An Evaluation of the Potential of NMR Spectroscopy and Computational Modelling Methods to

24 Pages·2017·2.31 MB·English

pharmaceutics
Review
An Evaluation of the Potential of NMR Spectroscopy
and Computational Modelling Methods to Inform
Biopharmaceutical Formulations
AkashPandya1,MarkJ.Howard2,MireZloh3,4andPaulA.Dalby1,*
1 DepartmentofBiochemicalEngineering,UniversityCollegeLondon,GordonStreet,LondonWC1E7JE,UK;
[email protected]
2 SchoolofChemistry,UniversityofLeeds,LeedsLS29JT,UK;[email protected]
3 FacultyofPharmacy,UniversityBusinessAcademy,Trgmladenaca5,21000NoviSad,Serbia;
[email protected]
4 NanoPuzzleMedicinesDesign,Business&TechnologyCentre,BessemerDrive,StevenageSG12DX,UK
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:11July2018;Accepted:17September2018;Published:21September2018
Abstract: Protein-based therapeutics are considered to be one of the most important classes of
pharmaceuticalsonthemarket. Thegrowingneedtoprolongstabilityofhighproteinconcentrations
inliquidformhasproventobechallenging. Therefore,significanteffortisbeingmadetodesign
formulations which can enable the storage of these highly concentrated protein therapies for
up to 2 years. Currently, the excipient selection approach involves empirical high-throughput
screening, but does not reveal details on aggregation mechanisms or the molecular-level effects
of the formulations under storage conditions. Computational modelling approaches have the
potentialtoelucidatesuchmechanisms,andrapidlyscreeninsilicopriortoexperimentaltesting.
NuclearMagneticResonance(NMR)spectroscopycanalsoprovidecomplementaryinsightsinto
excipient–proteininteractions. Thisreviewwillhighlighttheunderpinningprinciplesofmolecular
modelling and NMR spectroscopy. It will also discuss the advancements in the applications of
computationalandNMRapproachesininvestigatingexcipient–proteininteractions.
Keywords: formulation;excipients;aggregation;NMR;moleculardynamics;moleculardocking
1. Introduction
Protein-basedtherapeuticshavebecometheleadingclassofpharmaceuticalsonthemarketbysales.
Themostprominentaremonoclonalantibodies(mAbs),whichbelongtotheimmunoglobulinfamily[1].
Currently,mostofthemonoclonalantibodytherapiesapprovedareformulatedatproteinconcentrations
of>30mg/mL[2]andareadministeredparenterallyasliquidinjections(intravenousorsub-cutaneous
routes)[3].Duetoinstabilityinthegastrointestinaltract,capsularandtabletoraldosageformshavenotyet
beenfoundtobeapracticalwaytodeliversuchprotein-baseddrugs[4,5].Storageofbiopharmaceuticals
in the preferred liquid aqueous form must achieve a long shelf-life of up to 2 years at 4 ◦C to be
approved[6].However,thecontinuousthreatofaggregateformation,precipitation,chemicaldegradation
andothermodificationshasmadedesigningtheidealformulationverychallenging[7].
Aggregationisconsideredtobeoneofthemostpressingchallenges,andcanoccuratallstages
ofproteinpharmaceuticaldevelopment,manufacture,formulationandstorage. Thepropensityof
protein-basedproductstoaggregateduringvariousstagesofmanufacturinghasalsobeenreviewed
thoroughly[8,9]. Onestrategytoaddressthisproblemistounderstandthemechanismsthatdictate
aggregationpathwaysunderrelevantsolutionconditions. Thecurrentlyknownrangeofaggregation
pathwaysandmechanismshavebeenreviewedextensively[10,11],andarenotthefocusofthisreview.
Pharmaceutics2018,10,165;doi:10.3390/pharmaceutics10040165 www.mdpi.com/journal/pharmaceutics
Pharmaceutics2018,10,165 2of24
Understandingdetailedmechanismsistime-consumingandsotheindustryistypicallyunderpressure
totakeanempiricalapproachtoformulation,typicallythroughcombinatorialscreening,toobtain
productsthatarestabletoaggregation,alongwithotherdegradationpathways. Suchapproaches
oftenuseconvenientbiophysicalparameterdeterminationsasindicatorsofaggregationpropensity,
suchasthethermaltransitionmidpoint(T )forglobalconformationalunfolding,orthetemperature
m
(T )atwhichaggregationisfirstdetected. ThecapabilitiesandlimitationsoftheserapidT and
agg m
T measurementshavebeenexploredextensively,andhavebeenfoundtocorrelatewellwithrates
agg
ofaggregationatelevatedtemperatures[12,13]. However,thesecorrelationsdisappearforaggregation
kineticsunderlow-temperaturestorageconditions,becausetheaggregationmechanismsarenolonger
primarilydrivenbyglobalproteinunfolding[14–17].
Protein-basedpharmaceuticalsoftenrequiretheadditionofformulationexcipientstoensuretheir
stability. Themajorgroupsofexcipientsinclude: aminoacids;bufferingagents;sugars;osmolytesand
surfactants. Thereissignificantoverlapbetweencertainexcipientgroups,andmanyexcipientscarry
outmorethanonefunctioninformulations,includingcontrolofaggregation,vialsurfaceadsorption,
viscosity,shear-thinning,chemicalinstabilities,andcontainerinstabilities. Althoughpharmaceutical
excipientsarereadilyavailable,themechanismsofactionforeachvary. Anumberoftheoriessuchas
preferentialinteraction/hydration,volumeexclusion/crowding,cation-πinteractions,andelectrostatic
interactions,dispersiveandaromaticinteractionshavebeenhypothesised[18–20].
High-throughput(HTP)methodshavebeenusedinthepharmaceuticalindustryfordecades,
primarilyforidentifyingleadcompoundsindrugdiscovery,andseveralindepthreviewshavefocused
ontheirapplications[21–23]. Outlinedbelow(Table1)aresomeoftheprominenthigh-throughput
biophysical methods used to assist the excipient selection process for protein-based formulations.
AnextensivestudyontheuseofHTPmethodstodesignaformulationforahighlyconcentratedIgG
mAbisreportedhere[24].
Table1.Highthroughputbiophysicalmethodsusedforexcipientscreening.
