Complete Characterization of a Cysteine-

Poster Note 64 8 0 2
linked Antibody-Drug Conjugate Performed
on a Hybrid Quadrupole-Orbitrap Mass
Spectrometer with High Mass Range
Complete Characterization
Aaron O. Bailey , Eugen Damoc , Stephaneof
Houela Cysteine-linked
, Kai Scheffler , Jonathan L. Josephs
1
Antibody-Drug Con
2 1 3 1

Thermo Fisher Scientific, 1San Jose, CA, USA; 2Bremen, Germany; 3Dreieich, Germany
Aaron O. Bailey11,, Eugen
Aaron O. Bailey Eugen Damoc
Damoc22,, Stephane
Stephane Houel Houel11,, Kai
Kai Scheffler
Scheffler33,, Jonathan
Jonathan L.
L. Josephs
Josephs11
Thermo Fisher
Thermo Fisher Scientific,
Scientific, 11San
San Jose,
Jose, CA,
CA, USA;
USA; 22Bremen,
Bremen, Germany;
Germany; 33Dreieich,
Dreieich, Germany
Germany

ABSTRACT
ABSTRACT RESULTS
RESULTS
Wehave
We havemodified
modifiedthe
spectrometerto toadd
theinstrument
instrumentcontrol
addnative
nativeMS
controlsoftware
MScapability.
softwareof
capability.In
Inthis
ofaabenchtop
thisstudy
studywe
benchtopquadrupole-Orbitrap
quadrupole-Orbitrapmass
wedemonstrate
demonstratecomplete
complete
mass
DENATURING LC-MS,
DENATURING LC-MS, CYSTEINE-LINKED
CYSTEINE-LINKED ADC
ADC
spectrometer Intactprotein
proteinLC/MS
LC/MSanalysis
analysisconventionally
conventionallyinvolves
involvesusing
usingmobile
mobilephases
phaseswhich
whicharearecomprised
comprisedof of
characterizationof
characterization ofBrentuximab vedotin,aacysteine-linked
Brentuximabvedotin, cysteine-linkedADC,
ADC,which
whichrequires
requiresnative
nativeMS
MS Intact
conditionsfor
forintact
intactanalysis.
analysis.WeWedemonstrate
demonstratepreservation
preservationofofnon-covalent
non-covalentbonding
bondingof of organicand
organic andacidic/basic
acidic/basicpH, pH,often
oftensuited
suitedspecifically
specificallyfor
forreverse
reversephase
phasechromatography.
chromatography.This This
conditions strategycan
canbebeuseful
usefulfor
forachieving
achievinghigh
highresolution
resolutionprotein
proteinseparations.
separations.Conditions
Conditionssuchsuchasasthese,
these,
antibody subunits during electrospray ionization. HMR mode can be turned off
antibody subunits during electrospray ionization. HMR mode can be turned off for peptide for peptide strategy
mapping.WeWeuseusetrypsin
trypsinpeptide
peptidemapping
mappingapproach
approachwithwithHCD
HCDfragmentation
fragmentationto toachieve
achieve99%
99% however,are
however, arenot
notcompatible
compatiblefor forperforming
performingintact
intactanalysis
analysisononcertain
certainclasses
classesof ofcompounds
compoundswhich which
mapping. require preservation of non-covalent bonds to maintain structural integrity, such as cysteine-linked
coverageof
coverage ofthe
theBrentuximab
Brentuximabvedotin sequenceusing
vedotinsequence usingaasingle
singleLC-MS
LC-MSanalysis
analysisof ofaa90
90min
min require preservation of non-covalent bonds to maintain structural integrity, such as cysteine-linked
reversephase
phasegradient.
gradient.Finally,
Finally,we
wedemonstrate
demonstratethat thatsignature
signatureions
ionsspecific
specificfor
forHCD
HCD ADCs.We
ADCs. Wedemonstrate
demonstratethis thisphenomenon
phenomenonusingusingthe
thecysteine-linked
cysteine-linked ADC ADCBrentuximab
Brentuximabvendotin.
vendotin.
reverse Denaturing(reverse
(reversephase)
phase)LC/MS
LC/MSanalysis
analysisofofBrentuximab
Brentuximabvedotin resultsinindetection
vedotinresults detectionof ofroughly
roughly
fragmentationof
fragmentation ofBrentuximab
Brentuximabvedotin
vedotincan
canbe beutilized
utilizedto
toincrease
increaseMS/MS
MS/MSassignment
assignment Denaturing
confidence. sixunraveled
six unraveledforms (Figure3A-C).
forms(Figure 3A-C).WeWeobserved
observedaapreviously-reported 1 collisionally-inducedm/z
previously-reported1 collisionally-induced m/z
confidence. 718 fragment of the fragile vcMMAE linker-drug (Figure 3B). Upon deconvolution we also
718 fragment of the fragile vcMMAE linker-drug (Figure 3B). Upon deconvolution we also
observed a mass corresponding to light chain with addition of one vcMMAE
observed a mass corresponding to light chain with addition of one vcMMAE and a loss of and a loss of
INTRODUCTION
INTRODUCTION approximately762
approximately 762Da.
Da.

