Download Enabling Mass Spectrometric Analysis of Intact Proteins in Native

routine previously used was modified and adapted to ensure high mass accuracy across the full mass
range.
With the data collected on different types of samples and presented in this study we demonstrate the
successful analysis after implementation of the High Mass Range (HMR) mode, successful desolvation
and optimized critical hardware operation settings. This makes the instrument an ideal platform to
cover the three major workflows in BioPharma: intact mass analysis under denaturing and under native
conditions in HMR mode, subunit analysis (reduced mAb and or IdeS digested mAb) in protein mode
and peptide mapping in standard mode (see Figure 1).
Enabling Mass Spectrometric Analysis
Normal Mode
of Intact Proteins in Native Conditions
on A Hybrid Quadrupole-Orbitrap
Mass Spectrometer
fo
Peptide Mapping
Poster Note 64 8 04
Figure 1: Operating modes for the three major BioPharma workflows: Normal Mode,
Protein Mode and HMR mode
MAbPAC RP column
for denatured samples
SMART digest
MAbPAC SEC column
for native samples
Acclaim RP C18 column
HMR Mode
Intact Analysis
native & denatured
Kai Scheffler1, Eugen Damoc2, Aaron O. Bailey3, and Jonathan Josephs3
Thermo Fisher Scientific, 1Im Steingrund 4-6, Dreieich, Germany, 2Hanna-Kunath-Str. 14, Bremen, Germany,
Vanquish UHPLC
3
355 River Oaks Parkway, San Jose, CA, USA
B
C
D
Enabling Mass Spectrometric Analysis of Intact Protein
©2016 Genovis AB
Kai
Scheffler1,
Eugen
Damoc2,
Aaron O.
Bailey3,
INTRODUCTION
Analysis of proteins in native-like conditions free of organic solvents can allow proteins to preserve
non-covalent interactions and retain high degrees of folding. This effect has analytical benefits: greater
protein folding leads to reduced charge states leading to increased mass separation and increased
signal at higher m/z. This strategy has been utilized for analysis of antibodies and antibody drug
conjugates present in highly complex mixtures of different antibody/drug combinations [1].
Requirements for performing native MS on antibody samples include scanning towards 8000 m/z and
increased transmission optimization for large compounds. Such features are not compatible with
current commercially available quadrupole-Orbitrap instruments. Here we show results obtained after
successful implementation of modifications aimed at adding the capability to perform native MS
analysis without compromising performance of normal operation modes.
and
FabRICATOR®
(IdeS) digest
and/or
reduction
MAbPAC RP
column
Q Exactive BioPharma
• Protein Mode
• Enhanced Resolution Mode
• High Mass Range Mode ( m/z ≤8000)
Subunit Analysis
Jonathan
Josephs3,
Protein Mode
F
c
Thermo Fisher Scientific,
w
MATERIALS AND METHODS
RESULTS
Samples:
Samples
usedmany
in this
study that
are play
ammonium
hexafluorophosphate
Fishersome
Scientific,
part relate
numberto
There are
factors
a key role
in the analysis(AHFP,
of proteins,
of which
A0368370),
Trastuzumab(buffers,
(tradenamesolvents,
Herceptin, Roche,
UK).
sample and
preparation
additives)
while others relate to the mass
spectrometer’s source conditions as well as the physical environment inside the instrument
Chromatography:
A [2,3].
ThermoThe
Scientific™
Vanquish™
UHPLC
was usedHF
for all
LC/MS
experiments. For(Figure
native analysis,
50
Q Exactive
Plus
and system
Q Exactive
mass
spectrometers
2A) have
mM
ammonium been
acetateintroduced
buffer (99.99%,
used.option,
Reversedwhich
phase was
chromatography
was
previously
withSigma
the Aldrich)
Protein was
Mode
one of many
performed with water/0.1% formic acid and acetonitrile/0.1% formic acid on a Thermo Scientific™ MAbPac™ RP
advancements for intact protein analysis on the Orbitrap platform. For these two instruments
2.1x50 mm column.
an automated HCD gas control was introduced by using an electronically controlled valve for
re
T
a
a
H
e
in
F
to
h
is
d
T
a
o
a
1
L
Enabling Mass Spectrometric Analysis of Intact Protein
The analysis of intact proteins under native conditions is more challenging than under denaturing
conditions since the buffers used don’t contain any organic solvents. Performing electrospray from
aqueous buffer solutions produces larger solvent droplets size and desolvation is less efficient.
Moreover, for large proteins such as intact antibodies the required mass range for analysis under
1 range of more than 6000 m/z due
2 to a smaller number of accepted
native conditions requires a mass
charges. The increase of the upper mass range on the mass spectrometer was achieved by
implementing instrument control software changes. The analysis of molecules across the full mass
range including the detection of proteins under native conditions required the use of optimized
parameter settings to ensure efficient desolvation in the front region of the instrument, the efficient
transfer via multipoles, efficient trapping in the C-trap/HCD region and the sensitive injection and
detection in the Orbitrap mass analyzer. Critical parameters are the optimization of in-source
Analysis
of proteins
in native-like
conditions
free of
solventsprocess.
can allow
proteins
preserve
fragmentation
that strongly
influences
the support
of organic
the desolvation
Also,
for theto
transmission
non-covalent
interactions
and retain
of folding.
This effectto
hasensure
analytical
benefits:
efficiency specific
voltages
have high
beendegrees
evaluated
and optimized
robust
and greater
sensitive
protein
folding in
leads
to reduced
statesperforming
leading toanalyses
increased
mass
separation
and experiments
increased
conditions,
performance
the higher
mass charge
range when
under
native
signal
at
higher
m/z.
This
strategy
has
been
utilized
for
analysis
of
antibodies
and
antibody
drug
that have not been possible so far on this type of mass spectrometer. Also, the standard calibration
conjugates
presentused
in was
highly
complex
mixtures toofensure
different
antibody/drug
combinations
[1].
routine previously
modified
and adapted
high mass
accuracy across
the full mass
Requirements
for
performing
native
MS
on
antibody
samples
include
scanning
towards
8000
m/z
and
range.
increased
transmission
optimization
for
large
compounds.
