Download Structural Characterization of Antibody Drug Conjugates

Structtural Ch
haracte
erization of An
ntibody
y drug c
conjuga
ates
(A
ADCs) by a co
ombination of intact, middle
e-up an
nd botto
om-up
appro
oaches
s using CESI-M
MS
Dr. S
Stephen Loc
ck1, Nassur Said
S 2, Dr. Ra
abah Gahou
ual2,Dr. Alain
n Beck3, Dr. Yannis-Nico
olas Françoiss2 and Dr.
Emm
manulle Leiz
ze-Wagner2
1
SCIIEX, United Kingdom, 2Laboratoire
L
de
d Spectrom
métrie de Ma sses des Intteractions ett des Systèm
mes
(LSM
MIS), Université de Stras
sbourg, Stra
asbourg, France, 3Centre
e d’Immunologie Pierre Fabre, Saintt-Julien-enGenevois, Franc
ce
oduction
Intro
Materrials and Me
ethods
Chemic
cals: All chemiicals were Reag
gent Grade and were purchased
Antibo
ody drug conjuga
ates (ADCs) rep
present a rapidly
y growing class of
from Si gma Aldrich or alternative supp
pliers . Brentuxim
mab vedotin (BV
V)
biopha
armaceuticals. ADCs
A
are forme
ed by the conjug
gation of an actiive
roduced by Mille
enium Pharmacceuticals/Takeda and was buffe
er
was pro
1
3
drug sspecies to a monoclonal antibo
ody and often result in a mix of
exchang
ged with 200 m
mM ammonium acetate bufferr (pH 7.0) using
protein
ns containing a distribution of products co
ontaining differe
ent
Amicon filters before inttact analysis
3
numbe
ers of active drug
gs bound in different locations aro
ound the antibod
dy.
e Preparation: For middle-up a
analysis BV was cleaved at the
Sample
ary Electrospray
y Ionization (CE
ESI) is the integ
gration of capilla
ary
Capilla
hinge re
egion by proteolyysis using IdeS (FabriCATOR, G
Genovis) to obtain
electro
ophoresis (CE) and electrospra
ay ionization (E
ESI) into a sing
gle
Fc/2 frragments and o
one F(ab’)2 fra
agment.
2
Afte
er digestion was
processs in a single de
evice (Figure 1) . CESI-MS operrates at low nL/m
min
e was again b
buffer exchange
ed into 200 mM
M
completted the sample
flow rrates and offers
s several advan
ntages. These include increased
ammon ium acetate bufffer (pH 7.0) beforre CESI-MS ana
alysis.
ionizattion efficiency and a reductio
on in ion supprression. CESI-M
MS
separa
ates analytes by
b their charge
e and size and
d is, therefore, a
esalted IdeS cle
eaved ADC which
For botttom-up analysiss a sample of de
complementary separration mechanism
m to more traditional technique
es,
dergone buffer e
exchange was diiluted with RapiG
Gest and reduced
had und
as reverse phase
e LC.
such a
with Ditthiothreitol (DTT)). Before enzym
matic digestion, 10% of acetonitrile
ple. The reduced
d protein was th
hen digested with
was ad ded to the samp
document summa
arizes the work recently publishe
ed by the researrch
This d
3
med, isopropano
ol
trypsin overnight a seccond reduction sstep was perform
group at LSMIS at the
e University of Sttrasbourg . In th
his application no
ote
and formic acid ((1% v/v) were ad
dded and the fina
al solution diluted
(40%) a
SI-MS can be us
sed to characteriz
ze a gold standa
ard
we willl show how CES
using a
ammonium ace
etate (50 mM, pH 4.0) to produce a fina
al
ADC, brentuximab ved
dotin which is a monoclonal antiibody (mAb) which
concenttration of 2.2 µM
M.
3
d mono-methyl auristatin E (M
MMAE) which was
w
has a cysteine-linked
attach
hed without disru
upting the heav
vy and light cha
ain linkages of the
t
3
MS method: Forr the analysis o
of intact and digested samples a
CESI-M
mAb . We will show how CESI-MS will first be use
ed as a nanosprray
Bare fu sed silica OptiM
MS CESI cartridge (30 μm ID x 9
91 cm) which was
on device to iden
ntify the drug anttibody ratio of the sample. We will
w
infusio
d coolant regula
ated at 20 C was
thermosstatted using re circulating liquid
also sshow how CESI-MS can be used
d to study the ‘m
middle up’ structu
ure
used. F
digests a samp
ple was injected
For the analyssis of tryptic d
for this protein, e.g. by analyzing the
t
light chain, Fab and F(ab’)2
hydrodyynamically (10 psi, 60s) and peptides were separated using
nits. Finally a top
p down analysis of
o a tryptic diges
st will be performed
subun
conditio
ons shown in T
Table 1 using a background electrolyte of 10%
%
by CE
ESI-MS to fully se
equence the ADC
C.
