AlbertJ.R.HeckUtrechtUniversity

email:a.j.r.heck@uu.nlwww.hecklab.nl

BetterAnalyticsEnablesBetterBiologics

MonitoringGlycosylationandGlyco-engineeringbynativeMS

• Non-denaturing,higherm/z• Lesschargestates,equallysensitive

NativeMassSpectrometry

1000 2000 3000 4000 5000 6000 7000 8000 9000m/z

6420 6440 6460m/z

23+23+

24+

46+

45+FWHM 46+ : 0.70Resolution : 4600Mass: 148025.83 ± 1.53

FWHM 23+ : 1.25Resolution : 5100Mass: 148025.22 ± 0.56

50/50water/acetonitrile,1%f.acid

Aqueousammoniumacetate,pH=7

HeckNatureMethods2008

MassAnalyzersforNativeMS

Orbitrap Exactive EMR(JUMBO)modifiedforoptimaltransmissionofhighm/z ions

Roseetal.NatureMethods2012Snijder etal.AnnRevAnalChem 2014

1990

2012

• Orbitrap EMRappearstobeextremelypowerfulfortheanalysisofproteinassemblies

• Theimpactofthishighmassresolvingpoweratveryhighsensitivityistremendous;itopensupavenuestomeasuredynamic protein-proteininteractions,and(non)covalentbindingofsmallmoleculestoproteinassemblies

• Wide-rangingapplicationsmayincludethedirectanalysisofpost-translationalmodifications,e.g.phosphorylation,glycosylationonintactproteinsandproteinassemblies,co-factorbindingtonativeenzymes,nucleotidebindingtoDNArepairenzymes,andlipidbindingtoATPasesystems

• Representahighlycompetitiveoptionforaddressingthenecessitytoanalyseandthoroughlycharacterisebiopharmaceuticalproducts

ExploringanOrbitrapMassAnalyzer forNativeMassSpectrometry

Rosati etal.Angew Chemie 2012Rosati etal.mAbs 2013Dyachenko etal.AnalChem 2015

Snijder etal.JACS2014Snijder etal.NatureChem 2013

Viruses/nano-containers PharmaceuticalsProteincomplexesRoseetal.NatureMethods2012Wangetal.Mol Cell2016

Rosati etal.NatureProt 2014Yangetal.AnalChem 2013Yangetal.NatureComm 2016

Glycoproteins

T: FTMS + p NSI sid=200.00 Full ms [400.00-12000.00]

5400 5600 5800 6000 6200 6400m/z

5729.66 5958.87

6207.23

5517.37

6476.98

T: FTMS + p NSI sid=200.00 Full ms [400.00-12000.00]

5400 5600 5800 6000 6200 6400m/z

5729.66 5958.87

6207.23

5517.37

6476.98

GlycosylatedantibodiesonOrbitrap EMR25+

OnlyfewchargestatesNobackgroundnoiseVerysensitive,attomoles

SinglemutationsonIgG4leadtoverydistinctglycoprofiles

GlycosylationprofilesattheintactproteinleveloffourIgG4mutantsRosati etal.NatureProtocols2014

AidsinglycanassignmentsRevealsquantitativevalidityofthemeasurements

ChoppingupintactmAbs monitoredbyhigh-resolutionnativeMS

Rosati etal.NatureProtocols2014

Whatextrabringsglycoprofiling ofmAbs bynativeMS

Presenceofco-occurringsideproducts,possiblyduetostorage

Trastuzumab

Yangetal.MAbs.2017; 9:638-645

• Fullantibodywith2glycanchains• Fullantibodywithsingleglycanchains• Lightchainloss• Etc.

Bevacizumab

NativeMSofBevacizumab

Adcetris®ADC

DAR:--S-S--

--S-S--

Thedrugiscoupledtocysteineresidues,resultinginamixtureof9variantswithdifferentDARanddruglocalization.