Such
features
are
not
compatible
with
With the data collected on different types of samples and presented in this study we demonstrate the
current
commercially
available
quadrupole-Orbitrap
Here
we mode,
show results
obtained
after
successful
analysis after
implementation
of the Highinstruments.
Mass Range
(HMR)
successful
desolvation
successful
implementation
of modifications
aimed atThis
adding
the the
capability
to perform
MSto
and optimized
critical hardware
operation settings.
makes
instrument
an idealnative
platform
analysis
without
compromising
performance
of
normal
operation
modes.
cover the three major workflows in BioPharma: intact mass analysis under denaturing and under native
Mass
Spectrometry:
nitrogen
gas in the HCD cell for easier optimization of experimental conditions required for
Mass
spectrometers
in this wished
study are
Scientific™
Q Exactive™ Plus and Q Exactive™ HF
different
types ofused
analyses
tothe
runThermo
on a single
platform.
systems with BioPharma Option. The instruments were operated under Tune 2.8 instrument control software in
In
Normal
settings
factory-optimized,
suitable
analyses
and
ions
HMR mode, inMode
which pressure
the RF applied
to theare
C-trap
was increased from
2,400 Vfor
p-pmost
to 2,900
Vp-p for
better
are cooled
in the
(Fig.
trapping
gas
pressure
is 1 which
corresponds
trapping
of the high
m/zC-trap
ions. Also,
to 2B).
ensureThe
better
capture of
high-m/z
ions setting
in the Orbitrap
analyzer,
the initial
to a high
vacuum
pressure
deltafrom
(∆HV)
~3.1
e-5mbar.
Thethe∆HV
is defined
as the difference
central
electrode
voltage
was adjusted
−3.7ofkV
to −3.4
kV, while
setting
during detection
remained
unchanged
The
S-lens
RFon
level
was allowed
to be
increased
to a setting of 200 in HMR mode and set to
between(−5
HVkV).
with
HCD
gas
minus
HV with
HCD
gas off.
that
for allMode
experiments
shown trapping
here.
Inlevel
Protein
the default
gas pressure setting is 0.2 and that corresponds to a ∆HV
Kai Scheffler , Eugen Damoc , Aaron O. Bailey3, and Jonathan Josephs3, Thermo Fisher Scientific,
The combination of reduced C-trap and HCD cell gas pressures, and trapping ions in the HCD
There
are2.many
factors that
role in Plus/HF
the analysis
of proteins,
some ofand
which
relate to
Figure
A) Schematic
ofplay
the aQkey
Exactive
mass
spectrometers
differences
sample
preparation
solvents,
additives)
others
relateB) to
the Mode,
mass
in the trapping
path (buffers,
in the three
different
operatingwhile
modes
available:
Normal
spectrometer’s
source
conditions
as
well
as
the
physical
environment
inside
the
instrument
C) Protein Mode and D) HMR Mode. E) Illustration of improvement in signal intensity
[2,3].
The
Q
Exactive
Plus
and
Q
Exactive
HF
mass
spectrometers
(Figure
2A)
have
for +17 charge state of a mAb light chain comparing Protein Mode and Normal Mode.
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
A of analyses wished to run on a single platform.
different types
In Normal Mode pressure settings are factory-optimized, suitable for most analyses and ions
are cooled in the C-trap (Fig. 2B). The trapping gas pressure setting is 1region
whichwhere
corresponds
source
to a high vacuum pressure delta (∆HV) of ~3.1 e-5mbar. The ∆HV is defined
difference
CIDas
is the
applied
between HV with HCD gas on minus HV with HCD gas off.
Exactive Plus: setting is 0.2 and that corresponds to a ∆HV
In Protein Mode the default trapping gas Qpressure
Standard Orbitrap mass analyzer
HF:
which is 5x lower than in Normal Mode.Q Exactive
Additionally,
ions are transferred and cooled in the
Ultra High Field Orbitrap mass
analyzer
HCD cell and thus have a longer flight path
(Fig. 2C).
The combination of reduced C-trap and HCD cell gas pressures, and trapping ions in the HCD
cell prior to mass analysis extends the life time of protein ions resulting in increased signal
Trapping in Normal Mode:
intensities of isotopically resolved species
2E).
B
trapping gas(Fig.
pressure
setting 1
E 100
MAbPAC RP
column
N2
N2
Protein
Normal
ModeMode
MATERIALS AND METHODS
Samples:
Samples used in thisSMART
study digest
are ammonium hexafluorophosphate (AHFP, Fisher Scientific, part number
column
A0368370), and Trastuzumab (tradename Herceptin,MAbPAC
Roche,SEC
UK).
for native samples
Intact Analysis
Acclaim RP C18 column
Chromatography:
native & denatured
A Thermo Scientific™ Vanquish™ UHPLC system was used for all LC/MS experiments. For native analysis, 50
mM ammonium acetate buffer (99.99%, Sigma Aldrich) was used. Reversed phase chromatography was
performed with water/0.1% formic acid and acetonitrile/0.1% formic acid on a Thermo Scientific™ MAbPac™ RP
2.1x50 mm column.
HMR Mode
Mass Spectrometry:
UHPLC
Mass spectrometers used in thisVanquish
study are
the Thermo Scientific™ Q Exactive™ Plus and Q Exactive™ HF
systems with BioPharma Option. The instruments were operated under Tune 2.8 instrument control software in
HMR mode,FabRICATOR
in which the
RF applied to the C-trap
was BioPharma
increased from 2,400 V p-p to 2,900 Vp-p for better
®
Q Exactive
• Protein
Mode of high-m/z ions in the Orbitrap analyzer, the initial
trapping of the
high
m/z ions. Also, to ensure better
capture
(IdeS)
digest
MAbPAC RP
• Enhanced
Resolution
and/or
central electrode
voltage was adjusted
from −3.7
kV to −3.4
kV, Mode
while the setting during detection remained
column
• High Mass Range Mode ( m/z ≤8000)
unchanged (−5reduction
kV). 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.