Thecomplexity
The complexity of
ofmodern
modern therapeutic
therapeuticproteins,
proteins,such
suchas
asantibody-drug
antibody-drugconjugates
conjugates(ADCs),
(ADCs),
presentaagreat
present greatanalytical
analyticalchallenge
challengewhich
whichrequires
requireshigh
highresolution
resolutionchromatography
chromatographycombined
combined
withhigh
highresolution
resolutionmass
massspectrometry.
spectrometry.Complementary
ComplementaryMS MSapproaches
approachessuch suchasaspeptide
peptide Figure3.
Figure 3. Denaturing
DenaturingLC-MS
LC-MSanalysis
analysis
with (A)Unmodified
Unmodifiedsample
sample(1 (1ug)
ug)was
wasanalyzed
analyzedby byreverse
reversephase
phase
mapping and intact mass analysis are needed for complete characterization of
mapping and intact mass analysis are needed for complete characterization of therapeutic therapeutic (A)
chromatographycoupled
chromatography coupledtotoaaQQExactive
ExactivePlus
PlusOrbitrap
OrbitrapMS MS
proteins.Cysteine-linked
proteins. Cysteine-linkedADCs
ADCspresent
presentaaunique
uniquechallenge
challengeforforcharacterization
characterizationas asproper
proper operatingininHMR
HMRmodemodeandandproduced
producedseveral
severalpeaks. (B)The
peaks.(B) The
operating
intactanalysis
intact analysisrequires
requiresnative
nativeMS
MSconditions
conditionstotopreserve
preservestructurally-critical
structurally-criticalnon-covalent
non-covalent resultingaveraged
averagedMS MSspectrum
spectrumisisaacomplex
complexmixture
mixtureofof AA Reversephase
phasechromatography
chromatography
resulting Reverse
bindingbetween
binding betweenantibody
antibodychains.
chains.WeWehave
havemodified
modifiedcommercially-available
commercially-availableThermo
Thermo chargestate
charge stateenvelopes
envelopesas aswell
wellas
asaapreviously
previouslydescribed
described11 100
100

Abundance
ScientificTM
TM Q ExactiveTM
TM Plus and Q ExactiveTM TM HF OrbitrapTM TM mass spectrometers to vcMMAE-specificreporter
reporterfragment
fragmentionionatatm/z
m/z718. (C)Data
718.(C) Data

RelativeAbundance
Scientific Q Exactive Plus and Q Exactive HF Orbitrap mass spectrometers to vcMMAE-specific 80
perform native LC-MS experiments. In the present study, we demonstrate this capabilitywith
with analysiswith
analysis withReSpect
ReSpectdeconvolution
deconvolutionand andSliding
SlidingWindow
Window 80
Brentuximab
perform native LC-MS experiments. In the present study, we demonstrate this capability 60 Brentuximab
intactanalysis
analysisofofBrentuximab vedotin,aacysteine-linked
Brentuximabvedotin, cysteine-linkedADC (Figure1).
ADC(Figure 1).Additionally,
Additionally,we
wehave
have integration show roughly six covalently-structured forms ofof
integration show roughly six covalently-structured forms 60 vedotin
vedotin
intact unraveledcysteine-linked
unraveled cysteine-linkedADC.
ADC.We Wedetect
detectaaprotein
proteinspecies
species 40
40
performeddenaturing
denaturingLC-MS
LC-MSandandpeptide
peptidemapping
mappingon onthese
thesesame
sameinstruments
instrumentsto togenerate
generate