0

2

4

6

8

Whatextrabringsglycoprofiling ofmAbs bynativeMS

High-ResolutionNativeMassSpectrumofhumanErythropoietin

EPObackbone=18,235.99Da,MeasuredMwbynativeMSbetween26,000to33,000Da

Over230peakscouldbebase-lineresolved

Yang,Francetal.NatureComm 7(2016)

Lossof13sialic acids,heterogeneitystemslargelyfromsialic acid

High-ResolutionNativeMassSpectrumofhumanErythropoietinSialidase treated

Glycoproteomics:AbalancebetweenHigh-ThroughputandIn-Depth

YangY, Franc,Vetal.TrendsinBiotechnology (2017)

OverallglycoproteinprofilebynativeMSSite-specificprofileofPTMspersite

IntegratingHigh-ResolutionNativeMassSpectrometryandMiddle-DownProteomicsfortheAnalysisofGlycoproteins

“Thewholeisgreaterthanthesumofitsparts”Aristotle

Hybridmassspectrometryapproachesinglycoproteinanalysisandtheirusageinscoringbiosimilarity.YangY, etal.NatCommun.7(2016)13397.

Identifiedandquantified1)10glycoforms onN24,2)9glycoforms onN38,3)8glycoforms onN83and4)2glycoforms onS126

EachN-glycosylationsiteonrhEPO ismodifieduniquelyintermsofboththenumbersandrelativeabundancesofdifferentiallymodifiedglycoforms

Middle-downanalysisofhumanErythropoietin

Glycopeptides mayeasilylosetheirlabilesialic acidmoietyduringsamplepreparationandionization

Isthewholegreaterthantheparts

CombiningthedatatheoverallPTMcompositionofWTEPOcouldbefullyassigned

PTMheterogeneityinrhEPO productsislargelyoriginatingfromthevariabilityintheextentandoccupancyofsialylation onthevariousglycantreesoccurringinrhEPO

AnErythropoietinBiosimilarity Score

TheComplementSystemanditsActivation

n Complementactivationbyantibodiesboundtopathogensortumorsisacriticalfeatureofnaturalimmunedefenseandimmunotherapies

n Thecomplementsystemhelpstheabilityofantibodiesandphagocyticcellstoclearpathogensfromanorganism.

n Itispartoftheimmunesystemcalledtheinnateimmunesystemn Manyproteinsareinvolved,termedC1toC9,theyfunctioninacascadeof

events

n C1,isamulti-proteincomplexconsistingof22proteins;n C1q,C1randC1s

n C1qbindsasingleIgG Fcsegmentwithverylowaffinity

InitialstepintheactivationoftheComplementSystemC1q

[ABC]

n InastructureofIgG1(PDBentry1HZH),theFcsegmentsarearrangedinahexamericring.Weexaminedwhichaminoacidsmadecontactsandstartedtomanipulatethembysite-directedmutagenesis

IgG1antibodycrystalstructure

platform was composed of a continu ou s disk withsixpoorly resolv ed densities protru ding towardth e membrane and fou r discernible densities ontop, arrang ed as anincomplete h exagon(Fig . 4 , BtoE). We g enerated a model of th e C1 -antibodycomplexbydock ing th e 1 HZH crystal pack ing[adapted byFab rotation(fig . S5 )] intoth e lowerplatform and manu ally fitting C1 q h eadpieces(Fig . 4 F and fig . S6 ) (20). Th e fou r densities ontopof th e lower platform su g g ested incomplete(4 :6 ) C1 q h eadpiece binding to th e antibodyh examer (Fig . 4 C) and mayreflect flexibilityanddynamics of th e C1 q-IgG interactions.