BiophysicalMethod Details Limitations ApplicationReferences
Measuresshiftsinenergy
(wavelength)ofphotons
re-emittedafterinteractionwith Lowsensitivity.Outofthe
molecularvibrationalmodes. millionsofincomingphotons
Ramanspectroscopy Providesanempiricalsignature interactingwithmolecules, [18,25]
ofproteinstructure,thatcanbe thereisonlyonescattered
usedtomonitorchangesin Ramanphoton.
intramoleculardynamicsand
intermolecularinteractions.
Measuresthedifferencein
adsorptionofcircularly
polarisedlight.Far-UVCDcan
Areferenceproteinwith
determinetheabsoluteand
knownsecondarystructureis
relativecontributionsof
requiredtofitthe
Circulardichroism secondarystructuretypesin [18,26]
experimentaldata.Thequality
proteins.NearUVCDcanprobe
ofthefitalsodependsonthe
tertiarystructurecontent.
wavelengthsused.
Canprobechangesinprotein
structureinresponse
toformulation.
Measurestheheatemittedor
ITCcanbeusedtodetermine
absorbedduringthetitrationof
theexcipientmechanism
aproteinwithaligand.
Isothermaltitration directlyandindirectly.
Theamountofheatindicatesthe [18,27]
calorimetry(ITC) However,nostructural
proportionofexcipientthat
informationoftheprotein
bindstheproteinandits
isgiven.
associatedenthalpy.
Pharmaceutics2018,10,165 3of24
Table1.Cont.
BiophysicalMethod Details Limitations ApplicationReferences
Usefulforidentifying
Routinelyusedin
Pharmaceutics 2018, 10, x FOR PEER REVIEW excipientsthatpreferentially 3 of 25
high-throughputscreeningof
interactwithproteins,orthat
excipientsforformulations.
stUabseilfiusle ftohrr ioduengthifycrinogw ding
Determinestheimpactof
Differentialscanning efefexccitps.ieCntasn tnhoatt pbreeufesreedntitaolly
excipientsonthethermal [16,28–30]
calorimetry(DSC) Routinely used in high-throughput deintetecrtaoctt hweirthm percohteainniss, mors thoaft
stabilityoftheprotein,
screening of excipients for acsttiaobnil.isUe ntharboluegtho ccrhoawradcintegr ise
measuredasthemelting
Differential scanning formulations. Determines the impact of cheaffnegctess. Cspaencniofitc bteo utsheed to
temperatureandenthalpy [16,28–30]
calorimetry (DSC) excipients on the thermal stability of sedceotnecdta ortyheorr mteercthiaarnyissmtrsu ocft ure
ofunfolding.
the protein, measured as the melting ofapctrioonte. iUnns.able to characterise
temperature and enthalpy of unfolding. Thcheaenxgceist astpioecnifsico tuor ctheeo fthe
UsesaPCRthermocyclertoscan PCseRcoenqduairpym ore ntetrtciaanryp sotrtuecnttuiarel
thefluorescenceofextrinsic limofi ptrtohteeitnysp. eextrinsic
Differentialscanning dye-bindingtoproteinsas fluTohree esxcceintacteiodny seosuurcsee odf. the
[16,31–33]
fluorimetry(DSF) afunctionoftemperaturein UPnCabRl eeqtuoipchmaernatc cteanri speotential
Uses a PCR thermocycler to scan the
microtitreplates,anddetermine exlicmipiti etnhet mtypeceh eaxntriisnmsisc of
fluorescence of extrinsic dye-binding to
Differential scanning theirmeltingtemperatures. acftluioonreasncednccea ndyoensl uysdedet. e ct
proteins as a function of temperature in [16,31–33]
fluorimetry (DSF) teUrtniaarbyles ttor ucchtaurarectcehriasen ges.
microtitre plates, and determine their
excipient mechanisms of
melting temperatures.
action and can only detect
Given that high-throughput screening approachteerstiaaryr estreumctuprei rcihcaangl,esa. nd hence time-consuming,
andoftenonlyprovideindirectsurrogateevaluationsofwhetheraproteinislikelytobestableatlow
Given that high-throughput screening approaches are empirical, and hence time-consuming, and
temperatureover2years,thereisaneedtocreateamorefundamentalunderstandingofaggregation
often only provide indirect surrogate evaluations of whether a protein is likely to be stable at low
mechanismsandtheimpactofdifferentmodesofformulationunderthesestorageconditions. Thereis
temperature over 2 years, there is a need to create a more fundamental understanding of aggregation
alsothepotentialtousecomputationalmodellingapproachestobothelucidatesuchmechanisms,
mechanisms and the impact of different modes of formulation under these storage conditions. There is
andalsotorefinethemtocreatebettermodellingmethods.
also the potential to use computational modelling approaches to both elucidate such mechanisms, and
alTsoh etoc roemfinbein thateimon too fcrseuacteh baepttperro macohdeesllwinigt hmmetohroedds.e tailedbiophysicalanalyses,suchasNMRthus
hassignTifihcea cnotmpboitneantitoianl otof saudcvha anpcperothaechuens dweirthst amnodrien dgeatanidledp rbeiodpichtyasbiiclaitl yanoaflfyosrems, usulacthio ans NfoMrbRi othlougs ics.
A shcahse msigantiifcicsahnot wponteinntiaFli gtou ardev1anhcieg thhleig uhntdsetrhsteanddiifnfegr eanndt cparpedaibcitlaibtiielistya onfd folrimmuitlaattiioonn sfoor fbimoloolgeiccsu. lar
modAe lslcinhegmteactihcn siqhouwesna innd FNigMurRe .1N MhigRhslipgehctstr othsec odpiyffeprreonvt idcaepsasbtirluitcietsu raanlda nlidmfiutanticotniosn oafl imnsoilgehcutslairn to
molmecoudleellsinugn tdeechrniniqtueerrso agnadt iNonMtRh.r NouMgRh sopbescetrrovsactoiopny spirnocvliuddesi nsgtruthcteurcahle amndic afulnschtiifotnaanl dinssipgehctstr ianltoli ne
molecules under interrogation through observations including the chemical shift and spectral line
shape analysis that inform both on molecular structure in addition to intermolecular interactions.
shape analysis that inform both on molecular structure in addition to intermolecular interactions. Such
Suchinformationhasthepotentialtobeutilizedtovalidateandrefinemolecularmodellingapproaches
information has the potential to be utilized to validate and refine molecular modelling approaches
designedtopredictprotein-proteinandprotein-excipientinteractions,aswellasthepropensityof
designed to predict protein-protein and protein-excipient interactions, as well as the propensity of
proteinstoaggregate.
proteins to aggregate.

