Rapid Level 3 Characterization of Biologics using a CESI 8000 … Biologi… · through separation,...
Transcript of Rapid Level 3 Characterization of Biologics using a CESI 8000 … Biologi… · through separation,...
p 1
Rapid Level 3 Characterization of Biologics using a
CESI 8000 – Thermo Q-Exactive Platform
Comprehensive Qualitative and Quantitative Analysis of Biopharmaceuticals Using CESI-MS Technology Bryan Fonslow and Andras Guttman SCIEX Separations, Brea, CA
In the biopharmaceutical industry, comprehensive and
reproducible characterization of monoclonal antibody
therapeutics (mAbs) is crucial, as impurities and heterogeneity
can impact safety and/or efficacy. mAbs are large glycosylated
proteins (≈ 150 kDa) contain several other post-translational
modifications (PTMs). Peptide mapping of mAbs can provide
critical qualitative and quantitative information about impurities
and heterogeneity.1-2
Capillary electrophoresis (CE) in
conjunction with mass spectrometry has exceptional separation
efficiency and capabilities for peptide and glycopeptide mapping
(Characterization Level 3) of mAbs. The application of an
electrospray ionization (ESI) emitter integrating CE and ESI into
a single dynamic process (CESI) has already proven useful for
peptide and mAb analysis.3-7
In this Technical Note, we present data for the Level 3
Characterization of a leading, representative monoclonal
antibody, Trastuzumab (Herceptin). A Beckman Coulter CESI
8000 system, sold through SCIEX Separations, a part of AB
SCIEX, coupled to a Thermo Q-Exactive mass spectrometer was
used for the analyses. The results illustrate the benefits of the
integration of CE and ESI in a single dynamic process coupled
with high resolution mass spectrometry. Peptides, both large and
small (3 – 65 amino acids), were identified, separated, and
quantified. In particular, we identified and quantified degradative
hotspots like aspargine-deamidation, methionine-oxidation,
glutamic-acid-cyclization, and C-terminal lysine heterogeneity
from a mere 100 fmol (15 ng) of sample. A glycosylation hotspot
is highlighted by comprehensive characterization at Asn300
through separation, identification, and relative quantification of
glycopeptides. Collectively, these results indicate the capabilities
for CESI coupled with high resolution mass spectrometry to
quickly and comprehensively characterize therapeutic mAbs
using peptide and glycopeptide mapping from a small amount of
sample with the use of a single protease, trypsin.
Advantages of CESI 8000 for MS analysis
Low flow rate. With CESI flow rates around 20 nL/min,
ionization efficiency is maximized and ion suppression is
dramatically reduced, allowing strong ionization of modified
peptides and glycopeptides.5,7
Since a single, open
capillary tube is used for CESI, there are no difficult
nanofluidic connections to make between other capillaries or
pumps.
Separation efficiency. The inherent separation efficiency of
CE provides sharp peptide peaks for sensitive and
reproducible identification as well as relative quantitation.
Low sample requirements. CESI has small volume (< 250
nL) and mass (< 25 ng) requirements.
Sample carry-over. Using an open tubular format with no
solid support, all peptides elute from the capillary (both
hydrophobic and hydrophilic), supporting comprehensive
mAb sequence coverage and virtually eliminating sample
losses as well as any carry-over.
Comprehensiveness. Based on the 100% protein
sequence coverage routinely attained with this approach,
qualitative and quantitative analysis of mAb purity, stability,
and glycoform heterogeneity is possible from a single CESI-
MS run with the use of a single protease, trypsin.
Reproducibility. Migration times vary less than 30 sec (<
1.5% RSD) over the 45 minute separation within triplicate
runs. Relative abundance measurements of modified and
unmodified peptides vary less than 2% among triplicate
runs.
p 2
Orthogonality. CESI provides an orthogonal separation
mechanism to LC-MS-based methods for separation,
identification, quantification, and validation of sequence
variants as well as PTMs.
Experimental Design
Sample preparation: Trastuzumab (100 μg from 10 mg/mL
solution) was diluted and denatured using Rapigest followed by
reduction with DTT and alkylation by iodoacetamide. The sample
was then digested with trypsin at 37ºC overnight, dried, and
diluted three-fold with leading electrolyte (200 mM ammonium
acetate at pH 4), yielding a final concentration of 300 ng/μL of
the digested antibody.