acetic a
acid. For MS ana
alysis of tryptic d
digests, a SCIEX TripleTOF 6600
o
®
®
mass sp
pectrometer wass fitted with the N
NanoSpray III so
ource. Gas 1 and
2 were not used and th
he temperature o
of the interface w
was set at 150 ºC
C
e ionization at vvery low flow rate
es occurs by sim
mply applying the
because
ionsprayy voltage (1450 V). The curtain gas was set veryy low at 4 psi (se
et
automa
atically). MS Da
ata was acquired
d using a TOF ssurvey scan (m//z
00 amu) which trriggered MS/MS acquisition (m/z 100-2000 amu).
100-200
O
Ultra low
w flow ESI Interfa
ace.
Figure 1: OptiMS-
p1
Actio
on
Rinsse
Rinsse
Rinsse
Rinsse
Rinsse
Rinsse
Injecttion
Separa
ation
Volta
age
Time
(min)
3.5
1.5
3
5
2
60s
10s
35
2
Pressure
(psi)
100
100
100
100
75
10
5
0.1
10
Dirrection
Fo
orward
Fo
orward
Fo
orward
Fo
orward
Re
everse
Fo
orward
Fo
orward
Fo
orward
Fo
orward
Voltage
e
(kV)
0
0
0
0
0
0
0
20
1
Solution
0.1 M NaOH
H
Water
0.1 M HCl
10% Acetic acid
10% Acetic acid
Sample Via
al
10% Acetic acid
10% Acetic acid
10% Acetic acid
Table 1: CESI sepa
aration conditions used for the an
nalysis of tryptic
digestts.
e deconvoluted m
mass spectrum of the intact and
Based on charge state
osylated BV sho
own in Figure 2
deglyco
between
n 3.8 and 3.9.
the average DAR value was
A distribution off baseline resolvved species of BV
V
was ob
bserved whose m
masses corresp
ponding to the in
ntact mAb linked
with zerro to eight paylo
oads of drug . T
The mass accura
acies of intact BV
V
3
were in total agreementt with results repo
orted in the litera
ature .
ns as the intac
ocol and experimental condition
ct
Using tthe same proto
analysiss a middle-up d
digested sample
e was analyzed to elucidate the
location
n of the drug loa
ading on the mAb which had bee
en cleaved at the
hinge rregion (Figure 2
2). The raw da
ata highlighted multiply charged
For na
ano-spray infusio
on the CESI cap
pillary was flushe
ed with 10% ace
etic
protein peaks in three separate regions across the ma
ass range 2000 -
acid (5min, 50 psi) th
hen with sample
e (5min, 50psi) and MS data was
w
When deconvolute
ed these multiplyy charged protein
6000 D a (Figure 3). W
er each analysis the capillary was
s flushed with 10
0%
acquirred at 5psi. Afte
pes depicted in F
Figure 3 show that free Light Chain (LC) protein
envelop
acetic acid (10min, 50
0psi) to prevent carry over. Forr these nano-sprray
subunit (MW = 25040.1 ± 0.1 Da) had one drug moleccule linked to it as
on experiments MS data was ac
cquired using a maXis 4G syste
em
infusio
well
(Bruke
er). The maXis system was optimized for each analysis using the
t
5 kDa) had 0 - 4 molecules of d
drug linked to the
(approxximately 48 – 55
actual sample and ion
n funnels values
s ranged from 30
00 – 400 Vpp, the
t
protein and was presen t as a dimer. Th
he F(ab’)2 sub un
nit (approximately
ospray voltage ra
anged from 1200 – 1800 V, dryiing gas was set at
electro
08 kDa) had 0 - 8 drug molecule
es attached to the protein subunit.
97 – 10
1.5 L/m
min and the sourrce temperature was set at 150 °C.
As the drug units were
e spread across multiple regionss of the mAb the
as
two
on
glycatio
modification
ns.
The
smalle
er
Fab
subunit
ed to calculate th
he DAR for ADCs
IdeS miiddle up sample could not be use
Resu
ults
but doe
es give informatio
on as to the loca
ation of the drug molecules within
BV.