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simsuimlautiloantio,na,n danNd MNRMRsp sepcetrcotrsocsocpopyyt otoe leuluccididaattee pprrootteeiinn--eexxcciippiieennt tininteterarcatciotinosn tsot oinifnofromr mtheth reatriaotnioaln al
desdigensigonf pofr optreoitne-ibn-absaesdedfo fromrmuulalatitoionnss..
Pharmaceutics2018,10,165 4of24
We start below with an informative summary of the underpinning principles of computational
modellingandNMRspectroscopy.Basedontheseprinciples,thisreviewreportsonthecomplementary
insightsthatcanbeobtainedfromNuclearMagneticResonance(NMR)spectroscopyandcomputational
modellingmethods,toinformtherationaldesignofprotein-basedformulations.Wehaveaparticular
focusontheaggregationmechanismsobservedunderlow-temperaturestorageconditions.Theseinclude
thosemechanismsdrivenbyhot-spotinteractionsbetweenproteins,andbylocalfluctuationswithinthe
nativeproteinstructureensemblethatcanrevealaggregation-proneregions(APRs)[17,34–38]. Thus,
itwouldalsobeusefultoelucidateprotein-excipientinteractionswiththepotentialtomodifytheability
ofproteinstoself-associateviahydrophobichotspotsorAPRsonthesurface,orbylocalfluctuationsthat
revealburiedAPRs.
2. OverviewofMolecularModelling,MethodologiesandLimitations
2.1. MolecularDocking
Moleculardockingisacomputationalchemistrytoolthatisextensivelyusedindrugdiscovery
processes[39],withmorerecentapplicationsfoundinthedrugdevelopmentprocess. Thismethod
enables evaluation of intermolecular interactions between two molecules by predicting possible
bindingmodes. Thetargetmoleculeisabiopolymer,usuallyaprotein,whilethebindingtoatarget
can be predicted for either a small molecule (ligand-protein docking) as shown below in Figure 2
oranotherprotein(protein-proteindocking). Thedockingtypicallyrequirestheknownstructureof
thetargetmoleculeandseeksforanintermolecularcomplexwiththemostfavourablebindingpose
basedonthefitbetweentargetanditsboundmolecule, aswellasthestabilizationresultingfrom
intermolecular interactions. Molecular docking generally involves generating a number of viable
protein-ligand conformations by using search algorithms. Initially, a conformation of a complex
(bindingpose)ispredictedintheformoftheorientationoftwomoleculesrelativetoeachother. Thisis
followedbyestimationofthebindingenergiesforeachpose[40]. Asabsolutebindingaffinitiesare
difficult to predict, scoring functions have been developed to establish a ranking of the predicted
dockedposes. Thisrankingofthegeneratedconformationsisachievedbyusingthreemaincategories
ofscoringfunction. Force-fieldscoringfunctionsusemolecularmechanicsandareestablishedfrom
calculationsofatomicinteractionsincludingbondstretchingorbending,torsionalforces,vanderWaals
andelectrostaticinteractions[41]. Empiricalscoringfunctionspredictbindingenergiesfordocked
conformationsbyconsideringvanderWaals,hydrogenbonding,electrostaticsanddesolvationterms,
whose relative weightings have been optimized through continual validation and refinement [42].
Lastly,knowledge-basedscoringfunctionsoriginatefromexperimentalstructuralinformation[43].
Theentropiceffectscontribution,animportantcomponentofprotein-ligandbindingenergy,isusually
takenintoaccountthroughre-scoringoremployingothermethods[44]. Acombineduseofscoring
andre-scoringallowsselectionofthemostprobablebindingmodeforasingleligand,orrankingof
aseriesofligandsaccordingtotheirpredictedaffinityforaselectedtarget.
These scoring functions are usually implemented in software packages depending on their
intended use, along with the appropriate conformational sampling methods that can also include
differentlevelsofmolecularflexibilitywhengeneratingbindingposes. Rigidbodydockingallows
fornoconformationalchangesinthemoleculesofinterest,andisthusthefastest. Itissuitablefor
gaininginsightsintorelativeorientationsoftwodockedproteins,orforscanningaproteinsurface
with rigid small molecule ligands to identify binding hotspots. Notable docking programs that
facilitaterigidprotein-proteindockingareHex[45],andGRAMM-X[46]. Thesesoftwarepackages
sample protein-protein docked conformations based on a Fast Fourier Transform (FFT) algorithm.
RosettaDock [47] is also used to predict the lowest energy docked protein-protein complexes by
employingMonteCarloapproachestopositionmoleculesinrespecttoeachother,howeverittakes
intoaccountsomeflexibilitybyallowingtheside-chainstomove[47]. Protein-proteindockingcould
providepossibleinsightsintoaggregationbyidentifyingputativeprotein-proteininteractioninterfaces.
Pharmaceutics2018,10,165 5of24
Pharmaceutics 2018, 10, x FOR PEER REVIEW 5 of 25
Incontrast,flexibledockingresultsinaconformationalshiftinthemoleculeofinterest.Thismakes
In contrast, flexible docking results in a conformational shift in the molecule of interest. This makes
itfavorablewhenstudyingligand-proteincomplexes,andhasthepotentialtopredictandimprove
it favorable when studying ligand-protein complexes, and has the potential to predict and improve our
ourunderstandingofspecificinteractionsattheprotein-ligandinterface. Thewealthofligand-protein
understanding of specific interactions at the protein-ligand interface. The wealth of ligand-protein
docking software packages available have increased over the decades. Some prominent flexible
docking software packages available have increased over the decades. Some prominent flexible
dockingprogramsincludeAutoDock4[48],GOLD[49,50],andGEMDOCK[51]. Themajorlimitation
docking programs include AutoDock 4 [48], GOLD [49,50], and GEMDOCK [51]. The major limitation
for molecular docking is the incorporation of protein flexibility. It is a well-known fact that upon
for molecular docking is the incorporation of protein flexibility. It is a well-known fact that upon ligand
ligandbinding,aproteinundergoesconformationalchanges. Theflexibledockingsoftwarepackages
binding, a protein undergoes conformational changes. The flexible docking software packages
mentioned above overlook ligand-induced binding effects and thus treat the protein as rigid [52].
mentioned above overlook ligand-induced binding effects and thus treat the protein as rigid [52].