CESI-MS/MS: CESI-MS/MS was performed using a Beckman
Coulter CESI 8000 system – sold through SCIEX Separations, a
part of AB SCIEX – connected to a Thermo Q-Exactive mass
spectrometer. 50 nL of the sample (equivalent to 100 fmol or 15
ng of digested antibody) in 100 mM leading electrolyte was
injected by pressure into the bare fused silica capillary column.
Transient-isotachophoresis (t-ITP) was applied to focus the
sample components prior to the electrophoretic separation. 10%
acetic acid was used as background electrolyte (BGE) and a
voltage of 20 kV was applied between the inlet vial and the CESI
sprayer. Samples were analyzed in triplicate.
Data-dependent acquisition (DDA) was performed on the mass
spectrometer consisting of a high resolution Orbitrap scan at 70K
followed by several high resolution MS/MS scans at 17.5K. The
DDA parameters were as follows: 120 msec Orbitrap MS survey
scan from 150 - 1800 m/z; 60 msec DDA scans on the top 10
most abundant ions above a threshold intensity of 3.3x104; HCD
fragmentation with 27% normalized collision energy. The total
MS cycle times were all 1 sec or less. Dynamic exclusion was
set to 15 sec to match the sharp peaks (~ 30 sec wide) inherent
to CESI.
Data Analysis: Data analysis was performed using AB SCIEX
ProteinPilot™ (Peptide and modified peptide identification),
ProteinMetrics Byonic (Glycopeptide identification), and Thermo
Xcalibur (Peptide Quantitation). Thermo .RAW files were
converted to .MGF files using ProteoWizard MS Convert.
Database searches were performed with the Trastuzumab,
common contaminants, and reversed protein sequences.
Glycopeptides were also extracted manually and the MS/MS
spectra were checked for diagnostic glycans ions.
The CESI process
Transient isotachophoresis capillary zone electrophoresis (t-ITP-
CZE) is the separation method used for CESI-MS analysis of
proteolytically-digested biologics. With CZE, peptides are
separated by their charge-to-frictional drag coefficient, also
conveyed as their charge-to-hydrodynamic volume ratio. Thus,
peptides that differ in charge and/or size, either due to sequence
or PTM differences, are regularly separated, 8 as will be
illustrated by representative peptides herein. Prior to the CZE-
based separation, approximately 10% of the inlet end of the
capillary tubing is pressure-loaded with the sample. Peptide
analytes are stacked during the t-ITP process based on their
mobilities within an electric field and mobility gradient zone. The
application of t-ITP improves sensitivity by approximately 10-fold
over normal CZE methods since 10-fold more sample can be
loaded onto the capillary without degrading the inherent high
separation efficiency of CZE.
Electroosmotic flow (EOF) is generated at the charged wall of
the capillary from the application of an electric field. Even at pH
4 from the 10% acetic acid BGE, the acidic silanol groups on the
inner capillary wall maintain a bulk negative charge, and drive
EOF towards the capillary outlet. Notably, unlike pressure-driven
flow techniques such as liquid chromatography with parabolic
flow profiles, EOF has a flat flow profile, which minimizes band
broadening, achieving exquisitely sharp peaks. EOF drives all of
the peptides as separated peaks out of the CESI tip for
electrospray ionization at a flow rate around 20 nL/min. At this
flow rate, the benefits of nanoelectrospray ionization (nESI) are
realized.
The electrospray ionization voltage is applied at the CESI tip
through a conductive liquid of 10% acetic acid. To fabricate the
CESI sprayer tip, the exterior polyimide coating has been
removed and the fused silica has been chemically etched until
small ions can pass through the capillary wall. The small ions,
and thus current, that can pass through the CESI tip facilitate
application of the ESI voltage. The ESI process then transfers
peptides from the liquid phase into the gas phase. Notably, the
only liquid that is electrosprayed with CESI is from the CE
separation (no sheath-flow condition), maximizing sensitivity at
low nanoliter per minute flow rates.3
CESI-MS analysis of peptides
Tryptic peptides from mAbs have a large distribution of sizes and
solution charge states making them quite amenable to CE
separation. Peptides from Trastuzumab are indeed visibly
separated and distributed by CESI from 25 to 38 minutes during
a CESI-MS run (Figure 1). Although not visible in the base peak
electropherogram (BPE), peptides were also identified as late as
42 min, particularly glycopeptides, which will be described in
more detail later. The triplicate BPEs are quite reproducible both
in a qualitative comparison of profiles (Figure 1A) and a
p 3
quantitative comparison of migration times (Tables 1 & 2).