Subunit Analysis
©2016 Genovis AB
Data Analysis:
Protein Mode
A
0
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
For higher gas pressures, high charge states of the same protein decay
lower
regionfaster
wherethan
source
charge states. This is because center-of-mass collision energy
CID is applied
Kce=E*m/(M/z)
Q Exactive Plus:
with
M/z: the mass-to-charge ratio for
a given
charge
state
Standard
Orbitrap
mass analyzer
• Enhanced Resolution Mode
• High Mass Range Mode ( m/z ≤8000)
MAbPAC RP column
for denatured samples
20
10
D
Figure 1: Operating modes for the three major BioPharma workflows: Normal Mode,
Protein Mode and HMRSubunit
mode Analysis
Peptide Mapping
Protein Mode
Normal Mode
80
2
©2016 Genovis AB
and/or
reduction
90
(fixed on setting 1)
Figure 2.N A) Schematic of the Q Exactive Plus/HF mass spectrometers
and differences
70
x 5.1
60
in the trapping path in the three different operating modes available:
B) Normal Mode,
Trapping in Protein Mode:
50
C) Protein Mode and D) HMR Mode.
E) Illustration of improvement in signal intensity
C
trapping gas pressure default setting 0.2
40
for +17 charge state of a mAb light
chain
and Normal Mode.
(range
0.2-1) comparing Protein Mode
30
Relative Abundance
conditions in HMR mode, subunit analysis (reduced mAb and or IdeS digested mAb) in protein mode
The
of intact in
proteins
under
conditions
andanalysis
peptide mapping
standard
modenative
(see Figure
1). is more challenging than under denaturing
conditions since the buffers used don’t contain any organic solvents. Performing electrospray from
aqueous
buffer
solutions
produces
larger
solvent
size and
desolvation
is less
efficient.
Figure 1:
Operating
modes
for the
three
majordroplets
BioPharma
workflows:
Normal
Mode,
Moreover, for large proteins such as intact antibodies the required mass range for analysis under
Protein Mode and HMR mode
native conditions requires a mass range of more than 6000 m/z due to a smaller number of accepted
charges. The increase
of the
upper mass range on the mass spectrometer was achieved by
Normal
Mode
implementing instrument control software changes. The analysis of molecules across the full mass
Mapping
range including thePeptide
detection
of proteins under native
conditions
MAbPAC
RP columnrequired the use of optimized
for denatured
samplesof the instrument, the efficient
parameter settings to ensure efficient desolvation in the
front region
transfer via multipoles, efficient trapping in the C-trap/HCD region and the sensitive injection and
detection in the Orbitrap mass analyzer. Critical parameters are the optimization of in-source
SMART
digest the support of the desolvation process. Also, for the transmission
fragmentation that strongly
influences
HMR Mode
column
efficiency specific voltages have been evaluatedMAbPAC
and SEC
optimized
to ensure robust and sensitive
for native samples
Intact
Analysis
RP mass
C18 column
conditions, experiments
performance in theAcclaim
higher
range when performing analyses under
native
native
& denatured
that have not been possible so far on this type of mass spectrometer.
Also,
the standard calibration
routine previously used was modified and adapted to ensure high mass accuracy across the full mass
range.
With the data collected on different types of samples and presented in this study we demonstrate the
successful analysis after implementation of the High Mass Range (HMR) mode, successful desolvation
and optimized critical hardware Vanquish
operation
settings. This makes the instrument an ideal platform to
UHPLC
cover the three major workflows in BioPharma: intact mass analysis under denaturing and under native
conditions in HMR mode,® subunit analysis (reduced
mAb and or IdeS digested mAb) in protein mode
Q Exactive BioPharma
FabRICATOR
and peptide mapping
in standard mode (see Figure
1). Mode
• Protein
(IdeS) digest
and cooled in the
cell prior to mass analysis extends the life time of protein ions resulting in increased signal
RESULTS
intensities of isotopically resolved species (Fig. 2E).
Q Exactive HF:
m: mass of residual gas, nitrogen
Ultra High Field Orbitrap mass
E: ion energy inside the Orbitrapanalyzer
resulting in: Kce is proportional to the charge state z.
This explains observations of charge
envelope shifts on the m/z scale when comparing data
Trapping in Normal Mode:
100
acquired in different modes withtrapping
different
pressure
regimesE
in the
HCD cell and C-trap region,
gas pressure
setting 1
90
(fixed on
Protein Mode
as shown in one example in Figure
6. setting 1)
80
Normal Mode
N
Here we have investigated and implemented the new High Mass
Range (HMR) Mode that is
70
x
5.1
60
especially required for the analysis of proteins under native conditions
when samples are kept
Trapping in Protein Mode:
50
solvents
involved
at near
pH.
C in aqueous buffers with no organic
trapping
gas pressure
default setting
0.2 neutral
40
(range gas
0.2-1) pressure setting is 1 but
For HMR mode the default trapping
30 it can be slightly increased up
N
20 as protein complexes and
to 1.5
for even improved trapping of certain species such
10
heterogeneous large proteins (e.g. antibody drug conjugates). The
trapping path in HMR mode
0
in HMR
Mode: taking place in the 1355.5
with ion
cooling
HCD cell.
And also,
1356.0
1356.5 mass
1357.0
D is the same as in Protein ModeTrapping
trapping gas pressure default setting 1
m/z
detection is enabled ranging up(range
to m/z
8000 compared to m/z 6000 in the two other
modes.
1.0-1.5)
N
The trapping
gas pressure in all modes is set and saved in the tune files and since a method
allows for segmentation using different tune files different pressure settings can be used within
For
gas
pressures,
states
of theis same
protein
decay
than lower
onehigher
LC-MS
run.
In contrasthigh
the charge
mass range
setting
set in the
method
andfaster
the method
editor
charge
This nodes
is because
center-of-mass
collision
energy
allowsstates.
for several
with different
experiment
types
using different mass ranges within one
LC-MS run.
Kce=E*m/(M/z)
with
M/z: the mass-to-charge ratio for a given charge state
B
2
2
2
Relative Abundance
INTRODUCTION
which
is 5x lower than in Normal Mode. Additionally, ions are transferred
Data
Analysis:
TM BioPharma FinderTM 1.0 SP1 software.
