high resolution mass spectrometry of antibody drug...
TRANSCRIPT
High Resolution Mass Spectrometry of Antibody Drug Conjugates Using
the Orbitrap Mass Analyzer
INTRODUCTION
The complexity of modern therapeutic proteins presents
a great analytical challenge. Most often a whole set of
Kai Scheffler1, Eugen Damoc2, Aaron Bailey3, and Jonathan Josephs3
Thermo Fisher Scientific, 1Dreieich, Germany, 2Bremen, Germany, 3San Jose, USA
the Orbitrap Mass Analyzer
RESULTS
There are many factors that play a key role in the analysis
of proteins, some of which relate to sample preparation
Figure 7: A) The Sliding Window feature in BioPharma
Finder provides a composite spectrum based on stepwise
deconvolution results across the full RT range. B) Low
abundant species are represented more accurately and are
less prone to be underrepresented which is in particular useful
Figure 4: LC-MS analysis of intact Brentuximab vedotin
under denaturing conditions: (A) Reversed phase
chromatogram and the resulting averaged MS spectrum
provides a complex mixture of charge state envelopes as well
as vcMMAE-specific reporter fragment ion at m/z 718. (B) Dataa great analytical challenge. Most often a whole set of
analytical techniques is used to characterize these
proteins. With mass spectrometry alone several
complementary methods are required to analyze protein
drugs on the intact protein and on the peptide levels.
Native mass spectrometry on the intact protein level
allows also for the analysis of molecules which rely on
non-covalent interactions to preserve critical structural
features, such as antibody-drug conjugates (ADC). In
addition the use of 100% aqueous buffers in native MS
analysis produces lower charge states detected at
higher m/z values compared to analysis under
denaturing condition and thus improves mass separation
of heterogeneous mixtures [1]. Recent technical
advance-ments on the benchtop Orbitrap mass
spectrometry platform offer now complete
of proteins, some of which relate to sample preparation
(buffers, solvents, additives) while others relate to the
mass spectrometer’s source conditions as well as the
physical environment inside the instrument [2,3]. The Q
Exactive Plus and Q Exactive HF mass spectrometers
(Figure 2A) have previously been introduced with the
Protein Mode option, which was one of many
advancements for intact protein analysis on the Orbitrap
platform. For these two instruments an automated HCD
gas control was introduced by using an electronically
controlled valve for nitrogen gas in the HCD cell for easier
optimization of experimental conditions required for
different types of analyses wished to run on a single
platform.
In Normal Mode pressure settings are factory-optimized,
[ [ [ [ [
0
20
40
60
80
100
Relativ
eIntens
ity
8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8RT(min)
100
mAb
less prone to be underrepresented which is in particular useful
for low abundant, low and high drug loads of ADCs.
A
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6 7 8 9 10 11 12
Time (min)
0
20
40
60
80
100
Reversed phase LC-MS of Brentuximab vedotin yields ~9 masses of denatured species
500 1000 1500 2000 2500 3000 3500 4000 4500
m/z
0
20
40
60
80
100
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Reversed phase LC-MS
50
60
70
80
90
100
25041.47
(32.5 ppm)
2 x G0F
125678.81
(16.0 ppm)
1 x G0F
76675.44
(1.0 ppm)
2 x G0F
103272.01
(5.6 ppm)1 x G0F
54269.74
2 x G0F
148086.47
Rela
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as vcMMAE-specific reporter fragment ion at m/z 718. (B) Data
analysis with ReSpect deconvolution and Sliding Window
integration show roughly six covalently-structured forms of
unraveled cysteine-linked ADC.
A
B
Full MS spectrum
718.5121
762.5026
spectrometry platform offer now complete
characterization of the complex conjugates composed of
small molecule drugs attached to antibodies on a single
instrument platform. In this presentation relevant
workflows covering chromato-graphy, mass spectrometry
and data analysis for ADC characterization are laid out
and data obtained from two different types of ADCs are
presented.
