AlbertJ.R.HeckUtrechtUniversity
email:[email protected]
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