Data
analysis
was performed
Thermoflight
Scientific
HCD
cell and
thus havewith
a longer
path
(Fig. 2C).
The combination
of reduced C-trap and HCD cell gas pressures, and trapping ions in the HCD
A
cell prior to mass analysis extends the life time of protein ions resulting in increased signal
intensities of isotopically resolved species (Fig. 2E).
region where source
CID isand
applied
Figure 2. A) Schematic of the Q Exactive Plus/HF mass spectrometers
differences
in the trapping path in the three different operating modes available: B) Normal Mode,
Q Exactive Plus:
Standard
mass analyzer of improvement in signal intensity
C) Protein Mode and D) HMR Mode.
E) Orbitrap
Illustration
Q Exactive HF:
Ultra High Field
Orbitrap mass
for +17 charge state of a mAb light chain
comparing
Protein Mode and Normal Mode.
m/z
analyzer
E
100
90
Trapping in Protein Mode:
trapping gas pressure default setting 0.2
(range 0.2-1)
N2
40
30
20
10
0
Trapping
trapping gas pressure default setting 1
(range 1.0-1.5)
N2
region where source
CID is applied
50
Q Exactive Plus:
Standard Orbitrap mass analyzer
Q Exactive HF:
High
Field Orbitrap mass
inUltra
HMR
Mode:
analyzer
D
x 5.1
60
1355.5
1356.0
1356.5
1357.0
m/z
Trapping in Normal Mode:
trapping gas pressure setting 1
E
100
90
For higher gas pressures, high charge
states
decay faster thanProtein
lower
(fixed on setting
1) of the same protein
Mode
80
Normal Mode
N
charge states.
This is because center-of-mass collision energy
70
Kce=E*m/(M/z)
x 5.1
60
Trapping
in Protein
Mode:
50
M/z: the mass-to-charge ratio
for a given
charge
state
Cwith
Relative Abundance
2
trapping gas pressure default setting 0.2
m: mass of residual gas, nitrogen
(range 0.2-1)
E: ion energy inside the Orbitrap
N2
5070.9845
0.89
6048.98036
6048.9855
-0.85
5070.98903
5070.9845
0.89
6048.98036
6048.9855
-0.85
Figure 5. Trastuzumab analyzed under native (A) and denaturing
(B) conditions
7026.97170
7026.9619
1.39
resulting in highly similar deconvolution results (C).
60 Da
For calibration of the HMR mode enabling mass detection up to m/z 8000 we used ammonium
hexafluorophosphate (AHFP) in direct infusion mode.
Figure 3 displays the spectra
5923.3 obtained in Normal
5923.3
100
R=4359
versus HMR
mode with excellent mass accuracy
detected with low
5695.6 (Table 1) even for masses
80
In-source
CID
100
abundance at m/z higher than 6000, also shown in the mass accuracy test in Figure 4.
A 5 60displays
6169.9
Figure
the
spectra
obtained
for
intact
Trastuzumab
analyzed
in
native
and denaturing
z=+25
native
zoomand pH conditions (aqueous,
conditions40 resulting in different charge envelopes
5484.6 inherent to the solvent
near neutral pH for native; organic solvent content and low
pH for denaturing). Both types of spectra
6438.0
20
result in nearly identical results after spectra deconvolution (Fig. 5C).
70
Relative Abundance
C
B
Protein Mode
Normal Mode
80
0
3000
3085.6
4000
5000
m/z
6000
5900
7000
5950
m/z
3085.6
1005. Trastuzumab analyzed under native (A) and denaturing (B) conditions
Figure
R=3462
2962.2
3291.2
In-source CIDresults
80
80 in2904.1
resulting
highly similar
deconvolution
(C).
60 Da
30
20
resulting in: Kce is proportional to the charge state z.
10
0
This explains observations of charge
envelope
shifts on the m/z scale
when comparing data
Trapping
in HMR Mode:
1355.5
1356.0
1356.5
1357.0
Dacquired in different modes with different
trapping gaspressure
pressure default
setting 1in the HCD cell and C-trap
regimes
region,
m/z
(range 1.0-1.5)
as shown in one example in Figure 6.
N
Here we have investigated and implemented the new High Mass Range (HMR) Mode that is
especially
required
for the analysis
of proteins
under
native
conditions
are kept
For higher
gas pressures,
high charge
states
of the
same
protein when
decaysamples
faster than
lower
incharge
aqueous
buffers
with
no organic
solvents involved
at near
neutral pH.
states.
This
is because
center-of-mass
collision
energy
For HMR mode the default trapping gas pressure setting is 1 but it can be slightly increased up
ce=E*m/(M/z)
to 1.5 for even improved trapping of K
certain
species such as protein complexes and
with
M/z: the mass-to-charge ratio for a given charge state
heterogeneous
large
proteins
(e.g.
antibody
drug conjugates). The trapping path in HMR mode
m: mass of residual gas, nitrogen
is the same asE:inion
Protein
Mode
ion cooling taking place in the HCD cell. And also, mass
energy inside
thewith
Orbitrap
detection
ranging up to m/z
8000
compared
resultingisin:enabled
Kce is proportional
to the
charge
state to
z. m/z 6000 in the two other modes.
The
trapping
gas
pressure
in
all
modes
is set andshifts
saved
them/z
tune
files when
and since
a method
This explains observations of charge envelope
oninthe
scale
comparing
data
allows
for segmentation
usingwith
different
tunepressure
files different
pressure
beC-trap
used within
acquired
in different modes
different
regimes
in the settings
HCD cellcan
and
region,
one
LC-MS
run.