In Normal Mode pressure settings are factory-optimized,
suitable for most analyses and ions are cooled in the C-
trap (Fig. 2B). In Protein Mode the default trapping gas
pressure setting is 0.2 and that corresponds to a ∆HV
which is 5x lower than in Normal Mode. Additionally, ions
are transferred and cooled in the HCD cell and thus have a
longer flight path (Fig. 2C). The new High Mass Range
(HMR) Mode that is especially required for the analysis of
proteins under native conditions when samples are kept in
aqueous buffers with no organic solvents involved at near
neutral pH. For HMR mode the default trapping gas
pressure setting is the same as in Normal Mode and can
even be slightly increased for even improved trapping of
certain species such as protein complexes and
heterogeneous large proteins (e.g. antibody drug
conjugates). The trapping path in HMR mode is the same CONCLUSIONS
Figure 1: Operating modes for the three major
BioPharma workflows: Normal Mode, Protein Mode and
HMR mode Normal Mode
MAbPac RP column
for denatured samples
Peptide Mapping
components
integrated 0
20
40
60
80
100
Relativ
eIntens
ity
8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8RT(min)
0
20
40
60
80
Rela
tiveIntens
ity
8.0 8.2 8.4 8.6 8.8 9.0 9.29.4 9.6 9.8RT(min)
mAb + 2 drugs
mAb + 4 drugs
-100
-80
-60
-40
-20
0
20
40
60
80
100147082.012 148038.597
148996.413
146125.059
149953.100
148262.672149219.126
147306.160145166.647
146347.724 150910.692150179.154
145392.644 148396.374147434.934 151867.722146481.051 151133.091149347.719 152828.100150299.831 153321.699 154341.000
147081.809 148039.009
146124.209 148996.509
147305.809 148262.209149954.309
145169.709 146345.509 149220.509
147438.509 150916.709150173.309148485.909146481.309 149381.309145391.209 152463.109150529.609 151508.509
Mass
0
1
2 34
5
6
78 9
151865
152824153773
152000 153000 154000
7 8 9
BFigure 5: LC-MS analysis of intact Brentuximab vedotin
under native conditions:
A) Size exclusion chromatogram and mass spectrum
obtained from averaging 2 min chromatographic time which
includes all DAR forms (DAR 0-8). B) ReSpect deconvolution
result using the Sliding Window feature. A pattern of lower
abundance species were detected corresponding to a low
abundance loss of 762 Da from each glycoform at each DAR.
Based on the individual deconvolved abundances of the
G0F/G0F glycoform, an average DAR value of 4.07 was
calculated which is consistent with previous reports [4].
0
10
20
30
40
50(5.6 ppm)54269.74
(4.6 ppm)
148086.47
(28.1 ppm)2 x G0F
105907.20
(23.5 ppm)
20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 120000 130000 140000 150000
Mass
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24279.51
(-761.96 Da)
MATERIALS AND METHODS
Samples:
Samples used in this study are Trastuzumab (Roche,
UK; tradename Herceptin),Trastuzumab emtansine
conjugates). The trapping path in HMR mode is the same
as in Protein Mode with ion cooling taking place in the HCD
cell. And also, mass detection is enabled ranging up to m/z
8000 compared to m/z 6000 in the two other modes.
CONCLUSIONS
• The BioPharma option extends the capabilities of the
Q Exactive Plus and Q Exactive HF mass spectro-
meters allowing for mass detection up to m/z 8000 in
the new High Mass Range (HMR) Mode
• This new operating mode extends the instrument’s
capabilities to cover all three major workflows for
BioPharma characterization and allows for intact
mass analysis of larger proteins such as antibodies
and antibody drug conjugates under native
conditions.
• Native LC/MS intact mass analysis of Brentuximab
SMART digest
MAbPac RP
column
Intact Analysis
native & denatured
Subunit Analysis
FabRICATOR®
(IdeS) digest
and/or
reduction
Acclaim VANQUISH C18 column
MAbPac SEC column
for native samples
Q Exactive Biopharma
• Protein Mode
• Enhanced Resolution Mode
• High Mass Range Mode ( m/z ≤8000)
Vanquish UHPLC
Protein Mode
HMR Mode
©2016 Genovis AB
DAR2
DAR4
DAR6
2 x G0F
153355.40
A
B
5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bund
ance
G0F/G0FDAR
Mass
Accuracy
(ppm)
Relative
Abundance
DAR0 11.7 6.77
DAR2 23.1 69.23
DAR4 22.4 100.00
DAR6 40.5 69.75
DAR8 17.6 10.61
Average
Drug-to-Antibody
Ratio (DAR)
4.07
Brentuximab
vedotin
Buffer salts
50 mM
NH4OAc
isocratic
elution
0 2 4 6 8 10
Time (min)
0
20
40
60
80
100
Rela
tive A
bundance
Size Exclusion LC-MS
Figure 2: A) Schematic of the Q Exactive Plus/HF mass
spectrometers and differences in the trapping path in
the three different operating modes available: B) Normal
Mode, C) Protein Mode and D) HMR Mode. E) Illustration of
improvement in signal intensity for +17 charge state of a
mAb light chain comparing Protein Mode and Normal Mode.