Relative
performed whichcorresponds
which correspondstotoaalight
lightchain
chainwith
withaddition
additionofofone
onelinker
linker 20
20
complementarydatasets
complementary datasetsfor
forcomplete
completecharacterization.
characterization. drugand
drug andaaloss
lossofof762
762Da,
Da,which
whichisisalso
alsopresent
presentininthe
theraw
raw
00 6
spectrum.
spectrum. 6 77 88 99 1010 11
11 12
12
Time(min)
Time (min)
Figure1.
Figure 1. Schematic
Schematicfor
forConstructing
ConstructingCysteine-Linked
Cysteine-LinkedADC
ADC
BBrentuximab vedotinisisaacysteine-linked
rentuximabvedotin cysteine-linkedADC
ADCwhich
whichisisconstructed
constructedby bymodifying
modifyingananantibody
antibodywith
withvcMMAE,
vcMMAE,aa
preformedlinker-drug
preformed linker-drugcomprised
Saturaturated(8(8drugs)
comprisedofofaavaline-cirtuline-based
drugs)cys-linked
cys-linkedADCs
valine-cirtuline-basedlinker
ADCsareareheld
heldintact
intactwith
linkerand
withonly
andaamonomethyl
monomethylauristatin
onlynon-covalent
auristatinEEtoxic
non-covalentbinding.
binding.
toxicdrug.
drug.
BB
Denaturing LC-MS
Denaturing LC-MS
Saturaturated 100
100
90 SheathGas:
Sheath Gas:10
10
mAb
mAb Cys-linked 90
AuxGas:
Gas:55
Cys-linked Pairsofoflinker-drugs
linker-drugsareare 80
80 Aux
ADC Pairs AuxGas
GasTemp:
Temp:50ºC
50ºC
Abundance

drug ADC Aux

RelativeAbundance

attachedatatsites
sitesofof 70 718.5121
drug attached 70 718.5121 CapTemp:
Temp:320ºC
320ºC
linker-drug
linker-drug interchain-disulfide bonds 60 Cap
linker interchain-disulfide bonds 60 S-Lens:200
200
linker 50
S-Lens:
DrugtotoAntibody
AntibodyRatio
Ratio 50 In-sourceCID:
In-source CID:60
60
Drug
Relative

40 R=17500
17500
vcMMAE
vcMMAE (DAR)distribution
(DAR) distribution==0-8
0-8 40 R=
30
30
“linker”
“linker”
“drug”
“drug” 20 762.5026
762.5026
20
mAb SS
mAb
10
10
00
500
500 1000
1000 1500
1500 2000
2000 2500
2500 3000
3000 3500
3500 4000
4000 4500
4500
m/z
m/z

attachment
attachment
group
valine-
valine-
citrulline
spacer
spacer monomethylauristatin
monomethyl auristatinEE
CC
group citrulline
25041.47
25041.47 22xxG0F
G0F
(32.5 ppm) 125678.81
100 (32.5 ppm) 11xxG0F
G0F 125678.81
76675.44 (16.0ppm)
ppm)
MATERIALS AND
AND METHODS
METHODS
100 76675.44 (16.0
MATERIALS
90
90 (1.0ppm)
ppm)
80 (1.0
Intensity

80 2xxG0F
G0F
RelativeIntensity

70 (-761.96Da)
Da) 2
70 (-761.96 103272.01
60
60 11xxG0F
G0F 103272.01
(5.6ppm)
ppm) 22xxG0F
G0F
Brentuximabvedotin
Brentuximab vedotinwas
wasprepared
preparedfor forpeptide
peptidemapping
mapping(reduction,
(reduction,alkylation,
alkylation,andandtrypsin
trypsindigestion)
digestion)or orintact
intact 50 54269.74
54269.74 (5.6 148086.47
148086.47
50 2xxG0F
G0F (28.1ppm)
ppm)
analysis(no
(notreatment).
treatment).ForFordenaturing
denaturingLC-MS
LC-MSintact
intactanalysis
analysis11μgμgofofprotein
proteinsamples
sampleswere
wereseparated
separatedusingusingaa10
10 40 (4.6ppm)
ppm)
Relative