However,therearesoftwarepackagesdedicatedtoflexibleligand-flexibleproteindockingthatenable
However, there are software packages dedicated to flexible ligand- flexible protein docking that enable
side-chainflexibility,includingAutoDockVina[53]andFlexX[54]. Theprotein-liganddockingmay
side-chain flexibility, including AutoDock Vina [53] and FlexX [54]. The protein-ligand docking may
provideinformationnotonlyonmodeofbindingwithinaspecifiedactivesite,butitcanprovide
provide information not only on mode of binding within a specified active site, but it can provide
informationonpossibleinteractionsitesonthewholeproteinsurface[55],whichmayallowevaluation
information on possible interaction sites on the whole protein surface [55], which may allow evaluation
ofexcipientbindingontheproteinsurface(Figure2).
of excipient binding on the protein surface (Figure 2).
TThhee aaccccuurraaccyy ooff ddoocckkiningg pprreeddicictitoionnss ththaat tddoo nnoot taaccccoouunnt tfofor rpproroteteinin flfleexxibibiliiltiyty aanndd pprerseesnencec eof
of other components the solutions (water, ions, buffers) have always been debated. A common
other components the solutions (water, ions, buffers) have always been debated. A common solution is
tsoo lusetito nupis tmosoeltecuuplamr oldeycnualamridcys na(MmDics) (MsimDu)lsaimtiounlast iothnastt hcaatnc apnoptoentetniatilalyll yssaammppllee vvaarriioouuss prportoetienin
ccoonnffoorrmmaattiioonnss uuppoonn lliiggaanndd bbiinnddiinngg iinn eexxpplliicciitt pprreesseennccee ooffw waatteerrm mooleleccuulelessa annddb buufffefresr,sw, whhereerea am mosotst
ffaavvoouurraabbllee ddoocckkiinngg ppoossee ccaann bbee uusseedd aass aa ssttaarrttiinngg ccoonnffoorrmmaattiioonn ffoorr tthhee ssiimmuullaattiioonn eexxppeerriimmeennttss..

FFiigguurree 22.. AA sscchheemmaattiicc ooff tthhee mmoolleeccuullaarr ddoocckkiinngg pprroocceessss oonn aas siinngglleem muullttiimmeerriiccp prrootteeinin..
2.2. MolecularDynamics(MD)
2.2. Molecular Dynamics (MD)
Molecular dynamics (MD) is a powerful computational modelling tool, which enables the
Molecular dynamics (MD) is a powerful computational modelling tool, which enables the
followingofsubtleatomicmotionsofasystemofinterestasafunctionoftime[56]. All-atom(classical)
following of subtle atomic motions of a system of interest as a function of time [56]. All-atom (classical)
MDsimulationssampleconfigurationsbyintegratingNewton’slawofmotiontoalltheatomsinthe
MD simulations sample configurations by integrating Newton’s law of motion to all the atoms in the
systemsimultaneouslyoverafemtosecondtimestep. Atrajectoryisrecordedwithpreciseatomic
system simultaneously over a femtosecond time step. A trajectory is recorded with precise atomic
positions and velocities giving the user an indication of how the system evolves with time [57].
positions and velocities giving the user an indication of how the system evolves with time [57]. These
Thesepositions,definedbyCartesiancoordinatesofallatomsinasystem,allowcalculationofthe
positions, defined by Cartesian coordinates of all atoms in a system, allow calculation of the potential
potential energy of the system and forces that act on each atom. Molecular dynamics commonly
energy of the system and forces that act on each atom. Molecular dynamics commonly employs
employs molecular mechanics approximations and use of the force fields, sets of equations and
molecular mechanics approximations and use of the force fields, sets of equations and associated
Pharmaceutics 2018, 10, x FOR PEER REVIEW 6 of 25
constants to reproduce geometries of molecular systems [58]. These are calculated based on the some
commonly used force fields including CHARMM22 [59], CHARMM27 [60], AMBER [61] and GROMOS
[62]. There is a wide array of software packages available, including but not limited to Gromacs [63],
Amber [64], NAMD [65] and CHARMM [66], that have proven invaluable for the advancement of
molecular dynamics. A general summary of the molecular dynamics process is provided below in
Figure 3. A list of typically employed MD simulation times for various protein dynamics events are
listed below in Table 2. The main limitation of MD is the small time-step, relative to a typical
requirement for much longer simulation times. Longer simulations have been carried out on the
Pharmaceutics2018,10,165 6of24
millisecond time scale [67,68]. This may potentially place a strain on computational resources when
simulating larger systems, however the benefits outweigh limitations due to the wealth of information
tahsasto ccainat bede ocbotnasitnaendts atbooruetp trhoed suycsetegmeo omne tthreie astoofmmisotilce cleuvlaerl [s6y9s]t.e ms[58]. Thesearecalculatedbased
onthesomecommonlyusedforcefieldsincludingCHARMM22[59],CHARMM27[60],AMBER[61]
Table 2. The typical molecular dynamics (MD) simulations timescales that can observe various protein
andGROMOS[62]. Thereisawidearrayofsoftwarepackagesavailable,includingbutnotlimited
dynamics events.