Aregion where UK; tradename Herceptin),Trastuzumab emtansine
(Roche UK; tradename Kadcyla) and Brentuximab
vedotin (Seattle Genetics, US; tradename Adcetris).
All antifbody samples were prepared for intact mass
analysis by dissolving the dried substances according to
the manufacturer's instructions. For denaturing LC-MS
intact analysis 1 µg of protein samples were separated
using a 10 min gradient of 10-90% ACN in H2O and
0.1% formic acid (Thermo MAb-Pac RP; flow rate 250
µL/min). For native LC-MS intact analysis 10 µg of
sample was desalted online using size exclusion
chromatography with 50 mM NH4OAc using isocratic
elution at a flow rate of 300 µL/min and directly infused
into the mass spectrometer via electrospray ionization.Native MS intact protein analysis allows direct observation
of molecules which rely on non-covalent interactions to
preserve critical structural features, such as maintaining
• Native LC/MS intact mass analysis of Brentuximab
vedotin resulted in detection of intact ADC forms,
DAR0-8. ReSpect deconvolution and Sliding
Window integration showed an average DAR of
4.07, consistent with previous studies.
• Native LC/MS intact mass analysis of Trastuzumab
emtansine resulted in detection of intact ADC forms,
DAR0-8. ReSpect deconvolution and Sliding
Window integration showed an average DAR of
3.71, consistent with previous studies.
• The sliding window feature in BioPharma Finder
software is essential for accurate determination of
0
20
40
60
80
100
Re
lative
In
ten
sity
148000 149000 150000 151000 152000 153000 154000 155000 156000 157000 158000 159000 160000
Mass
147000
DAR0DAR8
2 x G0F
148086.53
2 x G0F
150720.06
153355.40
,
2 x G0F
155987.81
2 x G0F
158632.20
40
60
80
100
49+
53+52+
51+ 50+48+
60
80
100
Denatured
Figure 6: A) Mass spectra obtained for Trastuzumab
emtansine under denaturing and native conditions. B)
Calculation of DAR ratio based on C) the deconvoluted
spectra obtained using the Sliding Window feature in
BioPharma Finder software.
A
region where
source CID is
applied
Q Exactive Plus:
Standard Orbitrap mass
analyzer
Q Exactive HF:
Ultra High Field Orbitrap
mass analyzer
Trapping in Normal Mode:
trapping gas pressure setting 1
(fixed on setting 1)
Trapping in Protein Mode:
trapping gas pressure default setting 0.2
(range 0.2-1)
Trapping in HMR Mode:
trapping gas pressure default setting 1
(range 1.0-1.5)
1355.5 1356.0 1356.5 1357.0
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
x 5.1
Protein Mode
Normal Mode
B
C
D
E
N2
N2
N2
Chromatography:
A Vanquish UHPLC system was used for all LC/MS
experiments. For native analysis, 50 mM ammonium
acetate buffer (99.99%, Sigma Aldrich) was used. On a
size exclusion column. Reversed phase chromatography
was performed with water/0.1% formic acid and
acetonitrile/0.1% formic acid on a MAbPac RP 2.1x50
mm column.
Mass Spectrometry:
Mass spectrometers used in this study were the
commercially available Thermo ScientificTM Q ExactiveTM
Plus and Q ExactiveTM HF equipped with BioPharma
Option. The instruments were operated under Tune 2.8
instrument control software in HMR mode which allows
preserve critical structural features, such as maintaining
interchain associations which hold together cysteine-linked
ADCs. The use of 100% aqueous physiological pH buffers
in native MS analysis produces a fewer number and lower
charge states (increased m/z) compared to denaturing
conditions (Figure 3) and improves mass separation which
is beneficial in particular for heterogeneous mixtures. We
demonstrate this phenomenon using the cysteine-linked
ADC Brentuximab vedotin (Figures 4 and 5) and the
Asparagine-linked ADC Trastuzumab emtansine (Figure 6).
Based on mass spectra acquired under native condition
DAR values for both ADCs were determined that are well in
agreement with previously reported numbers [4,5].
software is essential for accurate determination of
DAR ratios.