analysis 40 (4.6 2 (28.1

30 24279.51 105907.20
mingradient
min gradientofof10-90%
10-90%ACNACNininHH2OOand and0.1%
0.1%formic
formicacid
acid(Thermo
(ThermoMAb-Pac
MAb-PacRP; RP;flow
flowrate
rate250
250μL/min).
μL/min).ForFor 30 24279.51 105907.20
(23.5ppm)
ppm)
2 20 (23.5
native LC-MS intact analysis 10 μg of sample was desalted online using size exclusion
native LC-MS intact analysis 10 μg of sample was desalted online using size exclusion chromatography chromatography 20
10
(WatersTMTM BEH SEC 4.6x150mm; 50 mM NH OAc isocratic elution, flow rate 300 μL/min) and directly presented 10
(Waters BEH SEC 4.6x150mm; 50 mM NH44OAc isocratic elution, flow rate 300 μL/min) and directly presented 00
totothe
themass
massspectrometer
spectrometervia viaelectrospray
electrosprayionization.
ionization.Peptide
Peptidemapping
mappingwas wasperformed
performedusingusing2.5
2.5μgμgofofsample
sample 20000 30000
20000 30000 40000
40000 50000
50000 60000
60000 70000
70000 80000
80000 90000
90000 100000
100000 110000
110000 120000
120000 130000
130000 140000
140000 150000
150000 160000
160000
separatedusing
separated usingaa90 90min
mingradient
gradientofof2-90%
2-90%ACN ACNininHH2OOandand0.1%
0.1%formic
formicacid
acid(Acclaim
(AcclaimRSLC
RSLC120 120C18;
C18;flowflowrate
rate Mass
Mass
2
250 μL/min). Commercially-available Orbitrap mass spectrometers (Q Exactive HF and
250 μL/min). Commercially-available Orbitrap mass spectrometers (Q Exactive HF and Q Exactive Plus) which Q Exactive Plus) which
weremodified
were modifiedtotoinclude
includeHigh
HighMass
MassRange
Range(HMR)
(HMR)mode modetotoallow
allowimproved
improvedhighhighmass
masstransmission
transmissionandandscanning
scanning
upuptotom/z
m/z8000.
8000.Native
Nativeintact
intactand
anddenaturing
denaturingMS MSdatadatawere
wereacquired
acquiredininHMR
HMRmodemodeatatsetting
settingofofR=15k
R=15kor or17.5k
17.5k
anddeconvolved
and deconvolvedusing
usingthe
theReSpect
ReSpectTM TM algorithm and Sliding Window integration in Thermo ScientificTM
algorithm and Sliding Window integration in Thermo ScientificTM
BioPharma Finder TM 1.0 SP1 software. Deconvolution species were identified automatically using the publicly-
BioPharma Finder 1.0 SP1 software. Deconvolution species were identified automatically using the publicly-
TM
availableFASTA
available FASTAsequence
sequencefor forBrentuximab
Brentuximabvedotin,
vedotin,aamass
masstolerance
toleranceofof5050ppm,
ppm,and
andaastatic
staticmodification
modificationofof
Gln>Pyro-Glufor forthe
theheavy
heavychain.
chain.Peptide
Peptidemapping
mapping datadatawere
wereacquired
acquiredby bydata
datadependent
dependentselection
selectionwith
withR=60k
R=60k
Gln>Pyro-Glu
oror70k
70kfor
forFull
FullMS
MSandandR=15k
R=15kor or17.5k
17.5kforforMS/MS.
MS/MS.Peptide
Peptidemapping
mappingdatadatawere
weresearched
searchedusing
usingthe
theMassAnalyzer
MassAnalyzer NATIVE INTACT
NATIVE INTACT LC-MS,
LC-MS, CYSTEINE-LINKED
CYSTEINE-LINKED ADC
ADC
algorithm in BioPharma Finder software with a tolerance
algorithm in BioPharma Finder software with a tolerance of 5 ppm. of 5 ppm. NativeMSMSintact
intactprotein
proteinanalysis
analysisallows
allowsdirect
directobservation
observationof ofmolecules
moleculeswhich
whichrely
relyon
onnon-
non-
Native
covalentinteractions
covalent interactionsto topreserve
preservecritical
criticalstructural
structuralfeatures,
features,such
suchasasmaintaining
maintaininginterchain
interchain
Figure2.
Figure 2. LC-MS
LC-MSInstrumentation
Instrumentationfor
forComplete
CompleteADC
ADCCharacterization
Characterization associations which hold together cysteine-linked ADCs. The use of 100% aqueous physiological
Allexperiments
experimentswere
wereperformed
performedusing
usingaaVanquish
VanquishUHPLC
UHPLCconnected
connectedtotoaaExactive
ExactiveHF
HFor
orQQExactive
ExactivePlus
Pluswith
with associations which hold together cysteine-linked ADCs. The use of 100% aqueous physiological
All pHbuffers
pH buffersininnative
nativeMSMSanalysis
analysisproduces
producesdecreased
decreasedcharge
chargestates
states(increased
(increasedm/z)
m/z)and
and
HighMass
High MassRange
Range(HMR)
(HMR)mode.
mode.
improvesmass
improves massseparation
separationof ofheterogeneous
heterogeneousmixtures.
mixtures. We Weperformed
performednative
nativesize
sizeexclusion
exclusionLC-LC-
MSand
MS andobserved
observed55distinct
distinctspecies
speciescorresponding
correspondingto tointact
intactBrentuximab
Brentuximabvedotin with0,
vedotinwith 0,2,
2,4,
4,6,6,
VanquishTM TM QQExactive
ExactivePlus/
Plus/ BioPharma
BioPharma or 8 vcMMAE linker-drugs (Figure 4). We measured an average drug-to-antibody
or 8 vcMMAE linker-drugs (Figure 4). We measured an average drug-to-antibody ratio of 4.07, ratio of 4.07,
Vanquish HFOrbitrap
OrbitrapTM
TM
FinderTM
TM
UHPLC
UHPLC
HF Finder which is consistent with a previously published studies reporting 3.9-4.2 drugs per
which is consistent with a previously published studies reporting 3.9-4.2 drugs per antibody .antibody 22.
MS
MS software
with HMR mode
software
with HMR mode
 PEPTIDE MAPPING, CYSTEINE-LINKED ADC
A fundamental component of biotherapeutic protein characterization is peptide mapping.

