4
Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry
intactHumira_ETD15_LP_HCD_240k
#
11-27
RT:
6.11-11.23
AV:
17
NL:
T:
FTMS+ pESIFullms22800.00@etd15.00 [200.00-4000.00]
500
1000
1500
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
1702.5
1703.0
1703.5
1704.0
17
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
R
R=101019
R=107362
R=97925
R=112194
R=103665
R=82023
y
91
7+
z = 6
c
125
8+
/c
·
125
8+
S Pump Plus was coupled to an
t was equipped with ETD (Figure 3).
Scientific™ BioBasic™ C4 column (150
.1 % FA, 2 % ACN in H
2
O, solvent B:
as 7 min 20–40 % B, 3 min 40–80 % B
in Deconvolution and ProSight software
e size of intact anti-bodies (~150 kDa)
has been significantly improved over the
ke mAbs show only very short transient
ross section. Thus, the method of choice
e shortest transient duration (48 ms)
igure 4).
FIGURE 8:
Summarized sequenc
using fragmentation techniques
conditions: trapping under h
glycosylation site.
FIGURE 7:
(A) CID spectrum an
antibody. (B) Zoom in into the
HUMIRA
showing the need for hi
(A)
(C)
(B)
CID
ETD
T:
2740
2760
2780
2800
2820
2840
2860
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
2794.98103
2848.70886
2743.24976
2800.91032
2854.81639
2749.10120
2767.72395
2820.90307
2787.42772
2840.04424
f intact HUMIRA. The insert shows a
harge states z=52,53,54. (B) Spectrum
+53
+54
+52
ex
+Hex
(C)
(D)
23380
23390
23400
23410
23420
23430
m/z
0
20
40
60
80
100
0
20
40
60
80
100
Relative Abundance
23407.63635
23421.61756
23433.66736
23393.62879
23435.63611
23407.58564
23419.59961
23428.60343
23390.64710
FIGURE 5
. (A) Full MS spectrum of intact light chain of HUMIRA. (B)
Zoom into +18 charge state of intact light chain. (C) Simulation of isotope
pattern of +18 charge state. (D) Isotope pattern of intact light chain after
deconvolution. (E) Monoisotopic mass (M) of the measured light chain of
HUMIRA obtained after deconvolution with Xtract.
(A)
(B)
z=18
measured
simulated
z=18
z=17
z=13
z=11
z=10
z=14
z=19
z=20
z=21
z=22
z=16
z=15
z=12
Isotope pattern after
deconvolution with Xtract
Monoisotopic mass (M) of the
intact light chain
after
deconvolution with Xtract
Δ
m=2.4 ppm
(E)
rap Elite hybrid ion trap-Orbitrap mass
source.
Humira_CID_59_120210173005
#
1
RT:
355.98
AV:
1
NL:
6.11E3
T:
FTMS+ pESIFullms21185.00@cid50.00 [325.00-4000.00]
400
600
800
1
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
551.31708
z=1
782.33112
z=?
971.26849
z=?
683.26221
z=2
452.24893
z=1
FIGURE 6:
HUMIRA heavy chain acq
averaged. Deconvoluted mass: Mr
demonstrate isotopic resolution of that
obtained after deconvolution using Xtra
HUMIRA_heavy_av_SIM
#
1
RT:
1556.88
AV:
1
NL:
3.30E3
T:
FTMS+ pESISIMms [1180.00-1190.00]
1181
1182
1183
1184
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
1183.9644
R=120204
1182.5933
R=116404
1183.5918
R=122204
1182.2444
R=121404
1181.6860
R=117204
1180.9857
R=133304
1300.5
1301.0
1301.5
1302.0
1302.5
m/z
0
20
40
60
80
100
0
20
40
60
80
100
Relative Abundance
1301.43152
1301.26538
1301.59814
1301.70923
1301.43034
1301.20754
1301.59741
1301.70878
1301.09615
1301.82014
1300.98475
1301.98718
NL:
3.84E5
FS_tripleSIM_20uS_1#19 RT:
7.66 AV: 1 T: FTMS + p ESI
SIM ms [1290.00-1320.00]
NL:
2.13E3
c
1027
h
1606
n
282
o
332
s
6
+H:
C
1027
H
1624
N
282
O
332
S
6
p (gss, s /p:40) Chrg 18
R: 120000 Res .Pwr . @FWHM
Δ
=0.9ppm
R=124.100
New
itrap
lyzer
C-trap
TransferMultipole
Reagent IonSource
Reagent 1
Heated Inlet
Reagent 2
Heated Inlet
HCDCollisionCell
uadrupoleMassFilter
3000
3500
/z
3
962.591
3085.98807
3151.64648
3220.13151
3291.67370
3366.46848
3444.75592
3526.72930
3703.00780
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
1304.56287
1235.95410
1381.19873
1174.17102
1467.49915
1565.29358
1118.31189
1956.39819
1677.03210
2134.19409
1806.14465
1067.56909
2347.60156
1021.25623
2610.80981