In
contrast
the
mass
range
setting
is
set
in
the
method
and
the
method
editor
as shown in one example in Figure 6.
allows
nodes with and
different
experiment
different
mass(HMR)
rangesMode
oneis
Here for
we several
have investigated
implemented
thetypes
new using
High Mass
Range
1 LC-MS
2withinthat
run. required for the analysis of proteins under native conditions when samples are kept
especially
B
A
2
C
3366.0
3444.2
3526.1
3612.1
In-source
3797.3
2848.3
60
40
100
40
2080
060
Relative Abundance
N2
40
native
3000
CID 100
0
100
100
80
80
60
60
40
40
20
z=+48
zoom
denatured
5923.3
5923.3
R=4359
5695.6
z=+25
6169.9
4000
5000
m/z
5484.6
20
Relative Intensity
Trapping in Normal Mode:
trapping gas pressure setting 1
(fixed on setting 1)
A
Relative Abundance
B
5070.98903
7026.97170
7026.9619
Table
1. Theoretical
and 1.39
Figure 4. HMR Mode spectral mass accuracy test using
measured masses (Figure 3) of
For
calibration
of the HMR mode The
enabling
mass test
detection up to m/z 8000 we used ammonium
ammonium
hexafluorophosphate.
displayed
ammonium hexafluorophosphate
hexafluorophosphate
(AHFP) in direct infusion mode. Figure 3 displays the spectra obtained in Normal
results in rms = 0.5 ppm.
for calibration of HMR mode
versus HMR mode with excellent mass accuracy (Table 1) even for masses detected with low
experimental
m/z
∆ Mass (ppm)
abundance at m/z higher than 6000, also shown in the mass accuracytheoretical
test in m/z
Figure
4.
670.02805
670.0280
0.07
Figure 5 displays the spectra obtained for intact Trastuzumab analyzed
in native
and denaturing
1159.02371
1159.0237
0.01
conditions resulting in different charge envelopes inherent to the solvent
and
pH
conditions
(aqueous,
2137.01504
2137.0155
-0.21
near neutral pH for native; organic solvent content and low pH for denaturing).
Both
types of spectra
3604.00204
3604.0029
-0.24
result in nearly identical results after spectra deconvolution (Fig. 5C). 4418.99481
4418.9929
0.43
6000
7000
Deconvolution
3000
4000
5000
4.7ppm
m/z
3085.6
148055.503
2962.2
3291.2
In-source
CID
2904.1
3366.0
2848.3
3444.2
3526.1
147848.203 147909.303
3612.1
3797.3
147850.188 147908.488
native
3080
6438.0
6000
80
zoom
m/z
3100
30 Da
5900
7000
5.0 ppm
148217.603
5950
m/z
3085.6
R=3462
z=+48
1.2 ppm
zoom
denatured
eins in Native Conditions on A Hybrid Quadrupole-Orbitrap
B
020
-20
0
-40
-60
-80
-100
3000
4000
denatured
148380.303
148542.203
148540.188
5000
m/z
6000
7000
148218.688
148379.888
3080
4.1 ppm
m/z
3100
-2 ppm 3
30 Da
Deconvolution
c, Im Steingrund 4-6, Dreieich, Germany, Hanna-Kunath-Str. 14, Bremen, Germany,
355 River
Oaks Par
in aqueous buffers with no organic solvents involved at near neutral pH.
For HMR mode the default trapping gas pressure setting is 1 but it can be slightly increased up
to 1.5 for even improved trapping of certain species such as protein complexes and
heterogeneous
large
proteins
(e.g. antibody
drug conjugates). Thedirect
trapping
path inexperiment
HMR mode
Figure 3. Full MS
spectra
of ammonium
hexafluorophosphate
infusion
the same
asHMR
in Protein
Mode with ion cooling taking place in the HCD cell. And also, mass
inisNormal
and
modes.
x10 in the two other modes.
detection is enabled ranging up to m/z 8000 compared to m/z 6000
The trapping gas pressure in all modes is set and saved in the tune files and since a method
Normal Mode
100
x5 within
allows for segmentation using different tune files different pressure settings can be used
and
the method
editor
1 80one LC-MS run. In contrast the mass range setting is set in the method
2
6048.9855
100
60allows for several nodes with different experiment types using different mass
ranges within one
-0.85ppm
80
40LC-MS run.
60
6863.9281
148056.888
-2.9
ppm
4.7ppm
148055.503
100
C
80
5.0 ppm
native
148217.603
Relative Abundance
Relative Intensity
eins in Native Conditions on A Hybrid Quadrupole-Orbitra
60
1.2 ppm
Figure 6. 40
Effect of in-source CID supporting desolvation/ declustering in Normal vs. HMR
148380.303
mode
20
147909.303
148542.203
0
-20
147848.203
147850.188
5923.4
100
147908.488
80
-40
Normal Mode
-60
In-source
CID 100eV
denatured
-80
single scan
(10 uscans)
-100
5923.4
NL:
2.06E7
148540.188
NL: 2.06E7
5695.4
zoom
60
148379.888
4.1 ppm
0.24ppm
80
Relative Abundance
60 0.07 ppm 0.01 ppm
100 670.0280 1159.0237
40
80
4418.9929
3604.0029
Normal Mode
-0.85ppm
5070.9845
2626.0123
x5
6048.9855
Relative Abundance
100
20
60
0
40
6374.9744
1000
2000
3000
20
0
4000
m/z
5000
6000
0.21ppm
100
Figure
4. HMR Mode spectralHMR
massMode
accuracy
test using
0.43ppm
4418.9929
ammonium
hexafluorophosphate.
The displayed
test
80
0.24ppm
3604.0029
ppm
results
inppm
rms0.01
= 0.5
ppm.
60 0.07
0.89ppm
670.0280
1159.0237
5070.9845
2626.0123
60
40
6048.9855
6863.9281
-0.85ppm
6863.9281
8000
7000
1.39ppm
7026.9619
20
-0.85ppm
6048.9855
theoretical m/z
experimental m/z
∆ Mass (ppm)
670.02805
670.0280
6374.9744
1159.023716863.9281
1159.0237
20
0
1000
2000
3000
4000
m/z
7678.9320
Table06000
1. Theoretical
and 7500
6500
7000
measured masses m/z
(Figure 3) of
ammonium hexafluorophosphate
for calibration of HMR mode
2137.0155
40
80
5000
6000
0.07
0.01
2137.01504
7000
3604.00204
2137.0155
8000
3604.0029
-0.21
-0.24
4418.99481
4418.9929
0.43
5070.98903
5070.9845
0.89
Table
1. Theoretical
6048.98036
6048.9855 and -0.85
Figure 4. HMR Mode spectral mass accuracy test using
measured
masses
3) of
7026.97170
7026.9619(Figure 1.39
ammonium hexafluorophosphate. The displayed test
ammonium hexafluorophosphate
For calibration
HMR mode enabling mass detection up to
8000 we
ammonium
results
in rms =of0.5the
ppm.
form/z
calibration
of used
HMR mode
hexafluorophosphate (AHFP) in direct infusion mode. Figure 3 displays the spectra obtained in Normal
theoretical m/z experimental m/z
∆ Mass (ppm)
versus HMR mode with excellent mass accuracy (Table 1) even 670.02805
for masses670.0280
detected with
low
0.07
abundance at m/z higher than 6000, also shown in the mass accuracy test
in
Figure
4.