onjugate Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrom

Native
exclusion MS intact protein
chromatography
NATIVE INTACT LC-MS, CYSTEINE-LINKED
Figure 4. Native intact LC-MS analysis
(A) Unmodified sample (10 ug) was analyzed using size
coupled analysis allows
to a Q Exactive
A
direct observation
Plus 100
ADC
Size exclusion chromatography
of molecules which rely on non-
Brentuximab
Figure 5. Peptide
Whereas
Linked
deviation
intactMapping
combinations,
of Cysteine-
mass analysis
ADC Brentuxmab vedotin
aims to detect the abundances and distributions of mass
peptide mapping allows highly sensitive analysis of site-specific
Reduced, alkylated, and trypsin-digested sample (2.5
sequence
covalent
Orbitrap interactions
MS operating to preserve
in HMR mode. critical structural features,
Buffer exchange 50 mM such as maintaining interchain ug) was separatedfeatures. Thegradient
using a 90 min vcMMAE linker-drug on Brentuximab vedotin poses particular
and eluted

Relative Abundance
80 vedotin physiological
occursassociations
online as ADCwhich holdas
forms elute together cysteine-linked ADCs. NH
a single peak, The use of 100% aqueous
4OAc into achallenges
Q Exactive HFwhen attempting
(equipped with HMR to identify drug conjugation sites. We prepared a sample for
mode)
followed by a second peak corresponding separated 60 isocratic
pH buffers in native MS analysis produces decreased charge states (increased m/z) and peptide
operating mapping
in Standard mode.using
Using reduction andcut
a mass accuracy alkylation to block non-drug-conjugated cysteines,
buffer salts. (B) Averaging 2 min chromatographic time elution buffer off of 5 ppm, we achieved 99% sequence coverage of
improves mass separation of heterogeneous mixtures. 40 We performed native size exclusion LC- followed by trypsin digestion. In one 90 min LC-MS gradient we were able to achieve 99%
produces a native intact MS spectrum which includes all salts both light and heavy chains. We detected known
MS and observed 5 distinct species corresponding 20to intact Brentuximab vedotin with 0, 2, 4, 6,
sequence
DAR forms (DAR 0-8). (C) ReSpect deconvolution and glycopeptides and coverage
were able to for both
detect light and heavy chains and detect peptides spanning all four
MMAE-
SlidingorWindow
8 vcMMAE linker-drugs
integration (Figurepeak
can accommodate 4). We
tailingmeasured 0 an average drug-to-antibody ratio of 4.07,
drug conjugation
conjugated sites.
peptides at all four HCD(red
cysteines fragmentation
circles) allowed detection of a peptide in the hinge
0 2 4 6 8 102
which
to report is consistent
quantitatively with
accurate a previously
abundances published studies
for the reporting 3.9-4.2 drugs per antibody . which are normally involved in interchain disulfide pairs
mixture of DAR forms which have diverse elution profiles.
Time (min) region of the heavy chain that is differentially modified with 0-2 vcMMAE drugs. As a result
in naked antibodies. A trypsin peptide sequence at the
A pattern of lower abundance species were detected hingeefficient elution
region of the heavyrequires sustained
chain (red asterisk) was delivery of high organic mobile phase.
corresponding to a low abundance loss of 762 Da from present in forms ranging from 0-2 vcMMAE
each glycoform at each DAR value (green arrows). (D)
Figure 4. Native intact LC-MS analysis A
Native LC-MS conjugations. A missed cleavage peptide contained
Figure 5. Peptide Mapping of Cysteine- up
Based on the individual deconvolved abundances of the Size exclusion chromatography to 3 conjugations.
(A) Unmodified sample (10 ug) was analyzed using size
G0F/G0F glycoform, we calculated an average DAR value Sheath Gas: 40 Linked ADC Brentuxmab vedotin
exclusion chromatography coupled to a Q Exactive Plus 100
Aux Gas: 5 Reduced, alkylated, and trypsin-digested sample (2.5
of 4.07, which is consistent with previous reports2. Brentuximab

*
Orbitrap MS operating in HMR mode. Buffer exchange 50Aux
mM Gas Temp: 125ºC ug) was separated using a 90 min gradient and eluted