Generally, some correlations are observed between the peptide
mass-to-charge ratios (m/z’s) with mass spectrometry and their
charge-to-drag coefficients with CE (Figure 2B). However, the
distribution of peptide peaks throughout the migration time and
m/z separation spaces facilitate comprehensive identification and
quantification of peptides. A few representative extracted peptide
electrophoretic peaks illustrate the ability to simultaneously
separate peptides of both very small and very large sizes (Figure
1C). Additionally, these extracted peptides illustrate the sharp
peaks inherent to CE separation (15 – 30 sec in width) that
facilitate identification and quantification of peptides.
Figure 1: Representations of CESI-MS peptide separations. (A) Triplicate base peak electropherograms (BPEs) of trypsin-digested Trastuzumab run by CESI-MS with a Thermo Q-Exactive. (B) Peptide mass map and (C) extracted ion electropherograms (XIEs) of representative small and large peptides from single CESI-MS runs.
Achieving 100% sequence coverage
A number of factors can contribute to achieving 100% sequence
coverage of proteins in bottom-up proteomics. In addition to the
already described efficient peptide separation and electrospray
ionization with CESI-MS, a protein database search is an
essential step in the process. The Paragon algorithm within
ProteinPilotTM
allows for fast, user-friendly identification of
proteins and over 300 PTMs simultaneously,9 making it an
excellent comprehensive characterization tool for proteolytically-
digested biologics analyzed by CESI-MS. As indicated in Figure
2, the database search result yielded 100% sequence coverage
for heavy and light chains in all three replicates. The ability to
separate and recover both very small and very large, as well as
hydrophobic and hydrophilic peptides from a single trypsin
digestion mixture contributed to the reproducible 100% sequence
coverage of heavy and light chains.
130115_CEMS_Tras-20kv5psi60sec60mi_01_OK 1/15/2013 6:06:11 PM
RT: 24.00 - 40.00
24 26 28 30 32 34 36 38 40
Time (min)
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
NL: 1.04E10
Base Peak m/z=
100.00-6000.00
MS
130115_CEMS_Tr
as-
20kv5psi60sec60mi
_01_OK
130115_CEMS_Tras-20kv5psi60sec60mi_01_OK RT: 24.00 - 40.00 Mass: 150.00 - 1800.00 NL: 1.04E10
24 26 28 30 32 34 36 38 40
Time (min)
500
1000
1500
m/z
RT: 24.00 - 40.00
24 26 28 30 32 34 36 38 40
Time (min)
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
130115_CEMS_Tras-20kv5psi60sec60mi_01_OK 1/15/2013 6:06:11 PM
RT: 24.00 - 40.00
24 26 28 30 32 34 36 38 40
Time (min)
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
NL: 1.04E10
Base Peak m/z=
100.00-6000.00
MS
130115_CEMS_Tr
as-
20kv5psi60sec60mi
_01_OK
130115_CEMS_Tras-20kv5psi60sec60mi_01_OK RT: 24.00 - 40.00 Mass: 150.00 - 1800.00 NL: 1.04E10
24 26 28 30 32 34 36 38 40
Time (min)
500
1000
1500
m/z
RT: 24.00 - 40.00
24 26 28 30 32 34 36 38 40
Time (min)
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
Di-, Tri-, Tetra-, & Penta-peptides
2nd longest peptide (25-mer)
Longest peptide
(63-mer)
B
C
A
p 4
Figure 2: Protein Pilot™ Software Database Search Results of CESI 8000 – Q-Exactive MS data reproducibly achieves 100% sequence coverage of Trastazumab heavy and light chains. The upper panel shows the 100% percent coverage of the identified heavy and light chains. The central panes show the two Protein Groups and representative peptide identifications that have been assigned to the heavy and light chains. The lower panes show the identified sequence coverages, which are color coded according to peptide identification confidence. Nearly 100% of the sequence coverage for the light chain is above 95% confidence (green). The lower confidence protein regions (yellow) inherent to two short peptides matches were further validated manually as both short peptides and missed cleavages on longer peptides. 100% sequence coverages were achieved for each of the CESI-MS analyses performed in triplicate.