Th e model su g g ested th at one Fab arm of eachantibody in th e h examer bou nd th e membrane-associated antig enwh ile th e oth er Fab arm waspositioned at th e h eig h t of th e platform (Fig . 4 F).Totest th e concept th at complement activ ation

mig h t onlyrequ ire monov alent binding , we g en-erated fu nctionally monov alent bispecific anti-bodies (20, 21) th at contained one specific and oneinnocu ou s Fab arm (i.e., Ig G1 -7 D8 /b1 2 andIgG1 -2 F8 /b1 2 , monov alently binding CD2 0 andEGFR, respectiv ely). Both antibodies indu cedefficient CDC of relev ant targ et cells (Fig . 4 ,G and H), wh ich for th e bispecific antibody2 F8 /b1 2 was strong ly enh anced relativ e to th eparental 2 F8 antibody. Th u s, for th is antibody-antig enpair, monov alent binding is better ableth an(h ig h -affinity) biv alent binding toaccom-modate th e Fc-Fc h examerization requ ired forefficient CDC.

Th e h exameric IgG-C1 binding model (Fig . 4 ,A and F, and fig . S6 ) rev ealed g eometrical re-straints th at cou ld explain th e strong antig enand epitope dependencyof complement activ a-

tion. Potent complement activ ationbymono-clonal antibodies is restricted tocertainantig ensand epitopes (12, 19, 22), presu mablybecau seantig ensize, density, and flu iditymayaffect ac-tiv ation(18, 22–26) and becau se IgG orientationresu lting from epitope g eometry imposes addi-tional stru ctu ral constraints (12, 19, 22, 25, 27).Polyclonal antibodies appear tobe less sensitiv eth anmonoclonal antibodies tosu ch constraints(24, 28, 29), potentiallybecau se binding of anti-bodies toa v arietyof antig ens or epitopes facil-itates clu stering of Fc segments, th erebyallowingefficient Fc-Fc assembly. Monov alent binding ofIgG molecu les inth e platform is consistent withearlier observ ations (30) and cou ld be env isag edtoprov ide more deg rees of freedom for th e Fcsegments, allowing th eir optimal positioning forC1 qrecru itment.

D EIgG1 -7 D8

H4 3 5 A

I2 5 3 A

N4 3 4 A

Q3 1 1 A

C1 q concentration (µg/mL)1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2

Antibody concentration (µg/mL)1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0%

lysi

sI2 5 3 YI2 5 3 D

H4 3 3 A

H4 3 5 RIrrel. mAb

IgG1

CH2

CH3

A B C

G

F

% ly

sis

0

2 0

4 0

6 0

8 0

1 0 0

No peptide

Control peptide

DCAWHLGELVWCT

0

2 0

4 0

6 0

8 0

1 0 0

IgG1 -7 D8 IgG1 -0 0 5

*

No mAb

S4 4 0 KK4 3 9 E +S4 4 0 K

IgG1 -0 0 5K4 3 9 E

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0

0

5

1 0

1 5

2 0

2 5

3 0

3 5

FIT

C R

b-an

ti-hu

-C1q

(M

ES

Fx10

-4)

C1 q concentration (µg/mL)

***

0

1 0

2 0

3 0

4 0

5 0

6 0

K3 2 2 A

S4 4 0 KK4 3 9 E/S4 4 0 KK4 3 9 E + S4 4 0 K

IgG1 -7 D8K4 3 9 E

Antibody concentration (µg/mL)