REFERENCES
[1] Rosati S. et al. In-depth qualitative and quantitative analysis
of composite glycosylation profiles and other micro-heterogeneity
on intact monoclonal antibodies by high-resolution native mass
spectrometry using a modified Orbitrap. mAbs. 2013;5(6):917-
924. doi:10.4161/mabs.26282.
[2] Fenn J.B. et al. Electrospray ionization for mass spectrometry
of large biomolecules. Science. 1989;246(4926):64-71.
[3] Fenn J..B, Electrospray wings for molecular elephants (Nobel
Lecture). Angew. Chem. Int. Ed. 2003. 42:3871–3894.
[4] Dabaene F. et al. Innovative native MS methodologies for
antibody drug conjugate characterization: High resolution native
100 ADC
Size Exclusion Chromatography
G0F/G1F
DAR
Mass
Accuracy
(ppm)
Relative
Abundance
DAR0 6.49 9.19
DAR1 21.69 34.26
DAR2 0.05 59.03
DAR3 6.81 100.00
DAR4 5.17 91.16
DAR5 6.69 67.42
Average
Drug-to-Antibody
Ratio (DAR)
3.71
2900 2950 3000 3050 31000
20
40
2500 3500 4500 5500 6500 7500m/z
0
20
40
60
80
100
0
20
40
Rela
tive
Ab
un
dan
ce
5950 6000 6050 6100 6150m/z
0
20
40
60
80
10026+
25+
Native
200 m/z
B
buffer salts
*
instrument control software in HMR mode which allows
for detection of a mass range up to 8,000 m/z. The S-
lens RF level was allowed to be increased to a setting of
200 in HMR mode and set to that level for all
experiments shown here. Resolution settings between
15,000 and 70,000 were applied.
Data Analysis:
Data analysis was performed with Thermo ScientificTM
BioPharma FinderTM 2.0 software using the ReSpectTM
algorithm and Sliding Window feature. DAR ratios were
calculated automatically based on deconvolution species
that were identified using the publicly-available FASTA
sequences for Brentuximab vedotin and Trastuzumab
emtansine, a mass tolerance of 50 ppm, and a static
antibody drug conjugate characterization: High resolution native
MS and IM-MS for average DAR and DAR distribution
assessment. Anal Chem. 2014. 86(21):10674-83.
[5] Lazar A.C. et al. Analysis of the composition of immunocon-
jugates using size-exclusion chromatography coupled to mass
spectrometry. Rapid Commun. Mass Spectrom 2005. 19:1806–
1814.
TRADEMARKS/LICENSING
© 2016 Thermo Fisher Scientific Inc. All rights reserved.
FabRICATOR is a trademark of Genovis AB. All other trademarks
are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these
Figure 3. Trastuzumab analyzed under native (A) and
denaturing (B) conditions resulting in highly similar
deconvolution results (C).
0
20
40
60
80
1005923.3
5695.6
6169.9
5484.6
6438.0
3080 3100m/z
3085.6
5900 5950m/z
5923.3
3000 4000 5000 6000 7000
m/z
3000 4000 5000 6000 70000
20
40
60
80
100
Rela
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3085.6
2962.23291.2
2904.13366.0
2848.33444.2
3526.13612.1
3797.3
m/z
zoom
zoom
z=+25
z=+48
native
denatured
60 Th
30 Th
R=3462
R=4359
Deconvolution
4.7ppm
A
B
0 2 4 6 8 10
Time (min)
0
50
5600 5800 6000 6200 6400 66000
20
40
60
80
100
25+
zoom
5950 6000 6050 6100 6150 62000
20
40
60
80
100
R=70,000
DAR5 6.69 67.42
DAR6 15.20 40.46
DAR7 6.28 24.28
DAR8 3.78 3.84
C
buffer salts
modification of Gln>Pyro-Glu for the heavy chain. products in any manner that might infringe the intellectual
property rights of others.
-100
-80
-60
-40
-20
0
20
40
60
80
100148055.503
148217.603
148380.303
147909.303147848.203 148542.203
148056.888
148218.688
148379.888
148540.188147908.488147850.188
-2.9 ppm
5.0 ppm
1.2 ppm
-2 ppm
4.1 ppm
native
denatured
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5950 6000 6050 6100 6150 6200m/z
0
20
40
60
80
100
148000 149000 150000 151000 152000 153000 154000 155000 156000
Mass
D* *
**
** ** *