toGromacs[63],Amber[64],NAMD[65]andCHARMM[66],thathaveproveninvaluableforthe
advancement oPfrmotoelienc uDlayrnadmynicasm Eivcse.ntA generalMsuDm Smimaruylaotfiothne Tmimoele Rcualnagred ynamics process is
providedbelowinVFibigrautrieon3a.l Amloitsitoonfs typicaFlleymetmospeclooynedds M(10D−15s)i mtou plaictoiosnectoimndess (f1o0r−1v2)a riousprotein
dynamicseventsarReolitsatteidonbaell omwoitnioTnasb le2.ThPeimcoasiencolinmdista (t1io0n−12o) ftMo nDainsotsheecsomndalsl (t1im0−e9)- step,relativeto
atypicalrequiremenLtofoorpm duycnhamlonicgse rsimulatPioicnotsiemcoens.dLs o(n10g−e1r2)s tiom mulialltiisoencsonhadvs e(1b0e−e3)n carriedouton
themillisecondLtiigmaensdc ablien[d6i7n,g6/8u].nTbhinisdminagy potentiaNllaynpolsaecceoandstsr a(1in0−o9)n tcoo smecpountadtsi onalresourceswhen
simulatinglargePrrosytesitnem fosl,dhionwg/euvnefrotlhdeinbge nefitsouMtwiecirgohselcimonitdast i(o1n0s−6d) utoe stoectohnedws ealthofinformation
thatcanbeobtainedaAbogugtrethgeatsiyosnt emontheatomisticlSeevceoln[d69s ]a.nd beyond

FFiigguurree 33.. AA sscchheemmaattiicc ooff tthhee mmoolleeccuullaarr ddyynnaammiiccss pprroocceessss..
Table2.Thetypicalmoleculardynamics(MD)simulationstimescalesthatcanobservevariousprotein
3. Overview of Nuclear Magnetic Resonance (NMR) Spectroscopy
dynamicsevents.
Nuclear Magnetic Resonance (NMR) spectroscopy detects nuclei of isotopes with spin angular
ProteinDynamicsEvent MDSimulationTimeRange
momentum (e.g., 1H, 15N, 13C and 19F) in a magnetic field with the goal to provide analytical information
regarding the strucVtuibrraalt ioannadl/moro tpiohnyssical natFuerme toosfe caonnyd sm(1o0l−ec15u)lteo upnicdoeserc oinnvdess(t1i0g−a1t2io)n. The resultant
Rotationalmotions Picoseconds(10−12)tonanoseconds(10−9)
NMR spectra contain highly defined resonances (often referred to as peaks) that reflect the chemical
Loopdynamics Picoseconds(10−12)tomilliseconds(10−3)
nature of specific atoms in the molecule of interest. NMR has been applied in a diverse range of
Ligandbinding/unbinding Nanoseconds(10−9)toseconds
pharmaceutical aPproptleiicnaftoioldnisn gth/autn ifnolcdluindge, characterMisaictrioosne coofn pdrso(t1e0i−n6s), tdorsuegco dnidsscovery and design [70].
Despite the power andA ugtgirleitgya toiof nNMR spectroscopy, linSeewcoinddthssa nodf breeysoonndance peaks become larger as
molecular weight increases. This due to a reduction in nuclear T2 relaxation times as the global tumbling
o3f. tOhev emrvoileewcuolef Nbeuccolmeaers Mslaogwneert.i cHRoewseovnearn, caell( Nis MnoRt )lSospte acstr oadscvoapnyces in NMR have allowed protein
observations to be made over a wide-range of molecular timescales e.g., domain shifts (microseconds
NuclearMagneticResonance(NMR)spectroscopydetectsnucleiofisotopeswithspinangular
momentum (e.g., 1H, 15N, 13C and 19F) in a magnetic field with the goal to provide analytical
information regarding the structural and/or physical nature of any molecule under investigation.
TheresultantNMRspectracontainhighlydefinedresonances(oftenreferredtoaspeaks)thatreflect
thechemicalnatureofspecificatomsinthemoleculeofinterest. NMRhasbeenappliedinadiverse
rangeofpharmaceuticalapplicationsthatinclude,characterisationofproteins,drugdiscoveryand
design[70].DespitethepowerandutilityofNMRspectroscopy,linewidthsofresonancepeaksbecome
largerasmolecularweightincreases. ThisduetoareductioninnuclearT relaxationtimesasthe
2
globaltumblingofthemoleculebecomesslower. However,allisnotlostasadvancesinNMRhave
allowedproteinobservationstobemadeoverawide-rangeofmoleculartimescalese.g.,domainshifts
Pharmaceutics2018,10,165 7of24
(microsecondstomilliseconds),sidechainmotions(picosecondstonanoseconds)andchangesinloop
regions(nanosecondstomicroseconds)[71,72].
For the purpose of this review, we are considering solution-state NMR methods where there
areseveralobservableNMRparametersincludingchemicalshifts(δ)andrelaxationtimes(T and
1
T ). A chemical shift of a nucleus in a molecule arises due to a nuclear shielding or de-shielding
2
effectoftheNMRstaticappliedmagneticfieldandistheresultofelectronssurroundingthenucleus.
Chemical shifts (δ) are measured in parts per million (ppm) [73,74]. The power of chemical shifts
providesstructuralinformationbydistinguishingchemically-equivalentnucleibutwheretheyare
indifferentmolecularenvironments. Forexample, theproton(1H)NMRofacetone(CH COCH )
3 3
willonlydisplayonepeakfromtwoequivalentmethylgroupsbutethanol(CH CH OH)provides
3 2
three1HNMRpeaksrepresentingthoseinthemethyl(CH ),methylene(CH )andhydroxyl(OH)
3 2
moietiesrespectively. Theseethanolpeaksareadditionallysubjecttofine-structuralsplittingfrom
spin-spin J-coupling, a concept taught to many undergraduates and found in NMR and organic
chemistry textbooks. This example is a simplistic view of the power within chemical shifts and it
cannotbestressedenoughthatstructuralandchemicalenvironmentinfluenceNMRpeakobservations.
Forexample,twoalanineresidueswithinaproteinwillbothhavemethylgroups,buttheirobserved
chemicalshiftswillbedependentontheirindividualstructuralenvironmentsandsodisplaydiverse
chemicalshifts. ChemicalshiftsareultimatelyderivedfromtheLarmorfrequencyofresonanceof
each NMR peak, where the distance (in Hertz) between any two peaks is dependent also on the
spectrometermagneticfieldstrength,becausetheLarmorfrequencyisproportionaltomagneticfield
strength. Whendefiningpeakseparationintermsofchemicalshift,a1ppmseparationforaproton
(1H)spectrumona600MHz(14.1Tesla)spectrometerequatestoafrequencyseparationof600Hz
whereasforan800MHz(18.7Tesla)spectrometeritwouldbe800Hz. Thisfielddependenceofpeak
separationcanbeextremelyusefulwhenmonitoringchemicalexchangeprocesses,aswillbedescribed
furtherbelow.