When CESI is used as
s a NanoSpray in
nfusion device th
he sample is simp
ply
4
ed to the MS dete
ector at low nL/m
min flow rates . Desalted intact BV
B
pushe
was a
analyzed in this mode in order to
t confirm the molecular
m
weight of
this AD
DC and measure
e the drug to antiibody ratio (DAR
R) which is the drrug
loading on the antibod
dy and typically ranges
r
from 0 to
o 8. An example of
ata achieved is shown
s
in Figure 2 which gives an
a overview of the
the da
analyssis of BV by CES
SI-MS.
ure 2: Overview of brentuximab vedotin
v
structural characterization
n
Figu
ussing sheathless CE-MS.
C
(a) Native MS infusion for average DAR
dete
ermination and drrug loaded distrib
bution assessme
ent, (b) middle-up
p
and bottom-up
p analysis.
Figure
e 3: (a) MS specttra corresponding
g to Native MS N
NanoESI infusion
n
of midd
dle-up BV. (b) Ch
harge state deco
onvoluted mass sspectra of (1) LCdrug co
onjugated subuniit, (2) Fab subunits with the incorrporation of 0 to 4
drug m
molecules and Fcc/2 homodimers and (3) F(ab)'2 ssubunits with the
incorp oration of 0 to 8 drug molecules.
p2
CESI--MS has previou
usly been used in the analysis of
o tryptic digests of
5
Identificcation of these drrug containing pe
eptides was confirmed by MS/MS
S
om
mAbs so the next sett of experiments performed on BV were the botto
data an
nalysis which hig
ghlighted the pre
esence of severa
al diagnostic drug
up an
nalysis of a tryptic digested sam
mple to determine the location of
fragmen
nt ions. One dru
ug loaded peptid
de was located o
on the light chain
modification sites (PTMs), the location
n of the drug link
ked to the mAb as
of the m
mAb (GEC) and
d the other pepttides were prese
ent on the heavy
d sequence of BV.
B
well as the amino acid
chain.
The tryptic protocol had been
The THTCPPC
CPAPELLG pepttide actually had
d the potential of
o
ed from the cla
assical approach
h to improve the
e overall digestion
adapte
olecules and thesse two different p
peptides migrated
containiing two drug mo
which was affected by
y the presence of
o the drug molec
cules bound to the
t
at differrent times.
3
mple had migratted
mAb . When injected all the peptide peaks in the sam
pillary and been detected in less
s than 35 minute
es.
to the end of the cap
clusions
Conc
y and efficiency
y of the CESI-M
MS analysis 100
0%
Due tto the sensitivity
seque
ence coverage could be obtained for BV in a siingle injection with
w
A CESI -MS protocol forr structural chara
acterization of AD
DC molecules ha
as
the id
dentification of the
t
peptides ba
ased on their accurate
a
molecular
developed.
been d
weightt as well as sequ
uence data from MS/MS analysis
s. The presence of
propertiies of an ADC co
ould be confirmed including:-
Ussing CESI-MS in two differen
nt modes severral
the orrganic solvent in the sample prep
paration did not seem to effect the
t
separa
ation of the tryp
ptic peptides with
h the detection of small (3 amino
nditions.
 DAR ratio calculation using native con
acids)) to large peptide
es (63 amino acid
d long) possible.
d Fc/2 using na
ano-spray infusio
on
 Drug distribution on the F(ab’)2 and
ed ‘middle-up’ sa
ample using nativ
ve
CESI--MS analysis of an IdeS digeste
ncluding N-glyco
opeptides) were
e detected in the
t
Modified peptides (in
PREEQYN
analyssis, e.g. TKYP
297
STY
YR was observ
ved to have 11
glycofo
forms. Regarding drug-loaded-pe
eptides, 4 were detected
d
(Figure 4)
this w
was aided by the presence off organic solve
ent in the samp
ple
preparration which prevented
p
loss of these hydrrophobic modified
condittions.
 100%
% Sequence covverage of the AD
DC and identificcation of the dru
ug
locatio
on as well as loccation of other PT
TMs of the ADC.
 Chara
acterization of d
drug loaded pe
eptides from an
nalysis of MS/M
MS
spect ra.
peptid
des.
e would like to re
efer readers to th
he
For furtther information on this topic we
3
entific publication on which this ap
pplication note iss based .
full scie
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Figure
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Documen
nt number: RUO-MKT-02-4400
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