4
Monoclonal Antibody and Related Product Characterization under Native Conditions using a Benchtop Exactive Plus EMR MS
NanoMate® (Advion USA)
Orbitrap Native MS Analysis o
The native mass spectrum of m
35,000 with the in-source CID vo
4-fold excess of JAM-A (20 μM) i
FIGURE 2. Orbitrap native MS detection of intact monoclonal antibody trastuzumab.
A. High-resolution, native MS showing complete mass spectrum and zoom of
corresponding 23+ charge state. B. Deconvoluted spectrum showing molecular
weights of each trastuzumab glycoform with low ppm mass accuracy.
,
itt
ith 5 i
A
detected: the intact free mAb (M
2.0 Da, blue) and 1:2 (MW 1983
thus confirmed that two JAM-A
the main G0F/G0F glycoforms.
intensities and proportions of m
b d t b 37% d 30%
rospray em ers w μm nner
Plus EMR Orbitrap MS system
o serve o e
an
, r
shows the corresponding mass
native conditions.
Figure 4. Orbitrap native MS d
A Deconvoluted mass spectr
1)
,000
o 140,000, depending on spectral
ity
.
B. Charge state distribution in
eV manually tuned for optimized
A
,
tion
00, manually tuned for optimized
sion and avoiding in-source
tation
B
(10 to 50 scans are averaged to
S/N ratio > 100)
Deconvolution software version 2.0
version 3.0
FIGURE 3. Orbitrap Native MS analysis of a monoclonal Antibody-Drug-Conjugate
(ADC). A. Native deconvoluted mass spectrum showing the determination of drug-
to-antibody ratio (DAR). B. Raw mass spectrum with the entire charge state
distribution of ADC under native conditions.
DAR4
DAR6
151163.2 ± 0.9 Da
= 3.8
DAR
=
8
8
0
n
DAR
A
nA
DAR
=
4.2
A
clonal Antibody Trastuzumab
DAR2
148527.8 ± 0.8 Da
153797.5 ± 0.7 Da
0
n
DAR
B
MS with resolution set at both
of the antibody are baseline
n be resolved by using a higher
mab glycoform were therefore
ge as shown in Figure 2B The
DAR8
DAR0
+2634.2 Da
+2635.4 Da
+2634.3 Da
+2635.1 Da
145893.6 ± 2.2 Da
156432.6 ± 1.8 Da
DAR8
,
.
62 Da, corresponding to a fucose
.
ody-Drug-Conjugate (ADC)
146000
144000
Mass (Da)
150000
148000
154000
152000
158000
156000
B
at a resolution of 35,000 and in-
the native deconvoluted mass
zero (grey), two (black), four (blue),
antibody (payloads) were detected
o the addition of two payloads
dy ratio (DAR) can be determined.
mined using MS peak intensities and
eement with hydrophobic