% ly

sis

% ly

sis

0

2 0

4 0

6 0

8 0

1 0 0

1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2

Fig. 1. C1q bind ing and complement activationby antibody hexamers. (A) IgG hexamer crystalpacking of IgG1-b12 (1HZH). The dashed enclosure indicates a single IgG molecule. The C1q binding residueLys322,located in the CH2 domain,is indicated in red. (B) Surface map depicting the Fc-Fc interface. Residuesinteracting with the Fc-binding peptide DCAWHLGELVWCT are indicated in blue. (C) The Fc-binding peptideinhibits CDC mediated by IgG1-7D8 (Rajicells) and IgG1-005 (Daudicells). Data are average values T SD(N = 3);one-way analysis of variance followed by Dunnett’s multiple comparison post hoc test:*P < 0.05,***P < 0.001. (D) C1q binding to CD20+ Rajicells opsonized with wild-type or mutated CD20 antibody IgG1-7D8. FITC,fluorescein isothiocyanate;MESF,molecules of equivalent soluble fluorochrome. A representativeexample is shown (N = 3). (E) CDC of Rajicells opsonized with wild-type and mutated IgG1-7D8. A represent-ative example is shown (N = 3). The absence of CDC without added C1q indicates classicalpathway activation.(F and G) CDC of K439E and S440K,abrogated in single point mutants,is restored in an IgG1-7D8 doublemutant [(F),Rajicells] and by mixing single mutants of IgG1-7D8 (F) or IgG1-005 [(G),Daudicells]. Rep-resentative examples are shown (N = 3). Amino acid abbreviations:A,Ala;C,Cys;D,Asp;E,Glu;G,Gly;H,His;I,Ile;K,Lys;L,Leu;N,Asn;Q,Gln;R,Arg;S,Ser;T,Thr;V,Val;W,Trp;Y,Tyr.

www.sciencemag.org SCIENCE VOL 3 4 3 1 4 MARCH 2 0 1 4 1261

REPORTS

I253

H433E345

I253H310

H433

Y436

Q438K439S440

E345

G385N434

Oligomeric statesofIgG determinedbynativeMSandSEC

When(IgG)6 signalsarenormalizedformolaritytheratioofhexamer tomonomercanbeestablished

Thesetechniquesareinexcellentagreement

Molarconcentration(MSdata)

1

1

Massconcentration(SECdata)

1

6

Oligomeric statesofIgG determinedbynativeMSandSEC

MakingselectedmutationsintheIgGs weareabletofinetunethehexamerization propensity

ThispropensitycanbereadoutbynativeMSand/orSEC

Thesetechniquesareinexcellentagreementwitheachother

DeJong,R.N.etal.Plos Biology(2016)

n UsingEMwewereabletodetectC1-hexamerinteractionatthemembranesurface

n Complementactivationisdirectlycorrelatedtohexamerpropensity

C1- IgG1hexamer antibodyEMstructureandactivation

0.0001 0.001 0.01 0.1 1 100

20

40

60

80

100

Neg control

IgG1-005E345RRGY

Antibody concentration (µg/mL)

% ly

sis

Diebolder etal.Science 2014

Bindingof(IgG)6 toC1qdeterminedbynativeMSandSEC

IgGs withhighhexamerization propensityselectivelyandstronglybindtoC1q

IgG glycosylationhasaneffectonthebindingtoC1q

Thisformsalreadya1.3millionDa24proteincomplex,whichcanbenicelyseparatedbynativeMSandwithsometweakingalsobySEC

Alsoherethesetechniquesareinexcellentagreementwitheachother

Wang,G.etal.(2016)Mol Cell,63,135–145

Bindingof(IgG)6 toC1qandtoAgdeterminedbynativeMS

IgGs withhighhexamerization propensityselectivelyandstronglybindtoC1q,butthatthusnoaffectantigenbindingsubstantially

Thisformsalreadya36componentsproteincomplex

Bindingof(IgG)6 toC1andtoAgdeterminedbynativeMS

ReconstitutionofthewholecomplexinvolvedinvolvedinComplementactivation

Thisformsthe2.3millionDa,40componentcomplex,Complementinitiationcomplex

Wang,G.etal.(2016)Mol Cell,63,135–145

AcknowledgementsRebeccaRoseSaraRosatiGuanbo WangAndrey DaychenkoYangYangFanLiuVojtech Franc

RobdeJongEwald vandenBremerJanineSchuurmanPaulParren

EduardDenisovAlexanderMakarov

Deniz UgurlarPietGros