Characterization Level 3 – Peptide Mapping
Peptide mapping of a therapeutic mAb with CESI-MS, similar to
LC-MS, involves identifying peptide sequences by tandem mass
spectrometry (MS/MS) and extracting peptide electrophoretic
peaks. The extracted ion electropherograms (XIEs) can be used
for sequence and PTM abundance comparison between different
biologics such as different mAb lots or between innovator,
biosimilar, and biobetter mAbs.
Both the BPEs and XIEs in Figures 1A and 1C illustrate the
characteristically sharp CE peaks that would be used for
comparative peptide mapping. That is, with the combined high
resolving powers of CESI in the separation dimension and high
resolution MS in the mass dimension, extracted peptide peaks
can be easily mapped between two or more samples. To best
illustrate the capabilities of peptide mapping with CESI-MS, the
identification and quantification of degradative hot spots on
peptides within Trastuzumab (Figure 3 and Table 1) are
highlighted. The common degradative PTMs that were identified
are N-terminal glutamate cyclization, methionine oxidation, C-
terminal lysine heterogeneity, asparagine deamidation,
tryptophan dioxidation, and lysine glycation. Additional less
abundant sites of the same degradative PTMs, among a few
others, were also identified by ProteinPilotTM
, but are not
described here.
The confident identification of these degradative PTMs are
illustrated by both well-matched high resolution MS/MS
fragmentation spectra in Figure 3 and very small precursor mass
errors (Table 1). For modifications with relatively large mass
shifts (i.e. oxidation, + 16 Da; dehydration, - 18 Da; glycation, +
162 Da, and C-terminal lysine loss, - 128 Da), these spectral
matches should provide unambiguous evidence for the PTMs.
For peptides containing small mass differences, such as
deamidated asparagine, which only differs in mass by one
Dalton, CESI can provide additional confidence to identifications.
The challenge of measuring a one Dalton mass shift on peptides,
even with high resolution MS, lies in the fact that isotopic
envelopes of unmodified and deamidated peptides can
overlap.10
Additionally, reverse-phase LC (RPLC) separations
often may not resolve these peptides since there is no change in
hydrophobicity from an asparagine to aspartic acid conversion;
however, there is a charge difference between Asn (neutral) and
Asp (acidic) that can be readily resolved by CE.11
Thus, the
solution-phase spatial resolution of the two peptides (Figure 3D)
created by the CE separation provides additional evidence that
the small mass differences measured by MS are from two unique
peptide species; and that the identification is not just form a
higher monoisotopic mass peak (from 13
C) within the same
isotopic envelope of the same unmodified peptide sequence.
Further, the small migration time shift (~ 30 sec) towards the
positively charged anode is expected from the change in peptide
charge from the neutral asparagine to the negatively charged
aspartic acid. That is, the more negatively charged peptide has a
stronger attraction towards the positively charged anode.
These separation advantages of capillary electrophoresis are
also observed with other PTMs that have more easily detectable
mass shifts by MS. For instance, the largest migration shift
observed for a degradative PTM was from the difference in
charge between the zwitterionic N-terminal glutamic acid
(positively charged under the acidic conditions used for CESI-
MS) to the neutral pyroglutamate (Figure 3A). The Glu to
pyroGlu migration shift was towards the anode, just as with
deamidation. Similarly, C-terminal lysine heterogeneity from the
loss of a positively charged lysine on the heavy chain can also
be confirmed by a large migration shift towards the anode
Table 1: Identification and relative quantification of Trastuzumab degradative post-translational modification hot spots
Modification Modified peptide sequence and associated mass shift
Monoisotopic mass [M+H]
+
Mass accuracy (ppm)
Average migration time (min)
1
Average relative abundance (%)
1,2
Methionine Oxidation K.DTLM(+15.9949)ISR.T 851.4291 0.33 37.95 ± 0.52 2.39 ± 0.98
Pyroglutamate formation -.E(-18.0106)VQLVESGGGLVQPGGSLR.L 1863.9923 0.95 31.41 ± 0.37 1.60 ± 0.19
Asparagine deamidation R.IYPTN(+0.9840)GYTR.Y 1085.5262 0.06 31.96 ± 0.37 15.57 ± 2.92
Lysine glycation K.VSNK(+162.0528)ALPAPIEK.T 1428.7944 -0.85 30.28 ± 0.33 0.28 ± 0.06*
Tryptophan dioxidation K.FNW(+31.9898)YVDGVEVHNAK.T 1709.7818 -1.62 30.32 ± 0.27 0.26 ± 0.03
C-terminal lysine loss K.SLSSPGK(-128.0950).- 660.3563 -0.80 34.39 ± 0.42 99.12 ± 0.10**
1Error was calculated as standard deviations from triplicate technical CESI-MS replicate runs.