1159.02371
1159.0237
0.01
Figure 5 displays the spectra obtained for intact Trastuzumab analyzed
and denaturing
2137.01504 in native
2137.0155
-0.21
3604.00204
3604.0029
-0.24
conditions resulting in different charge envelopes inherent to the solvent
and pH conditions
(aqueous,
4418.99481
4418.9929
0.43
near neutral pH for native; organic solvent content and low pH for denaturing). Both types of spectra
5070.9845
0.89
result in nearly identical results after spectra deconvolution (Fig. 5C). 5070.98903
6048.98036
6048.9855
-0.85
7026.97170
7026.9619
1.39
Relative Abundance
Figure 5. Trastuzumab analyzed under native (A) and denaturing (B) conditions
For calibration of the HMR mode enabling mass detection up to m/z 8000 we used ammonium
resulting in highly similar deconvolution results (C).
60 Da
hexafluorophosphate (AHFP) in direct infusion mode. Figure 3 displays the spectra obtained in Normal
versus HMR mode with excellent mass accuracy (Table 1) even for masses detected with low
5923.3
5923.3
100 at m/z higher than 6000, also shown in the
abundance
mass accuracy test in FigureR=4359
4.
5695.6
Figure 5 80displays the spectra
obtained
In-source
CID for
100intact Trastuzumab analyzed in native and denaturing
A 60resulting
conditions
in different charge envelopes inherent
6169.9to the solvent and pH conditions
z=+25(aqueous,
native
near neutral pH for native; organic solvent content and low pH for denaturing).
Both types of spectra
zoom
5484.6
40
result in nearly identical results after spectra deconvolution (Fig. 5C).
6438.0
20
0
5900conditions
5950
Figure 5. Trastuzumab
analyzed
under
and denaturing
(B)
3000
4000
5000native (A)
6000
7000
m/z
m/z
3085.6
3085.6
resulting
similar deconvolution
results (C).
100 in highly
60
Da
R=3462
2962.2
3291.2
In-source CID 80
80
2904.1
100
60
2848.3
3366.0
3444.2
5923.3
zoom
5695.6
denatured
z=+48
5923.3
R=4359
80
3526.1
In-source CID 100
40
Enabling
Mass Spectrometric
Analysis of Intact Proteins in Native Conditions on A
2B
3612.1
A
nce
60
20
40
0
native
3000
4000
z=+25
6169.9
3797.3
5000
5484.6
zoom
6000
7000
3080
3100
148056.888
20
-2.9 ppm
0
100
6170.3
5923.3
NL: 9.83E7
NL: 1.33E8
in-source80CID
x10
0.89ppm
Trapping gas pressure
setting of 1 for both modes
Peaks in this box at m/z >6000 can
not be detected in Normal Mode
on a regular instrument and are
shown here for demonstration
purposes only to display the full
charge envelope.
Figure 6. Effect of
supporting
desolvation/ declustering in Normal vs. HMR
5923.3
6177.1
HMR Mode
mode
60
zoom
5923.4
5923.4
In-source CID 200eV
100
40
NL: 2.06E7
NL: 2.06E7
5695.4
single scan (10 uscans)
80
20
Normal Mode
zoom
60
In-source CID 100eV
0
6000
7000
5900
5950
6000
40 5000
m/z
m/z
single scan (10 uscans)
Figures 6 and 7 depict the20 influence and importance of source fragmentation supporting
Trapping gasand
pressure
desolvation
declustering required
to obtain the correct pattern of glycoforms. The data in these
0
6170.3
5923.3
100
setting
of 1 for
bothacquired
modes in nanospray
two
figures
were
mode, requiring significantly higher in-source CID settings
NL: 1.33E8
NL: 9.83E7
for native analyses. Due to neither
gases available
in the nanospray source nor a heated probe, the
80
5923.3
HMR Modeprocess needs to be
desolvation
supported with 6177.1
source fragmentation taking place inside the mass
60
zoom
In-source CID
200eV step implemented between the S-lens and the Q00 (Figure 2A).
spectrometer,
a potential
40
single scan (10 uscans)
Peaks in this box at m/z >6000 can
not be detected in Normal Mode
on a regular instrument and are
shown here for demonstration
purposes only to display the full
charge envelope.
20
Figure 7. Intact Trastuzumab analysis under native conditions with increasing in-source
CID settings highlighting the 0importance
of this parameter
on desolvation/
declustering.
5900
5950
6000
5000
6000
7000
m/z
m/z
In-source
6187.1 fragmentation supporting
6187.1
Figures
6 and1007 depict the influence
and importance
of source
100
NL: 1.58E6
CID
(eV)
6172.7
desolvation and declustering required to obtain the correct pattern
of glycoforms. The data in these
60
50 were60acquired in nanospray mode, requiring
two figures
significantly higher in-source CID settings
20
20
for native analyses. Due to neither gases available in the nanospray source nor a heated probe, the
6170.4 taking place inside the mass
100
100fragmentation
desolvation process
needs to be 6170.4
supported with source
NL: 3.70E7
spectrometer,
a potential
step implemented between the60S-lens and the Q00 (Figure 2A).
100
60
20
20
Figure 7. Intact
under native
with increasing in-source
6170.4
6170.4
100Trastuzumab analysis
100conditions
NL: 1.06E8
CID settings highlighting the importance of this parameter on desolvation/ declustering.