Relative Abundance
occurs online as ADC forms elute as a single peak,
80
NH4OAc vedotin
Cap Temp: 275ºC into a Q Exactive HF (equipped with HMR mode)
B 100 followed by a second peak corresponding separated 60 isocratic operating in Standard mode. Using a mass accuracy cut
90 buffer salts. (B) Averaging 2 min chromatographic time S-Lens: 200 buffer
40 elution off of 5 ppm, we achieved 99% sequence coverage of
In-source CID: 100
80 produces a native intact MS spectrum which includes all salts both light and heavy chains. We detected known
20 R= 17500
Relative Abundance

70 DAR forms (DAR 0-8). (C) ReSpect deconvolution and glycopeptides and were able to detect MMAE-
60
Sliding Window integration can accommodate peak tailing 0 conjugated peptides at all four cysteines (red circles)
0 2 4 6 8 10
to report quantitatively accurate abundances for the Time (min) which are normally involved in interchain disulfide pairs
50
mixture of DAR forms which have diverse elution profiles. in naked antibodies. A trypsin peptide sequence at the
40 A pattern of lower abundance species were detected
hinge region of the heavy chain (red asterisk) was
30 corresponding to a low abundance loss of 762 Da from present in forms ranging from 0-2 vcMMAE
20 each glycoform at each DAR value (green arrows). (D) Native LC-MS conjugations. A missed cleavage peptide contained up
10
Based on the individual deconvolved abundances of the to 3 conjugations.
G0F/G0F glycoform, we calculated an average DAR value Sheath Gas: 40
0
of 4.07, 5400
which is 5600
consistent with previous reports 2. Aux7200
Gas: 5 7400

*
5200 5800 6000 6200 6400 6600 6800 7000 7600
m/z Aux Gas Temp: 125ºC
B 100 Cap Temp: 275ºC
DAR4 S-Lens: 200
C 90
80
In-source CID: 100
DAR6 R= 17500
Relative Abundance

70 DAR2
60
Figure 6. Hinge Region Peptide of Cysteine-linked ADC is Site of Multiple Conjugations
(A) Our data analysis in BioPharma Finder software resulted in detection of peptides which covered the hinge region
50
of the heavy chain (red asterisk). A faithfully-trypsin-cleaved THT-KPK peptide was detected with 0-2 vcMMAE
40
2 x G0F
conjugations at cysteines (red circles) normally involved in interchain disulfide pairs. (B) Addition of vcMMAE to
30 153355.40 peptides dramatically increases hyphobicity which results in poor elution and increased retention time. MS/MS
20 , analysis of the (C) 1 linker-drug (both positional isomers) and (D) two linker-drug forms in BioPharma Finder allowed
100 2 x G0F 2 x G0F clear sequencing of y-ions in the hinge peptides, and thus facilitated automatic detection.
10 50720.06 155987.81 DAR8
80 DAR0
0
Relative Intensity

60 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600
m/z A B 100
40 (-762.25 Da)
2 x G0F 2 x G0F
DAR4 158632.20
C

%B
20 148086.53 50

0
147000 DAR2 152000 DAR6 Heavy chain 0
148000 149000 150000 151000 153000 154000 155000 156000 157000 158000 159000 160000
Figure 6. hinge
Hinge Region Peptide of Cysteine-linked ADC
30 is40
Site(min)
of60Multiple
70 80 Conjugations
*
100
Mass region peptide 0 10 20 Time50 90 100 110 120
(A) Our data analysis in BioPharma Finder software resulted 0invcMMAE
detection of peptides which covered the hinge region

Relative Abundance
50

of the heavy chain (red asterisk). A faithfully-trypsin-cleaved

0 THT-KPK peptide was detected with 0-2 vcMMAE
D THTCPPCPAPELLGGPSVFLFPPKPK
100

G0F/G0F Mass Accuracy 2 x G0F

Relative 50 1 vcMMAEdisulfide pairs. (B) Addition of vcMMAE to
conjugations at cysteines (red circles) normally involved in interchain
DAR (ppm) 153355.40
Abundance peptides dramatically increases hyphobicity which results in poor elution and increased retention time. MS/MS
, Average analysis of the (C) 1 linker-drug (both positional isomers)
0
100 and (D) two linker-drug forms in BioPharma Finder allowed
100 DAR0 2 x G0F 11.7 6.77 Drug-to-Antibody
2 x G0F clear sequencing of y-ions in the hinge peptides, and thus 50 facilitated
2 vcMMAE automatic detection.
50720.06 155987.81 DAR8
DAR0 0
80 DAR2 23.1 69.23 Ratio (DAR) 0 20 40 60 80 100 120
Relative Intensity

4.07
Time (min)
60 DAR4 22.4 100.00 A B 100
40
2 x G0F
DAR6 40.5 (-762.25 Da)
69.75 2 x G0F C 100
D 100
158632.20 80 80

%B
148086.53
20 DAR8 17.6 10.61 60 THT-KPK 50
60
THT-KPK
0 Predicted
Relative Abundance

