreconomicsfor buildingaviablecommercialdrugbusinessviathisapproach.Overtheyears,however,scientistshave figuredouthowtogetcellstoproducesignificantlylargeamountsofaspecific protein,bymanipulatingtheirDNAviarecombinantDNAtechnology.Thedevelopmentof thiscapabilitywasthekeyinenablingthebiopharmaceuticaldrugindustryto flourish.Overallthisprocessofmakingproteinbiopharmaceuticaldiffergreatlyfromtheclassicalprocess usedtomakesimpleorganicdrugmoleculescalledpharmaceuticals,see Fig.1.5.Cellularand molecularbiologistcannowproduceproteinbiopharmaceuticalsatconcentrationsexpressed intheculturemediavolumeasgreatas10g/L [67].Nevertheless,thechallengesofdoingthis

FIG.1.5 Asimplecomparisonillustratingdifferencesintheprocessofmakingapharmaceuticalversusmakinga biopharmaceutical:(A)Coarseoutlineofthesequentialchemicalreactionsformakingapharmaceutical,usingaspirin asanexampleand(B)acoarseoutlineofthebasicstepsformakingabiopharmaceutical,whichconsistsof first synthesizingapieceofDNAcontainingthecorrectnucleotidesequencecodeformakingthedesiredbiopharmaceutical’spolypeptidechain(s),theinsertionofthisDNAintoaninitialsmallcollectionofcells(themicroscale factoriesformakingthebiopharmaceutical)usingrecombinantDNAtechnology,thelarge-scalegrowthofthesecells duringwhichthecell’sinternalproteinsynthesizingnano-machine(theribosome,acomplexcellularorganelle composedofmanyproteinsandseveralpiecesofRNA)aredirectedtosynthesizethetargetbiopharmaceutical, illustratedhereaseitherinterferonbeta-1a(IFNb)oramonoclonalantibody(mAb).Notethatthespace-filling molecularmodelsofaspirin,IFNb andmAbhaveallbeendisplayedroughlyonthesamearbitraryscaletohelp providethereaderwithanapproximateperspectiveonhowtheywouldrelativelycomparetoeachotheronthebasis ofsize.ThedashedcirclehighlightingpartofthestructureofIFNb correspondstothecarbohydrate-containing portionofthisbiopharmaceuticalthatplaysadominantroleingivingrisetoitsmicroheterogeneityshownin chapter2inFigure2.3whencoupledwithotherposttranslationalmodifications(PTMs). Reproducedwithpermission fromBerkowitzSA [116]

successfullyaresigni ficant.Forcingacelltoproduceunusuallylargeamountsofasingle proteinpresentsuniqueproblemstothecell.Particularlyintermsofmakingsurethatall theproteinmoleculesareproperlyfoldedandhaveconsistentphysical,chemical,andbiologicalproperties.Hence,toachievethisgoalrequirestheconstantanddiligentmonitoringand characterizationoftheproteinbiopharmaceutical’sHOS.

Theprocessof finding,developing,andobtainingregulatoryapprovalofaprotein biopharmaceuticalthatismadeusingrecombinantDNAtechnologyproceedsthrougha sequenceofkeyactivitiesorbasicphasesofactivitythatisoutlinedin Fig.1.6.Successof

FIG.1.6 Acoarseoverviewofthebasicareasandsequenceofmajoractivitiesinvolvedincommercializinga proteinbiopharmaceutical.Therelativelengthofeachblockarrowisroughlyassociatedwiththelengthoftime typicalspentateachstage,fromresearchthroughcommercialization.Overall,thecostinthisprocesscaneasilybein excessofoneormorebilliondollarsandcanrequiremorethanandecadetodevelop [68].Thesenumberscanvary significantlyfromcompanytocompanyandfromdrugtodrug.Asaresulttheyareonlyapproximate. 1.Thecomplexityofproteinstructureandthechallengesitposesindevelopingbiopharmaceuticals

thisprocessishighlydependentonbiophysicalcharacterizationworkassociatedwithmonitoringtheconsistencyofthephysicochemicalpropertiesoftheproteindrug,confirmingthe absenceofchangesinthedrug’sHOS(whichmightgiverisetosmallunwantedsubpopulationsofalteredmolecules),andinassessingthepotentialimpactthatPTMsmighthaveona drug’sstructure.

Duringthe firstpartofthischapterwehavedealtwiththeverybasicpropertiesofprotein structure.Intheremainingsections,wewilldiscusshowthesepropertiesareresponsiblefor manyofthepotentialproblemsthatareofgreatconcerntoalargerangeofbiopharmaceuticalscientists.Inaddition,wewillbrieflylookatsomeofthemorenoveltypesofprotein biopharmaceuticalsthathaveandarebeingdevelopedthatfurtherchallengethetaskof biophysicallycharacterizingthesecomplexdrugs.

1.4.1ImpactofPTMsontheHOSofproteinbiopharmaceuticals

Thecomplexchemicalcompositionofproteins,consistingof20chemicallydifferentnaturallyoccurringbuildingblocks(i.e.,aminoacids)effectivelyempowerthecellwiththe neededcomponents(chemistryset)tomakethenecessaryarrayofproteinsitneedstoproperlyfunction.However,theseaminoacidsalsoofferarangeofchemicallydifferenttargets thatcanundergochemicalchanges,viadirectchemicalreactionsorthroughtheparticipation ofvariousenzymaticreactions.Thechemicalchangesthataproteinbiopharmaceuticalcan incurofferopportunitiestoaltertheHOSofthesemolecules,impactingtheconsistencyof manufacturingorworse,causeadverseeventswhenadministratedtoapatient.As mentionedin section1.1.4,thesechemicalchanges,whethertheyoccurinvivoorinvitro, arecollectivelyreferredtoasPTMs.ManyPTMsplayrolesinthebiologicalfunctionofaproteininvivo,whileothersarearesultofnormaldegradationoraging.Hence,theideathata givenproteinexistsasasingledefineduniquechemicalentityismisleading.Infact,nearlyall proteinbiopharmaceuticalsexistasacollectionofhighlysimilarvariantforms.Therangeof thesevariantsandtheiramountsinthe finalbiopharmaceuticaldrugproductisdetermined bythenatureofthecell-lineused,thecellcultureconditions(e.g.,rawmaterialsandholdtimeswithinthebioreactor),theresolutionpropertiesofthepurificationprocess,aswellas ourabilitytodetectandcharacterizethem.Thecollectionofhighlysimilarproteins, variant forms or proteoforms [69] ofeffectivelythesameproteinthatcharacterizeabiopharmaceutical

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