150
60
60
In-source
6187.1
6187.1
100
100
NL: 1.58E6
20
CID (eV) 20
6172.7
6170.4
6170.4
10060
10060
NL: 9.21E7
50
200
60
60
20
20
6170.4
100
20
100
60
5000
6000
6170.4
100
20
7000
m/z
6100
60
NL: 3.70E7
6200
m/z
6300
Further parameters20that were found critical in optimizing source
conditions for native analysis using
20
6170.4were the capillary
6170.4
size exclusion chromatography
(SEC)
temperature
(also referredNL:
to1.06E8
as transfer
100
100
tube) and the probe heater temperature (also referred to as Aux heater temperature). Figure 8
150
60
60
shows one example of two different temperature settings resulting in differences in charge state
20
20
distribution.
6170.4
100
6170.4
100
NL: 9.21E7
Figure 8. Intact Trastuzumab analysis under native conditions with different probe heater and
200
60
60
capillary temperatures.
z=+25
z=+25
Probe heater temp./
transfer capillary temp.
20
5923.3
100
80
5000
20
5695.6
6000
m/z
7000
6169.9
100
6100
80
Relative Abundance
20
0
Figure 3. Full MS0.21ppm
spectra of ammonium hexafluorophosphate direct
infusion
experiment
6000
6500
7000
7500
2137.0155
HMR Mode 0.43ppm
100
m/z
in Normal and HMR modes.
Relative Abundance
7678.9320
Relative Abundance
7026.9619
Relative Abundance
0
1.39ppm
40
Relative Abundance
20
148218.688
40
5923.3
6200
m/z
6300
60
60
zoom source
Further parameters that
were found critical in optimizing
conditions for native analysis using
5484.6
40
40
275 °
C / 275 °
Cchromatography
size
exclusion
(SEC) were
the capillary temperature
(also referred to as transfer
6438.0
20
20
z=+22
tube) and the probe heater temperature (also
6730.8 referred to as Aux heater temperature). Figure 8
0
0
5923.3
shows one example100of two different
settings 100
resulting in differences
in charge state
5923.3 temperature
6169.9
Hybrid
Quadrupole-Orbitrap
Mass Spectrometer
distribution.
80
80
Relative Abundance
Relative Abundance
3 355 River Oaks Pa
c, Im Steingrund 4-6, Dreieich, Germany, Hanna-Kunath-Str. 14, Bremen, Germany,
-2 ppm
6438.0
60
60
zoom
°
175 °
C8./ 175
C Trastuzumab
5695.6
Figure
Intact
analysis
under native
conditions
with different probe heater and
40
40
6730.8
capillary temperatures.
6170.4
6170.4
Re
100
100
NL: 1.06E8
tube) and the
probe heater temperature (also referred
to as Aux heater temperature).
Figure 8
shows
of two different temperature settings
resulting in differences in charge state
150 one example
60
60
distribution.
20
20
6170.4
Figure 8.
Trastuzumab analysis under
capillary
temperatures.
200
z=+25
60
Probe heater temp./
20
transfer capillary temp.
5000
6000
60
z=+25
7000
6169.9
m/z
6100
zoom
80
6200
m/z
60
6300
Relative Abundance
Further
parameters
that were
found5484.6
critical in optimizing source conditions
for native analysis using
40
40
275
C / 275 °
C
°
6438.0
size exclusion chromatography
(SEC) were the
capillary temperature
(also referred to as transfer
20
20
z=+22
6730.8
tube) and the probe heater0 temperature (also referred
to as Aux0 heater temperature).
Figure 8
5923.3
5923.3 6169.9
100
100
shows one example of two
different temperature settings resulting
in differences in charge state
80
80
6438.0
distribution.
60
60
zoom
175 °
C / 175 °
C
5695.6
40
40 with different probe heater and
Figure 8. Intact Trastuzumab
analysis under native
conditions
6730.8
20
capillary temperatures. 20
5484.6
7051.3 z=+20
z=+25
z=+25
7403.8
5923.3
0
100
5000
5695.6
80
6000
6169.9
60
m/z
zoom
5923.3
0
100
8000
80
7000
Relative Abundance
Probe heater temp./
transfer capillary temp.
5900
5920
5940
5960
m/z
60
Relative Abundance
5484.6
40
40
Figure
9. Intact
SEC-MS analysis under native
conditions with different resolution
275
C / 275
C Trastuzumab
°
°
6438.0
20
20
z=+22
settings.
6730.8
Base Peak Chromatogram
0
0
100
100
90
5923.3 6169.9
100
buffer salts
100
50
5923.3
R=17.5k
ap Mass Spectrometer
0 80
0
100 60
Relative Abundance
175 °
C / 175 °
C
50
0
100
50
0
100
40
20
0
2
4
Time (min)
6438.08
6
5695.6
R=17.5k
80
80
zoom
7051.3
5000
6000
R=70k
60
40
50
20
40
0
30
z=+20zoom/
7403.8
7000
R=35k
70
60
6730.8
5484.6
R=35k
10
overlay
8000
20
m/z
5900
5920
R=70k
5940
5960
m/z
arkway,
San Jose, CA, USA
Figure 9. Intact Trastuzumab SEC-MS analysis under native conditions with different resolution
50
0
2000
settings.
3000
4000
5000
m/z
6000
10
7000
0
8000
Base Peak Chromatogram
100
0
0
2
4
6
8
5920
5930
m/z
100
buffer salts
50
5910
10
5940
90
R=17.5k
80
R=35k
characterization.
Mass
Relative Abundance
Relative Abundance
A
100
New Lot
60
80
REFERENCES
• This new operating mode extends the instrument’s capabilities to cover all three major workflows for
BioPharma characterization.
[1] Rosati S, van den Bremer ET, Schuurman J, Parren PW, Kamerling JP, Heck AJ. In-depth
• Desolvation/declustering
conditions,
transfer glycosylation
and trapping profiles
have been
optimized
to allow for
qualitative and quantitative
analysis ions
of composite
and other
micro-heterogeneity
improved
in HMR
modeby
forhigh-resolution
resolution settings
asmass
high as
70k.
on intactsensitivity
monoclonal
antibodies
native
spectrometry
using a modified
Orbitrap. mAbs. 2013;5(6):917-924. doi:10.4161/mabs.26282.