40

Relative Abundance
40
147000 148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000 160000 Heavy chain spectra 0

*
20
hinge region peptide
100
0 20 10 20 30 40
Time50
(min)60 70 80 90 100 110 120
Mass
00 vcMMAE

Relative Abundance
0 50
100 y5
0 100 y5
D 80 Experimental
THTCPPCPAPELLGGPSVFLFPPKPK
100
801 vcMMAE
G0F/G0F Mass Accuracy Relative y13 50
DAR (ppm) Abundance 60 spectra 60 y13
Average 0

PEPTIDE MAPPING, DAR0 CYSTEINE-LINKED ADC

40 100
y6 y8 40 y
11.7 6.77 b3 y9
Drug-to-Antibody 20 y7
y14 y17
50
20
2 vcMMAE
y7
y8 y11 y14 y17
0
A fundamental component of DAR2
biotherapeutic
23.1protein characterization
69.23 is peptide
Ratio (DAR) mapping. 0
500 1000 1500 0 0 20 40 60 80 100 120
m/z 500 1000 1500
Whereas intact mass analysisDAR4 aims to detect
22.4 the abundances and distributions of mass m/z (min)

4.07
Time
100.00
deviation combinations, peptide DAR6mapping 40.5
allows highly69.75
sensitive analysis of site-specific C 100
D 100
sequence features. The vcMMAE linker-drug
17.6 on Brentuximab vedotin poses particular 80 80
DAR8 10.61 60 THT-KPK THT-KPK
challenges when attempting to identify drug conjugation sites. We prepared a sample for Predicted
60
Relative Abundance

40
Relative Abundance

40
peptide mapping using reduction and alkylation to block non-drug-conjugated cysteines, 20 spectra 20
followed by trypsin digestion. In one 90 min LC-MS gradient we were able to achieve 99% 0 0
y5
sequence coverage for both light and heavy chains and detect peptides spanning all four 100 100 y5
drug conjugation sites. HCD fragmentation allowed detection of a peptide in the hinge 80 Experimental 80
y13
region of the heavy chain that is differentially modified with 0-2 vcMMAE drugs. As a result
60 spectra 60 y13

PEPTIDE MAPPING, CYSTEINE-LINKED ADC

40 y8 40
y6 y
efficient elution requires sustained delivery of high organic mobile phase. 20
b3
y7
y9
y14 y17 20
y8 y11 y14
y7 y17
A fundamental component of biotherapeutic protein characterization is peptide mapping. 0
500 1000 1500
0
m/z 500 1000 1500
Whereas intact mass analysis aims to detect the abundances and distributions of mass m/z

deviation combinations, peptide mapping allows highly sensitive analysis of site-specific

sequence features. The vcMMAE linker-drug on Brentuximab vedotin poses particular
challenges when attempting to identify drug conjugation sites. We prepared a sample for
peptide mapping using reduction and alkylation to block non-drug-conjugated cysteines,
followed by trypsin digestion. In one 90 min LC-MS gradient we were able to achieve 99%
sequence coverage for both light and heavy chains and detect peptides spanning all four
drug conjugation sites. HCD fragmentation allowed detection of a peptide in the hinge
region of the heavy chain that is differentially modified with 0-2 vcMMAE drugs. As a result
efficient elution requires sustained delivery of high organic mobile phase.

2 Complete Characterization of a Cysteine-linked Antibody-Drug Conjugate Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrometer with High Mass Range
 (A) Our data analysis in BioPharma Finder software resulted in detection of peptides which covered the hinge region
of the heavy chain (red asterisk). A faithfully-trypsin-cleaved THT-KPK peptide was detected with 0-2 vcMMAE
conjugations at cysteines (red circles) normally involved in interchain disulfide pairs. (B) Addition of vcMMAE to
peptides dramatically increases hyphobicity which results in poor elution and increased retention time. MS/MS
analysis of the (C) 1 linker-drug (both positional isomers) and (D) two linker-drug forms in BioPharma Finder allowed
Figure 7. HCD Signature Fragment Ions for vcMMAE Linker-Drug
clear sequencing of y-ions in the hinge peptides, and thus facilitated automatic detection.
The light chain C-terminal peptide SFN-GEC is a conjugation site for vcMMAE. This modified peptide was
CONCLUSIONS
automatically identified by BioPharma Finder (left side top panel). Further manual inspection produced additional
fragment assignments for vcMMAE signature ions (right side top panel). Theoretical masses (top panel) were •We have modified the control software in Q Exactive Plus and Q Exactive HF mass spectrometers
A Bmasses
100
calculated manually and matched to experimental (bottom panel) within 5ppm (green boxes). A cleavage site to add native MS capability.
for the loss of 762 Da is shown (black box; theoretical monoisotopic mass = 762.5017). We observed a high