2Relative abundances were calculated using peak areas of modified and unmodified peptides of the same sequence and charge state unless otherwise noted.
*The peak area of unmodified peptides K.APLPAPIEK.A was used for the relative abundance calculation. **The +1 charge state of the modified peptide and the +2 charge state of the unmodified peptide were used for the relative abundance calculation.
p 5
(Figure 3C). Importantly, all modified peptides identified had
very good migration time reproducibility, with standard deviations
less than 30 seconds. Thus, these migration time variations are
compatible with peptide mapping experiments. Of course these
separation capabilities are also applicable to PTM relative
quantification. Similar to the higher identification confidence of
deamidated peptides by CESI separation, quantification of
deamidated peptides is also improved. Quantification of peptides
relies on extracting ion masses over time. Since the isotopic
envelopes no longer overlap between unmodified and
deamidated peptides when spatially separated by CESI, their
XIEs also do not overlap in time.
Figure 3: Peptide mapping of Trastuzumab degradative PTM hotspots. MS/MS spectra identification and XIEs of modified and unmodified peptides for (A) N-terminal glutamate cyclization (pyroGlu), (B) methionine oxidation (MetOx), (C) C-terminal lysine heterogeneity, (D) asparagine deamidation, (E) tryptophan dioxidation, and (F) lysine glycation. XIE scales were adjusted to show the quantification of the lower abundance peptide electrophoretic peak. The amino acid site and type of PTM are shown in each panel for both modified and unmodified peptides.
130115_CEMS_Tras-20kv5psi60sec60mi_01_OK 1/15/2013 6:06:11 PM
28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0 32.5 33.0 33.5 34.0 34.5 35.0
Time (min)
0
5
10
15
20
25 NL: 3.79E9
m/z= 542.7730-542.7770 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_01_OK
NL: 3.79E9
m/z= 543.2665-543.2670 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_01_OK
26 27 28 29 30 31 32 33 34
Time (min)
0
2
4
6
8
10 NL: 2.67E9
m/z= 559.9380-559.9400 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_01_OK
NL: 7.88E7
m/z= 570.5985-570.6035 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_01_OK
26 27 28 29 30 31 32 33 34
Time (min)
0.0
0.1
0.2
0.3
0.4
0.5
NL: 1.00E10
m/z= 419.7500-419.7600 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_01_OK
NL: 1.00E10
m/z= 476.9340-476.9375 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_01_OK
130115_CEMS_Tras-20kv5psi60sec60mi_03 1/15/2013 8:47:17 PM
29 30 31 32 33 34 35 36 37 38 39
Time (min)
0
2
4
6NL: 3.15E9
m/z= 941.50-941.51 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_03
NL: 3.15E9
m/z= 932.50-932.51 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_03
28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0 32.5 33.0 33.5 34.0 34.5 35.0
Time (min)
0
1
2
3NL: 5.87E9
m/z= 418.22-418.23 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_03
NL: 5.87E9
m/z= 426.21-426.22 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_03
27 28 29 30 31 32 33 34 35
Time (min)
0
2
4
6NL: 2.44E9
m/z= 394.725-394.735 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_03
NL: 2.44E9
m/z= 660.350-660.360 MS
130115_CEMS_Tras-
20kv5psi60sec60mi_03
Re
lativ
e a
bu
nd
an
ce
(%
)
Time (min)
N-terminal GluN-terminal PyroGlu
Met255
Asn55
Asn55 deamidation
Trp280
Trp280
dioxidation
Lys329 glycation
Lys329
A
B
C
D
E
F
MetOx255
C-terminal Lys450
present
C-terminal Lys450
loss
p 6
This is observed as two well-resolved XIE peaks for the
deamidated and unmodified peptides (Figure 3D). However,
even if peptides remain unresolved by CE, there are still benefits
for relative quantification over LC-based techniques. CESI is a
low flow rate method that essentially eliminates ion suppression.