• Critical
parameters
for online
LC-MS
analyses
native conditions
capillary and
probe for mass
[2] Fenn
JB, Mann
M, Meng
CK,
Wong under
SF, Whitehouse
CM. are
Electrospray
ionization
heater
temperatures
as biomolecules.
key factors in supporting
desolvation/declustering.
spectrometry
of large
Science. 1989;246(4926):64-71.
[3] Fenn JB, Electrospray wings for molecular elephants (Nobel Lecture). Angew. Chem. Int. Ed. 2003.
42:3871–3894.
REFERENCES
TRADEMARKS/LICENSING
[1] Rosati S, van den Bremer ET, Schuurman J, Parren PW, Kamerling JP, Heck AJ. In-depth
qualitative
quantitative
composite
profiles and other
© 2016and
Thermo
Fisher analysis
ScientificofInc.
All rightsglycosylation
reserved. FabRICATOR®
is a micro-heterogeneity
trademark of Genovis AB.
on intact
monoclonal
antibodies
high-resolution
native
mass
spectrometry
using a modified
All other
trademarks
are theby
property
of Thermo
Fisher
Scientific
and its subsidiaries.
This information
Orbitrap.
2013;5(6):917-924.
is notmAbs.
intended
to encourage usedoi:10.4161/mabs.26282.
of these products in any manner that might infringe the intellectual
[2] Fenn
JB,rights
Mannof M,
Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass
property
others.
spectrometry of large biomolecules. Science. 1989;246(4926):64-71.
[3] Fenn JB, Electrospray wings for molecular elephants (Nobel Lecture). Angew. Chem. Int. Ed. 2003.
42:3871–3894.
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 products in any manner that might infringe the intellectual
property rights of others.
zoom
60
20
0
100
80
60
Old Lot
40
R=35k
40
20
0
R=35k
40
20
0
100
80
20
5500
m/z
6000
0
6500
5860
5880
5900
5920
m/z
5940
5960
B
Relative Abundance
2xA2G0F
100
80
60
40
20
0
-20
-40
-60
-80
-100
148055.632
New Lot
1xA2G0F/1xA2G1F
148218.219
2xA2G1F
148379.910 1xA2G1F/1xA2G2F
148540.955
1xA2G0/1xA2G0F
147909.041
147599.568
148705.783
147762.623 147909.046
148543.700
148056.349
Mass
148218.427
148381.229
Old Lot
CONCLUSIONS
• We have successfully implemented the High Mass Range Mode on the Q Exactive Plus and Q
Exactive HF mass spectrometers allowing for mass detection up to m/z 8000.
• This new operating mode extends the instrument’s capabilities to cover all three major workflows for
BioPharma characterization.
• Desolvation/declustering conditions, ions transfer and trapping have been optimized to allow for
improved sensitivity in HMR mode for resolution settings as high as 70k.
• Critical parameters for online LC-MS analyses under native conditions are capillary and probe
heater temperatures as key factors in supporting desolvation/declustering.
REFERENCES
www.thermofisher.com
[1] Rosati S, van den Bremer ET, Schuurman J, Parren PW, Kamerling JP, Heck AJ. In-depth
©2016 Thermo
Fisher Scientificanalysis
Inc. All rights
reserved. FabRICATOR
is a trademark
Genovis
AB. micro-heterogeneity
qualitative
and quantitative
of composite
glycosylation
profilesof and
other
other monoclonal
trademarks are antibodies
the property ofbyThermo
Fisher Scientific
and its
subsidiaries.
This information
onAllintact
high-resolution
native
mass
spectrometry
using isa presented
modified
as
an
example
of
the
capabilities
of
Thermo
Fisher
Scientific
products.
It
is
not
intended
to
encourage
use of these products in any manners that
Orbitrap. mAbs. 2013;5(6):917-924. doi:10.4161/mabs.26282.
might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available
[2] Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass
in all countries. Please consult your local sales representative for details.
spectrometry of large biomolecules. Science. 1989;246(4926):64-71.
[3] Fenn JB, Electrospray wings for molecular elephants (Nobel Lecture). Angew. Chem. Int. Ed. 2003.
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Old Lot
CONCLUSIONS
• Critical parameters for online LC-MS analyses under native conditions are capillary and probe
60
40
148381.229
• Desolvation/declustering conditions, ions transfer and trapping have been optimized to allow for
improved sensitivity in HMR mode for resolution settings as high as 70k.
Figure 10. Comparison of two different lots of Trastuzumab showing significant variation
in the pattern of the glycoforms, a known issue in production of biopharmaceuticals.
80
148218.427
5950
Time (min)
100
Figure 9 shows
the SEC-LC/MS
analysis of intact Trastuzumab
under native conditions acquired
70
R=70k
50
R=17.5k
on the Q Exactive
Plus in HMR mode at different60 resolution settings of 17.5k, 35k and 70k.
0
50
100
Increasing the resolution setting in this casezoom/
is not40 aiming at achieving isotopic resolution but
R=35k
50
overlay
allowing to resolve
possible sodium and potassium
30 adducts [1] arising e.g. from salts and/or
0
100
20
formulation buffer
and
thus significantly improving mass
accuracy after deconvolution.
R=70k
50
Figure 10 displays
glycoform pattern variability from10 two different lots of Trastuzumab analyzed
0
0
2000
3000
4000
5000
6000
7000
8000
5910
5920 and
5930 reported
5940
5950
with SEC-LC/MS, which ism/z not desired but has been observed
previously.
The
m/z
possibility for such a variation is inherent to the production process of biopharmaceuticals and
shows the capabilities of mass spectrometry to easily pick up these variations since the analyses
of glycoform patterns on the intact antibody level are very reproducible and reliable, considering
the use of consistent optimized parameter settings for all experiments in a study.
100
148056.349
heater temperatures as key factors in supporting desolvation/declustering.
• We have successfully implemented the High Mass Range Mode on the Q Exactive Plus and Q
Exactive HF mass spectrometers allowing for mass detection up to m/z 8000.
5923.3
100
20
5695.6
80
conditions with different
probe heater and
NL: 9.21E7
60
5923.3
100
6170.4
100
native
Relative Abundance
100
Intact
-100BioPharma
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