NMR is a spectroscopic process that involves excitation and energy transfer between nuclear
spinstateswhichsubsequentlyneedtorelaxandreturntoequilibrium. ThisNMRrelaxationisof
fundamentalinteresttoNMRspectroscopistsasthetimeconstants(T andT )andrates(R andR )
1 2 1 2
oftheassociated mechanismsprovidea wealth ofinformationaboutthemolecules beingstudied.
Indeed,nuclearrelaxation,andinparticularrelaxationdispersion,hasfounduseasatooltostudy
protein folding and enzyme kinetics where chemical exchange events of interest, between species
ormolecularenvironments,canbequantifiedthroughtheirinfluenceonNMRrelaxationprocesses,
asdescribedin[75–77]. EachNMR-activenucleusinamoleculewillpossessauniquesetofrelaxation
timesfrommanyfundamentalmolecularandbulksolutionpropertieswhichisbeyondthisreview.
However,itisusefultonotethatmolecularsizeandshape,proximityandtheisotopeofthenearest
NMR-active nucleus, in addition to temperature, viscosity and the NMR magnetic field strength,
allinfluencenuclearrelaxationinquantifiableways. Thespin-latticemechanisminvolvesanuclear
spinexchangingenergywithitssurroundings(i.e.,lattice),andthereafterreturningtoitsgroundstate
withtime-constantT andrelaxationrateR . Thesecondspin-spinrelaxationmechanisminvolvesthe
1 1
lossofphasecoherenceandsubsequentlossofbulkmagnetizationthatunderpinstheNMRsignal,
withtime-constantT andrelaxationrateR . Forbothprocesses,R =1/T . Althoughtheprocesses
2 2 x x
givingR andR areindependentevents,theR andR relaxationratesconvergeforsmallmolecules
1 2 1 2
e.g.,acetoneandethanol,whereasforlargerspeciessuchasproteinsinsolutionR andR arefoundto
1 2
besignificantlydifferent. Furthermore,proteinrelaxationisextremelypowerfulandcandifferentiate
theshapeandinternalmotionsofamoleculeandreviewsareavailableonthissubject[78,79].
ThelinewidthoftheindividualNMRsignalsisproportionaltoR andthereforetothecorrelation
2
(timeofthemolecule. ThecrucialpointisthatR hasaproportionalitytothecorrelationtime(τ );
2 c
thetimeforamoleculetorotatethrough1radian(i.e.,molecularmotion)whichissubsequentlyrelated
tomolecularsizeandshape. Assumingasphere,correlationtimecanbeestimatedfrom[4πηr3/3k T]
b
whereη—viscosityofthesolvent,k —Boltzmannconstant,T—temperatureandrtheradiusofthe
b
Pharmaceutics2018,10,165 8of24
sphere. Asamoleculebecomeslarger,rincreasesandsoτ increases,whichthenmakesR increase
c 2
and is finally observed in NMR as an increase in linewidth. This is the fundamental reason why
proteinNMRspectraarebroaderthansmallmoleculespectra. Increasingthetemperatureand/or
loweringtheviscositywoulddroplinewidthbutmanyproteinsarelimitedintheiroperationalsolvent
conditionsandthermallimits. TakingthisconceptofmolecularsizeinfluencingNMRastepfurther,
a ligand that binds to a protein would experience a significantly different correlation time, that is
dictatedbythesizeofthecomplex,comparedtowhenthesamemoleculeisfreesolution. Inmany
cases,ligandbindingisnotpermanent,butdynamicandisdefinedbyanequilibriumwithanaffinity
thatcanfurtherinfluencetheNMRobservation[80].
Whenamoleculeisinequilibriumbetweentwoormorestates,therateofexchangedetermines
whetherthechemicalshiftsofeachofeachstateisvisibleorthechemicalshiftofasingletime-averaged
stateisobserved. Theobservationisthusdictatedbytheequilibriainvolvedinexchangeaswellas
thedifferenceinnuclearspinrelaxationratesR andR . Thesetwolimitsareknownasslowandfast
1 2
exchangeinthechemicalshifttimescalebutthereisanintermediateconditionwhereallresonances
become broad and difficult to detect. At this condition, known as coalescence, the exchange rate
constantiscomparabletothedifference(inHz)betweentheLarmorfrequenciesofthetwostates.
Thistiesinwiththeearlierconceptofchemicalshiftsbetweenpeaksbeingfrequencydependentandas
therelationshipbetweenchemicalshiftandtheequilibriumisnotbasedonpartspermillion(ppm)but
thefundamentalHertzdistancebetweenNMRresonances. Therefore,changingNMRfieldstrengths
canhelpmovetheobservationbetweentheslow,intermediateandfastexchangeregimes.
3.1. Limitations
The potential for NMR spectra to become very challenging to interpret is very real when
increasingthemolecularweightoftheprotein. Thenumberofprotonresonancesscalesproportionally
withmolecularweight, butthespectralwidthremainsconstant, leadingtoincreasedcrowdingof
peaks. In addition, line width increases with the molecular weight, leading to a further decrease
inresolutionandinsignalintensitywiththeconsequencethat,resonanceoverlapincreasesrapidly
with molecular size, until individual lines become too difficult to resolve. A commonly utilized
workaround is to expand the NMR spectra from one to more dimensions (2D, 3D, 4D, etc.) to
createmorespecialrepresentationofresonancepeaks. Alternatively,datacanbeobtainedathigher
magneticfields(e.g.,at14.1,18.8,21.1and23.5Teslathatequatesto600,800,900and1000MHz1H
resonantfrequencies)toimproveresolution. Otherapproachesincludetheadditionofalternative
NMR-active nuclei than 1H, such as 15N and/or 13C in addition to reducing spin-spin (R ) based
2
relaxationbydeuteration; theprocessofreplacing1Hwith2H.Asdiscussed,R isresponsiblefor
2
linebroadeningandreducingthisprocesswillcreatenarrowerlines. However,asdiscussedfurther
below,TROSYNMRcanbeusedtoselectslowerNMRrelaxationpathwaystoandthereforeobserve
narrowerlines,whichthenextendstherangeofmolecularweightforwhichpeakscanberesolved.