Thus, unresolved modified and unmodified peptide peak areas
better represent their true relative abundances. Of the
degradative PTMs identified on Trastuzumab, this characteristic
is applicable to relative quantification of methionine, tryptophan
oxidation, and lysine glycation.
The relative abundances of the degradative PTMs are shown in
Table 1. Notably, relative abundances generally varied less than
0.2% among the triplicate CESI-MS runs. However, for
modifications that can also occur during sample preparation
(methionine oxidation and asparagine deamidation), their relative
abundances varied less than 3% among triplicate runs. Relative
quantification of these degradative PTMs was straightforward.
Quite simply, the peak area of the modified peptide was divided
by the sum of peak area of itself and the unmodified peptide.
The relative quantification of lysine glycation required a bit more
consideration. Trypsin is unable to cleave at the glycated version
of the lysine, but otherwise cleaves the non-glycated lysine as
expected. Thus, different peptide sequences were used to
perform relative quantification. The most abundant version of the
same portion of the glycated lysine peptide sequence was used
for the relative abundance calculation. However, lysine glycation
characterization would be more straightforward by peptide
mapping since differences in trypsin cleavage would not need to
be considered.12
Characterization Level 3 – Glycopeptide Mapping
Therapeutic mAb glycoforms are typically characterized at
Levels 1 and 2 through intact and reduced molecular weight MS
measurements, respectively, and at Level 4 as cleaved glycans
with LC or CE separations.13-14
N-glycosylation is usually
localized on a consensus asparagine residue in the IgG1 Fc
domain, but can also be localized in variable regions, non-
consensus regions, or even on glutamine.15-16
Thus, site specific
localization of glycans is becoming an essential mAb
characterization step. Characterization levels 1, 2, and 4 are not
well-suited for confirmation of N-glycosylation localization. Even
glycopeptides identified by LC-MS by Level 3 characterization
often remain unseparated by RPLC, leaving the potential for
misassignment of glycan structures and variability in relative
abundance measurements. With CESI-MS, comprehensive
glycoform analysis is achieved at characterization Level 3
through efficient separation and electrospray ionization of
glycopeptides.
Examples of the glycopeptide separations are shown in Figure
4A and B and their average migration times are listed in Table 2.
Migration times of glycopeptides were also reproducible, with the
majority having around 30 seconds of variability within triplicate
CESI-MS runs. As mentioned earlier, the CE separation is based
on the charge-to-frictional drag ratio of peptides. One of the keys
to glycopeptide separation and characterization with CESI-MS is
that each sugar unit adds enough addition frictional drag to the
corresponding glycoforms to be separated by CESI.
Glycopeptides with high mannose: M5 (also referred to as
Man5), afucosylated biantennary complex: A2, A2G1 and A2G2
(also referred to as G0, G1, G2), fucosylated biantennary
complex: FA2, FA2G1 and FA2G2 (also referred to as G0F,
G1F, G2F), and similar uncharged glycans are separated by this
mechanism. For the fucosylated (Figure 4A) and afucosylated
(Figure 4B) series, the separations are illustrated as resolved
peaks. Additionally, although not obvious from Figure 4, the
migration times between afucosylated and fucosylated versions
of the same glycan structures also have different migration
times. For instance, A2 and FA2 (also referred to as G0 and
G0F) had average migration times of 35.00 and 34.72 min (Table
2), respectively. In the instances of the A1, FA1, and FA2
glycoforms (also referred to as G0-GlcNAc, G0F-GlcNAc and
G0F), addition of 5-N-acetyl--neuraminic acid (Neu5Ac)
induces a larger migration shift due to changes in the
glycopeptide charge from the addition of the acidic Neu5Ac
residue. This mechanism is quite similar to the migration time
shifts observed with the degradative PTMs when the modified
peptide is more acidic than the unmodified peptide (Figures 3A,
C, & D).
As with other modified peptides, the XIEs of glycopeptides are
dependent on their identification through the combination of their
high mass accuracy precursor and fragment ion measurements.