%B
abundance ion at m/z 1366.6189 (orange box, asterisk) 50 which corresponds to the peptide-retaining fragment pair of a
762 Da loss with an additional loss of 2 protons, presumably due to formation of a seven-membered aromatic ring.
•Native LC/MS intact analysis of Brentuximab vedotin resulted in detection of intact ADC forms,
DAR0-8. ReSpect deconvolution and Sliding Window integration showed an average DAR of 4.07,
Heavy chain 0
consistent with previous studies.
*
100
hinge region peptide 0 10 20 30 40
Time50
(min)60 70 80 90 100 110 120
0 vcMMAE

Relative Abundance
50
762.5017 +1 506.3593 +1 321.2178 +1
0
100
152.1075 +1 •Acquisition of MS/MS spectra with HCD fragmentation on Q Exactive Plus and Q Exactive HF
THTCPPCPAPELLGGPSVFLFPPKPK
S 50 1 vcMMAE Orbitrap mass spectrometers followed by data analysis with BioPharma Finder resulted in 99%
0
100 sequence coverage from a single 90 min gradient using 5 ppm mass tolerance.
50 2 vcMMAE
0
*1366.6213 +1
•Addition of vcMMAE linker drug dramatically increases peptide hydrophobicity and retention time.
0 20 40 60 80 100 120
Time (min)
718.5119 +1 •Signature HCD fragment ions of linker-drug may allow additional means for identifying drug-
C 100
D 100
conjugated peptides.
80 80
60 THT-KPK 60
THT-KPK
Predicted
REFERENCES
Relative Abundance

40 100
a3 -H2O 2+ Relative Abundance 40
20 spectra 20 1366.6189
80
Relative Abundance

0 152.1070 0
100 y5 506.3584 100 1. Janin-Bussat MC, Dillenbourg M, Corvaia N, Beck A, Klinguer-Hamour C. Characterization of
60 y5
80
321.2170
b6 Experimental 80 antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis.
40
60 y13 718.5105
spectra 60 y13
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Feb 15;981-982:9-13.
b4 y3 -H2O
40 20 a2 y8 40 y
b3 y6 y9
20 y14 y17 y8 y11
0
y7 20
y7
y14 y17 2. Debaene F, Boeuf A, Wagner-Rousset E, Colas O, Ayoub D, Corvaïa N, Van Dorsselaer A, Beck A,
0 0
500 1000 500
m/z
1500 1000 m/z 500 1500 1000 1500 2000 Cianférani S. Innovative native MS methodologies for antibody drug conjugate characterization: High
m/z
resolution native MS and IM-MS for average DAR and DAR distribution assessment. Anal Chem.
2014 Nov 4;86(21):10674-83.

© 2016 Thermo Fisher Scientific Inc. Waters™ is a trademark of Waters Corporation. ReSpect™ is a trademark of
Positive Probability Ltd. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its
subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the

CONCLUSIONS
intellectual property rights of others.

•We have modified the control software in Q Exactive Plus and Q Exactive HF mass spectrometers
to add native MS capability.

•Native LC/MS intact analysis of Brentuximab vedotin resulted in detection of intact ADC forms,
DAR0-8. ReSpect deconvolution and Sliding Window integration showed an average DAR of 4.07,
consistent with previous studies.

•Acquisition of MS/MS spectra with HCD fragmentation on Q Exactive Plus and Q Exactive HF
Orbitrap mass spectrometers followed by data analysis with BioPharma Finder resulted in 99%
sequence coverage from a single 90 min gradient using 5 ppm mass tolerance.

•Addition of vcMMAE linker drug dramatically increases peptide hydrophobicity and retention time.

•Signature HCD fragment ions of linker-drug may allow additional means for identifying drug-
conjugated peptides.

REFERENCES
1. Janin-Bussat MC, Dillenbourg M, Corvaia N, Beck A, Klinguer-Hamour C. Characterization of
antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Feb 15;981-982:9-13.

2. Debaene F, Boeuf A, Wagner-Rousset E, Colas O, Ayoub D, Corvaïa N, Van Dorsselaer A, Beck A,

Cianférani S. Innovative native MS methodologies for antibody drug conjugate characterization: High
resolution native MS and IM-MS for average DAR and DAR distribution assessment. Anal Chem.
2014 Nov 4;86(21):10674-83.

© 2016 Thermo Fisher Scientific Inc. Waters™ is a trademark of Waters Corporation. ReSpect™ is a trademark of
Positive Probability Ltd. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its
subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the
intellectual property rights of others.

www.thermofisher.com
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