Suchamethodisatitsmostpowerfulwhencombinedwithdeuteration.
3.2. Protein-ObserveMethods
Asmentioned,thechemicalshiftissensitivetothemolecularenvironmentaroundthenucleus
andwhenaligandorexcipientinteractswithaprotein,physicalcharacteristicsforbotharealtered.
Thebindingevencreatesachangeinelectrondensityandsoinfluencesthemostprominentobservable
NMR parameter, chemical shift. As a result, chemical shift mapping (CSM) or chemical shift
perturbation (CSP) methods are potential modes of investigation in protein-observe NMR [81].
CSM/CSP methods compare two protein NMR spectra, such as with and without the addition of
aligand,andtrackanychangesinchemicalshiftand/ordisappearanceofpeaks. Thesechangeswill
identifyanyareasoftheproteinthatareinfluencedbythebindingevent,withthelargestchanges
being typically observed in the region around the binding site. In addition, careful experimental
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Pharmaceutics 2018, 10, x FOR PEER REVIEW 9 of 25
designcanutilizeasuiteofNMRspectra,obtainedoverarangeofligandconcentrations,toprovide
isothermsfromwhichdissociationconstantsforthebindingprocesscanbedetermined.
High molecular weight protein species such as whole mAb, Fab fragment and Fc region can be
HighmolecularweightproteinspeciessuchaswholemAb,FabfragmentandFcregioncanbe
expressed with isotopic labelled nuclei, which enable greater resolution of peaks through the collection
expressedwithisotopiclabellednuclei,whichenablegreaterresolutionofpeaksthroughthecollection
of multidimensional heteronuclear correlation spectra. There are several labelling protocols that exist
of multidimensional heteronuclear correlation spectra. There are several labelling protocols that
for NMR studies. Uniform 15N isotopic enrichment is the simplest form of labelling a protein. The
existforNMRstudies. Uniform15Nisotopicenrichmentisthesimplestformoflabellingaprotein.
protein is expressed in E. coli (BL21) [82] grown on minimal growth media and supplemented by
TheproteinisexpressedinE.coli(BL21)[82]grownonminimalgrowthmediaandsupplementedby
15NH4Cl and unlabelled glycerol/glucose that is purified using standard methods to provide a +90%
15NH Clandunlabelledglycerol/glucosethatispurifiedusingstandardmethodstoprovidea+90%
enrich4ed protein product as shown in the schematic below in Figure 4. Heteronuclear single quantum
enrichedproteinproductasshownintheschematicbelowinFigure4. Heteronuclearsinglequantum
coherence (HSQC) NMR spectra are 2D NMR experiments recorded to show all nitrogen-hydrogen
coherence(HSQC)NMRspectraare2DNMRexperimentsrecordedtoshowallnitrogen-hydrogen
correlations, which typically are dominated by backbone amide groups from the protein [83]. In the
correlations,whichtypicallyaredominatedbybackboneamidegroupsfromtheprotein[83]. Inthe
case of studying excipient–protein interactions, a series of HSQC NMR spectra can be recorded of the
caseofstudyingexcipient–proteininteractions,aseriesofHSQCNMRspectracanberecordedof
protein in presence and in the absence of the ligand. Binding effects of these interactions can be
theproteininpresenceandintheabsenceoftheligand. Bindingeffectsoftheseinteractionscanbe
investigated by overlaying the series of 15N HSQC spectrums. If there is an interaction between the
investigatedbyoverlayingtheseriesof15NHSQCspectrums. Ifthereisaninteractionbetweenthe
excipient and protein, the peaks on the spectra will be found to track from their initial position when
excipientandprotein,thepeaksonthespectrawillbefoundtotrackfromtheirinitialpositionwhen
no ligand was present [84].
noligandwaspresent[84].
More complex labelling approaches exist, such as 2H/15N/13C triple labelling which uses bacteria,
Morecomplexlabellingapproachesexist,suchas2H/15N/13Ctriplelabellingwhichusesbacteria,
yeast or insect cells as an expression system supplemented by 15NH4Cl, 13C-glucose and deuterated
yeastorinsectcellsasanexpressionsystemsupplementedby15NH Cl,13C-glucoseanddeuterated
water (D2O). This form of labelling enables detailed mapping of whole4 structural changes in the protein
water (D O). This form of labelling enables detailed mapping of whole structural changes in the
upon exci2pient binding as it can offer significant coverage of the protein backbone and side chains.
proteinuponexcipientbindingasitcanoffersignificantcoverageoftheproteinbackboneandside
Deuteration replaces the aliphatic and aromatic protons for 2H across the protein to reduce the
chains. Deuterationreplacesthealiphaticandaromaticprotonsfor2Hacrosstheproteintoreducethe
significant nuclear relaxation effect of 1H nuclei that facilitate line broadening. The proportion of 1H to
significantnuclearrelaxationeffectof1Hnucleithatfacilitatelinebroadening. Theproportionof1H
2H depends greatly on the carbon source used and whether H2O or D2O is present [85]. However, using
to2HdependsgreatlyonthecarbonsourceusedandwhetherH OorD Oispresent[85]. However,
specifically labelled methyl group probes (13CH3) in the study of2 high m2olecular weight proteins has
usingspecificallylabelledmethylgroupprobes(13CH )inthestudyofhighmolecularweightproteins
proven useful in expanding the molecular weight l3imit reached with perdeuterated proteins [86].
hasprovenusefulinexpandingthemolecularweightlimitreachedwithperdeuteratedproteins[86].
Methyl groups tend to occur in the hydrophobic cores of proteins and provide an excellent probe for
Methylgroupstendtooccurinthehydrophobiccoresofproteinsandprovideanexcellentprobefor
conformational modifications [87,88]. Several protocols for methyl group labelling are available [89–
conformationalmodifications[87,88].Severalprotocolsformethylgrouplabellingareavailable[89–92].
92].

FFiigguurree 44.. AA sscchheemmaattiicc ddeeppiiccttiinngg tthhee bbaacctteerriiaall pprroodduuccttiioonn ooffr reeccoommbbininaanntti siosototoppiciacalllylyl albabelelleldedp rportoetieni,n,
aanndd rreeccoorrddiinngg ooff aann NNMMRR ssppeeccttrruumm..