The glycopeptides identified also have very high mass
accuracies (Table 2), all well below ± 5 ppm error. To illustrate
the quality of MS/MS spectra that were used to identify the
glycopeptides, the spectrum of the lowest abundance glycoform
identified as A2G2S1 and FA2G1 as the highest as shown in
Figure 4B. As with the majority of identified glycopeptide spectra,
a few peptide sequence-dependent b- and y-ions are matched,
but the spectrum matches mostly originate from glycan
signatures: an intact glycopeptide mass measurement, multiple
sugar unit losses from the glycopeptide, and multiple glycan
fragment ions. The fact that even the lowest abundance
glycopeptide has a high intensity, high quality matching spectrum
indicates the qualities of the other higher intensity spectra are
also high. The relative quantification values for the identified
Trastuzumab glycans are shown in Table 2. Most notably, the
A2G2S1 glycan was quantified at 0.06% relative abundance.
p 7
Thus, between the most abundant FA2G1 glycoform at 54.49%
and the A2G2S1 glycoform, that represents three orders of
magnitude quantification of mAb glycosylation. Within this 1000-
fold dynamic range, 20 glycoforms were identified and quantified
as glycopeptides. Additionally, the majority of glycopeptide
relative abundance measurements were reproducible, with CVs
less than 10%. Thus, the combination of CESI and MS allow for
both separation and high confidence, high sensitivity
identification of glycopeptides, facilitating the confident
identification and quantification of mAb glycosylation.
Conclusions
We have presented a new platform combining CESI, a robust
and highly efficient separation and nanoelectrospray technique
connected on-line with a high accuracy and resolution mass
spectrometer for qualitative and quantitative analysis of
monoclonal antibody therapeutics. With this new CESI design,
peptides were separated and electrosprayed directly into the
mass spectrometer by making the terminal end of the separation
capillary a sprayer. The droplets formed during the
nanoelectrospray process in the CESI design are small and
result in highly efficient ionization. With flow rates around 20
nL/min, ion suppression was minimized, providing higher
sensitivity to analyze less abundant glycopeptides present in the
same sample. The sensitivity and dynamic range of the
approach for glycopeptides was illustrated by the relative
quantification of as low as 0.06% and three orders of magnitude
range of glycoform identification and quantification of 20
glycopeptides, respectively. Also notable, CESI-MS revealed
degradative PTM hotspots in the mAb down to 0.3% relative
abundance including methionine oxidation, asparagine
deamidation, cyclization of N-terminal glutamate to
pyroGlutamate, heavy chain C-terminal lysine heterogeneity,
tryptophan oxidation, and lysine glycation. The CESI-MS
analysis of Trastuzumab presented herein represents an
attractive workflow when rapid, comprehensive, reproducible
verification of a biologic’s primary sequence and glycosylation
profile are required, particularly when mass-limited.
Acknowledgments
We thank Dr. Alain Beck at Pierre Fabre, France for providing Trastazumab; Jean-Marc Busnel of Beckman Coulter, Anna Lou of SCIEX separations, and Zhiqi Hao and David Horn of Thermo Fisher Scientific for generation of the data; Chris Becker and Eric Carlson of ProteinMetrics for access to Byonic; and all for comments on the Technical Note.
References
1. iosimilar iobetter and e t eneration ntibody haracterization by ass pectrometry eck anglier-
ianf rani an Dorsselaer Anal. Chem. 2012, 84, 4637−4646
2. Simultaneous Quantitative and Qualitative Analysis of Proteolytic Digests of Therapeutic Monoclonal Antibodies using a TripleTOF® System. AB SCIEX Technical Note 7460213-01.
3. High Capacity Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry: Coupling a Porous Sheathless Interface with Transient-Isotachophoresis, Busnel, J.M. et al., Anal. Chem., 2010, 82, 9476-9483.
4. Optimization and Evaluation of a Sheathless Capillary Electrophoresis Electrospray Ionization Mass Spectrometry Platform for Peptide Analysis: Comparison to Liquid Chromatography Electrospray Ionization Mass Spectrometry, Faserl, K., Sarg, B., Kremser, L., and Lindner, H.; Anal. Chem., 2011, 83, 7297-7305.
