3
Thermo Scientific Poster Note
•
PN-64091-ASMS-EN-0614S
tibody-drug conjugates (ADCs),
s under their native conditions
ht
t
t
ith
Chip-based infusion conditions
Instrumentation
TriVersa NanoMate® (Advion USA)
FIGURE 2. Orbitrap native MS dete
A. High-resolution, native MS show
corresponding 23+ charge state. B.
weights of each trastuzumab glyco
Direct-Infusion Native MS Conditions
op mass spec rome er w
les were introduced using an
nization in the positive mode to
Range mass spectrometer.
,
system
Ionization voltage 9kV)
1.6-1.8
Gas pressure (psi)
0.3 – 0.6
Th ESI Chi R i t f
f 400
l t
itt
ith 5 i
A
re set according to the type of
r determination of molecular
rried out using Thermo
l
EMR MS l
l
i ht
e
p cons s s o an array o
nanoe ec rospray em ers w μm nner
diameters.
MS conditions
Instrumentation
Exactive Plus EMR Orbitrap MS system
us
, mo ecu ar we g
pm mass deviation range allowed
solution. The number of drug
complexes was also assessed
of the detected species.
(Figure 1)
Mass Range
350 – 20,000
Resolution
17,500 to 140,000, depending on spectral
complexity
ble technique for
s, reaching a high level of
intact monoclonal antibodies
Target value
3 x 10
6
Microscans
10
Maximal injection time (ms)
300
Insource CID energy (eV)
60 to150 eV manually tuned for optimized
of the molecules, glycoform
(dimer, trimer, tetramer), thus
haracterization tool. [2,3]
ectrometer with an extended
,
desolvation
S-lens level (%)
100 to 200, manually tuned for optimized
transmission and avoiding in-source
fragmentation
B
of high-mass ions for the
ures under native conditions.
uipped with a TriVersa
ce
Trapping gas pressure setting factor
4
Spectra average
Enabled (10 to 50 scans are averaged to
achieve S/N ratio > 100)
D t A l i
a a na ys s
Software
Protein Deconvolution software version 2.0
Sp2 and version 3.0
Deconvolution parameters
FIGURE 3. Orbitrap Native MS anal
(ADC). A. Native deconvoluted ma
to-antibody ratio (DAR). B. Raw m
distribution of ADC under native c
Number of iterations
4
Noise compensation
On
Minimum adjacent charges
1 to 3
15116
A
Results
High-Resolution Native MS Analysis of Intact Monoclonal Antibody Trastuzumab
DAR2
148527.8 ± 0.8 Da
Trastuzumab was analyzed on the Exactive Plus EMR MS with resolution set at both
17,500 and 35,000 (Figure 2A). The major glycoforms of the antibody are baseline
resolved at 17,5000 resolution. An interference peak can be resolved by using a higher
resolution, 35,000. Molecular weights of each trastuzumab glycoform were therefore
measured with good mass accuracy in the low ppm range as shown in Figure 2B The
DAR0
+2634.2 Da
+2635.4 D
145893.6 ± 2.2 Da
,
.
mass differences between species are +146 Da and +162 Da, corresponding to a fucose
or to the addition of multiple hexose units, respectively.
Orbitrap Native MS Analysis of a Monoclonal Antibody-Drug-Conjugate (ADC)
Brentuximab Vedotin
lonal antibody-drug conjugate
), the mAb/antigen complexes of
antibodies were introduced
R Orbitrap mass spectrometer.
146000
144000
15000
148000
B
The brentuximab vedotin mass spectrum was recorded at a resolution of 35,000 and in-
source CID voltage was set to 75 eV. Figure 3A shows the native deconvoluted mass
spectrum of the deglycosylated ADC. Populations with zero (grey), two (black), four (blue),
six (red), and eight (green) molecules loaded onto the antibody (payloads) were detected
with a mass difference between peaks corresponding to the addition of two payloads
re monitored by native MS. The
ing amounts (1:1, 1:2, 1:4, 1:8) of
osylated humanized IgG
ximab and trastuzumab) and nine
were mixed together prior to
(+2,634 Da). For each set of peaks, the drug-to-antibody ratio (DAR) can be determined.
Relative ratios of each detected compound were determined using MS peak intensities and
served to estimate the mean DAR (4.2), which is in agreement with hydrophobic
chromatography data (data not shown).
buffer exchanged against 150
deglycosylated brentuximab
AM-A were injected at 5 μM, and
the Exactive Plus EMR Orbitrap