The increased relaxation already mentioned for proteins increases with increasing magnetic
The increased relaxation already mentioned for proteins increases with increasing magnetic field
fieldstrength,whichcreatesadditionalissues. Consequently,severalprotein-NMRmethodological
strength, which creates additional issues. Consequently, several protein-NMR methodological
advanceshavebeenutilizedtoaddressthischallenge,themostpopularbeingTransverserelaxation-
advances have been utilized to address this challenge, the most popular being Transverse relaxation-
optimised spectroscopy (TROSY) [93,94]. TROSY which uses spectroscopic means to reduce the
optimised spectroscopy (TROSY) [93,94]. TROSY which uses spectroscopic means to reduce the
observedT relaxationbyselectingtheslowerrelaxationpathwayforobservation. Remember,T is
observed T22 relaxation by selecting the slower relaxation pathway for observation. Remember, 2T2 is
related to line width and accessing the slowest relaxing pathway will produce narrower resonance
Pharmaceutics 2018, 10, x FOR PEER REVIEW 10 of 25
Pharmaceutics2018,10,165 10of24
lines. Therefore, TROSY accompanied by various isotopic labelling techniques especially deuteration
allows the study of biomolecules above 25–30 kDa [95]. The effect of TROSY on the relaxation rate of
relatedtolinewidthandaccessingtheslowestrelaxingpathwaywillproducenarrowerresonance
an excipients and proteins is demonstrated in Figure 5.
lines. Therefore,TROSYaccompaniedbyvariousisotopiclabellingtechniquesespeciallydeuteration
TROSY, when combined with higher magnetic fields (e.g., equal to or greater than 18.8 T–800 MHz
allowsthestudyofbiomoleculesabove25–30kDa[95]. TheeffectofTROSYontherelaxationrateof
1H) and the introduction of cryogenic probes has enabled NMR fingerprinting using 1H/2H, 15N 13C
anexcipientsandproteinsisdemonstratedinFigure5.
isotopes at natural abundance of full mAb, Fab, Fc and other therapeutic proteins possible [96–99].

FFigiguurere 55. .(a(a) )TThhee NNMMRR spspeectcrturumm fofor raa ssmmaalll lmmooleleccuulele lilgigaanndd wwilill lddeeppicict taa nnaarrrrooww lilnineewwididthth dduuee ttoo a
loanlgoenrg terrantrsavnesrvseer rseelarxelaatxioanti otinmteim. (eb.) (Ibn) cIonnctoransttr,a as tp,raopteriont ehinash aa sshaosrhteorr tterarntrsavnesrvsee rrseelarxealatixoant itoinmtei maned
thaunsd ath bursoaadb roliandewlinidetwhi dist hshisoswhno.w (nc.) (Tc)RTORSOYS Yprporloonlognsg sthteh etrtarannsvsveersrsee rreellaaxxaattiioonn ttiimmeess aanndd ththeererebbyy
imimpprorovvese sththe epproroteteinin sisgignnaal linin ththee ssppeecctrturumm. .
3.3. LiTgaRnOd-SOY,bwsehrveen Mcoemthbodins edwithhighermagneticfields(e.g.,equaltoorgreaterthan18.8T–800MHz
1H)andtheintroductionofcryogenicprobeshasenabledNMRfingerprintingusing1H/2H,15N13C
Ligand observe methods exploit the dependence of relaxation rates on molecular size described
isotopesatnaturalabundanceoffullmAb,Fab,Fcandothertherapeuticproteinspossible[96–99].
earlier in this review, specifically via the difference in molecular weight between a small ligand and a
si3g.3n.ifLicigaanntdly-O labrsgerevre pMroettehiond.s Nuclear Overhauser Effects (NOEs) are defined as the change in intensity
of NMR resonances caused by dipole-dipole coupling. The sign and the magnitude of the NOE is
Ligandobservemethodsexploitthedependenceofrelaxationratesonmolecularsizedescribed
dictated by the hydrodynamic radius (r6) and the correlation time (τc). This makes NOEs available to
earlierinthisreview,specificallyviathedifferenceinmolecularweightbetweenasmallligandand
detect intramolecular and intermolecular interactions. Large molecules tend to tumble slower and so it
asignificantlylargerprotein. NuclearOverhauserEffects(NOEs)aredefinedasthechangeinintensity
is anticipated that a negative NOE will be observed. In contrast ligands tumble fast resulting in a
ofNMRresonancescausedbydipole-dipolecoupling. ThesignandthemagnitudeoftheNOEis
positive NOE [100]. Two prominent NOE based NMR methods include Saturation Transfer Difference
dictatedbythehydrodynamicradius(r6)andthecorrelationtime(τ ). ThismakesNOEsavailableto
(STD) and WaterLOGSY (Water Ligand-Observed via Gradient spectcroscopy.
detectintramolecularandintermolecularinteractions. Largemoleculestendtotumbleslowerand
STD NMR involves recording a STDon spectrum, whereby only the 1H protons of a protein and not
soitisanticipatedthatanegativeNOEwillbeobserved. Incontrastligandstumblefastresultingin
the excipient (ligand) are selectively saturated by a narrow selective 1H NMR pulse typically placed
apositiveNOE[100].TwoprominentNOEbasedNMRmethodsincludeSaturationTransferDifference
between −1 and −3 ppm. The saturation is transferred throughout the protein via spin diffusion and
(STD)andWaterLOGSY(WaterLigand-ObservedviaGradientspectroscopy.
onto any bound ligand, resulting in a reduction in the intensity of the ligand resonances as shown in
STDNMRinvolvesrecordingaSTD spectrum,wherebyonlythe1Hprotonsofaproteinand
Figure 6a. In order to detect this transfer, otnhe ligand must dissociate from the protein. Therefore, the
nottheexcipient(ligand)areselectivelysaturatedbyanarrowselective1HNMRpulsetypicallyplaced
dissociation constant (KD), which describes the affinity of an excipient to the protein, has to be
between−1and−3ppm. Thesaturationistransferredthroughouttheproteinviaspindiffusionand
ontoanyboundligand,resultinginareductionintheintensityoftheligandresonancesasshown