2
Structure Characterization of Intact Monoclonal Antibody Using an Orbitrap Tribrid Mass Spectrometer
Waters_mAb_100C
#
193-233
RT:
8.54-9.49
AV:
41
NL:
2.38E6
T:
FTMS+pESIsid=80.00 Fullms [1500.00-4000.00]
2200
2300
2400
2500
260
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
2604
2559.38
2556.53
2515.96
2513.31
2474.17
2471.36
2436.20
2319.52 2358.98
2283.56
waters_mab_rosa_285c
#
168-199
RT:
8.61-9.59
AV:
32
NL:
4.70E4
T:
FTMS+pESIsid=80.00 Fullms [1500.00-4000.00]
2200
2300
2400
2500
260
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
260
2559.39
2516.02
2513.17
2473.81
2433.54
2431.03
2391.89
2353.78
2321.92
2215.98
waters_mab_225c
#
186-223
RT:
8.82-9.74
AV:
38
NL:
1.02E6
T:
FTMS+pESIsid=80.00 Fullms [1500.00-4000.00]
2200
2300
2400
2500
26
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
260
2559.36
2556.54
2515.98
2513.20
2474.06
2433.55
2394.38
2354.23
2215.49
2297.37
waters_mab_225c
#
181-216
RT:
8.67-9.53
AV:
36
NL:
1.07E6
T:
FTMS+pESIsid=80.00 Fullms [1500.00-4000.00]
2200
2300
2400
2500
260
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
2604.
2559.36
2556.54
2515.98
2513.20
2474.06
2433.55
2394.38
2354.22
2215.49
2297.36
Overview
Purpose:
Structural characterization of intact mAb method coupling capillary LC with
new generation Orbitrap mass spectrometer.
Methods:
An intact monoclonal antibody (mAb) molecule mass was measured by
Thermo Scientific™ Orbitrap™ Fusion™ MS. Mass spectra of the intact protein were
analyzed by Thermo Scientific™ Protein
Deconvolution
™ software. Both Thermo
Scientific™ Orbitrap Elite™ MS and Orbitrap Fusion MS systems were used for
mAb
top-
down analysis. ETD and HCD MS/MS data were analyzed by Thermo Scientific™
ProSightPC
™ 3.0 software.
Results:
mAb was accurately measured with low ppm mass error and multiple
glycoforms were accurately identified. About 70% backbone sequence coverage for
light chain and 20% for heavy chain was achieved using top-down analysis.
Introduction
Monoclonal antibodies (mAbs) are increasingly being developed for the detection and
treatment of diseases. The quality of the antibodies is crucial for both safety and
efficacy. Due to the heterogeneity of mAb products, thorough characterization is very
challenging. Among the analytical tools used for the analysis of therapeutic mAb, high
resolution accurate mass (HRAM) mass spectrometry (MS) has become increasingly
important for the determination and quantification of the various glycoforms,
confirmation of amino acid sequence and post-translational modifications, and
detection of minor impurities and high order structure. In this study, new methods
coupling capillary LC with a new generation Orbitrap mass spectrometer were
developed and optimized for structural characterization of an intact mAb.
Methods
Sample Preparation
A monocolonal antibody (mAb) (NIST and purchased from Waters) were diluted with
0.1% FA in water for intact protein analysis. The reduced monoclonal antibody was
prepared in 6 M Guanidine , 100 mM DTT, and heated at 60
C for 30 min prior to top-
down analysis.
Liquid Chromatography
Thermo Scientific™
Dionex
™
ProSwift
™ RP
-10R monolithic columns (200 µm x 25 cm
or 1 mm x 5 cm) were used for desalting prior to intact mass measurement. 0.1%
formic acid in H
2
O (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) were
used as the LC solvents. The column was heated to 60 °C and the flow rate was 800
nL/min. After injection of 100 ng mAb, an 8 min gradient was used to elute mAbs from
the 200 µm x 25 cm column (0.0min, 10%B; 1.0min, 90%). For the 1 mm x 5 cm
column, the flow rate was 80 µL/min using the same gradient.
A Thermo Scientific™
Dionex
™
PepSwift
™ 500 µm x 5 cm column was used for heavy
and light chain separation. Flow rate was 10 µL/min. 2 µg of reduced antibody was
injected, a 3 to 5 min 5%B to 25%B and 5 min to 11 min 25%B to 40% gradient was
used to separate the heavy and light chain.
Mass Spectrometry
The samples were directly injected from the capillary LC to either an Orbitrap Fusion or
Orbitrap Elite mass spectrometer. The Orbitrap Fusion MS was used for both antibody
intact mass measurement and top-down analysis. An Orbitrap Elite MS was also used
for top-down analysis. For the intact antibody, various parameters including ion transfer
tube temperature, in-source activation energy, s-lens values, AGC target and others
were optimized. For top-down analysis, the ETD and HCD were performed in separate
runs to achieve maximum combined sequence coverage. For ETD MS/MS, the
activation time was 5 to 50 msec while for HCD MS/MS, normalized collision energy
level was 10%-25 %. For the source conditions, the spray voltage was set to 4kV, the
sheath gas flow rate was set at 8, the capillary temperature was 225
°
C, the S-lens
level was set at 55 and in-source CID was set at 45 eV. Resolution was set at 120,000
and AGC target was set to 1E6 for top-down MS/MS while maximum IT was set at 250
ms
.
Data Analysis
The MS full spectra for intact mAb were analyzed using Protein Deconvolution software
using ReSpect
TM
or Xtract deconvolution. The top-down ETD and HCD spectra were
analyzed using ProSightPC 3.0 software.
FIGURE 1. Experiment par
a) Example mAb analysis
Results
Intact mAb Mass Measure
Several mass spectrometer e
source fragmentation energy,
probe were optimized for inta
cell pressure (Figure 1a), sou
target 5e4 and 1e5 were use
significant impact on the inta
the capillary temperature pla
shows the mass spectra for t
100 ºC to 285 ºC As the tem
envelope for the intact mAb
of magnitude sensitivity drop.
highest temperature. 225 ºC
and the sensitivity as a opera
X:\2013\...\073013_watermAb_100SID_5e4
073013_watermAb_100SID_5e4
#
431-475
RT:
11.56-12.51
AV:
45
NL:
T:
FTMS+ pNSIsid=100.00 Fullms [1500.00-4000.00]
1500
2000
2500
3000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
2800.7183
z=?
2910.4874
z=?
2698.9671
z=?
2968.6
z=?
3029
z
2604.2826
z=?
30
2515.9953
z=?
2391.6792
z=?
2217.9855
z=?
1861.9974
z=?
RT:
5.96 - 17.96
6
7
8
9
10
11
12
13
14
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
11.80
11.91
11.97
12.06
12.36
12.54
12.91
11.58
13.37
10.97
7.00 7.31
6.70
7.87 8.49
1
10.15
4mTorr
b) Capillary temperature op
100 ºC
285 ºC
225 ºC
150 ºC