5. Ultra-Low Flow Electrospray Ionization-Mass Spectrometry for Improved Ionization Efficiency in Phosphoproteomics, Heemskerk, A.A.M. et al., Anal Chem., 2012, 84, 4552-4559.
6. Improving the Comprehensiveness and Sensitivity of heathless apillary Electrophoresis−Tandem ass Spectrometry for Proteomic Analysis, Wang, Y and Fonslow, B.R. et al., Anal. Chem. 2012, 84, 8505-8513.
7. Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry, Gahoual R. et al., mAbs, 2013, 5, 1-12.
8. Electrophoretic mobility for peptides with post-translational modifications in capillary electrophoresis, Kim, J., Zand, R., Lubman, D., Electrophoresis, 2002, 23, 782–793.
9. The Paragon Algorithm, a Next Generation Search Engine That Uses Sequence Temperature Values and Feature Probabilities to Identify Peptides from Tandem Mass Spectra, Shilov IV et al., Mol. Cell. Proteomics, 2007, 6, 1638-1655.
10. Accurate Identification of Deamidated Peptides in Global Proteomics Using a Quadrupole Orbitrap Mass Spectrometer, Nepomuceno, A.I. et al., J Proteome Res. 2014, 13, 777-85.
11. Characterization of deamidated peptide variants by micro-preparative capillary electrophoresis and mass spectrometry, Gennaro, L.A. and Salas-Solano, O., J. Chrom. A, 2009, 1216, 4499–4503.
12. Unveiling a Glycation Hot Spot in a Recombinant Humanized Monoclonal Antibody, Zhang, B. et al., Anal. Chem. 2008, 80, 2379-2390.
13. Performing native mass spectrometry analysis on therapeutic antibodies, Thompson, N.J., Rosati, S., and Heck, A.J.R., Methods, 2014, 65, 11-17.
14. Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection, Hamm, M., Wang, Y, and Rustandi, R.R., Pharmaceuticals, 2013, 6, 393-406.
15. Impact of variable domain glycosylation on antibody clearance: An LC/MS characterization, Huang, L. et al., Anal. Biochem., 2006, 349, 197–207.
16. Glutamine-linked and Non-consensus Asparagine-linked Oligosaccharides Present in Human Recombinant Antibodies Define Novel Protein Glycosylation Motifs, Valliere-Douglass, J.F. et al., J. Biol. Chem., 2010, 285, 16012–16022.
p 8
Figure 4: XIEs of glycopeptides identified by CESI-MS. From glycopeptide identifications by MS/MS, peaks were extracted for standard (A) fucosylated and (B) afucosylated mAb glycoforms for relative quantification. A representative glycopeptide MS/MS spectrum is shown for the lowest abundance A2G2S1 glycoform. Ions annotated in green represent glycan fragmentation. Further glycopeptide characterization is shown in Table 2. Glycan structure interpretation followed the CFG protocol.
p 9
Table 2: Characterization of Trastuzumab heavy chain glycosylation hot spot N300 through identification and relative quantification. Glycan structure interpretation followed the CFG protocol.
Glycopeptides identified as R.EEQYN(Glycan)STYR.V
mAb glycan abbreviation
Glycan mass (Da)
Monoisotopic mass [M+H]
+ (Da)
Mass accuracy (ppm)
Average migration time (min)
1
Average relative abundance (%)
1,2
FA2G1 (G1F) 1606.5867 2796.0987 -0.41 34.92 ± 0.43 54.49 ± 1.45
FA2 (G0F) 1444.5339 2634.0459 0.25 34.72 ± 0.42 17.28 ± 0.97
FA2G2 (G2F) 1768.6395 2958.1515 -0.01 35.19 ± 0.43 8.59 ± 0.40
A2 (G0) 1298.476 2487.9880 0.67 35.00 ± 041 5.58 ± 0.31
A2G1 (G1) 1460.5288 2650.0408 0.93 34.88 ± 0.43 4.07 ± 0.09
FA1G1 1403.5073 2591.0037* 3.04 34.45 ± 0.41 1.52 ± 0.06
FA2G2S1 2059.7349 3249.2469 0.78 34.45 ± 0.42 1.41 ± 0.11
FA1 1241.4545 2430.9665 0.40 34.76 ± 0.44 1.25 ± 0.06
M5 (Man5) 1216.4228 2405.9349 0.09 38.18 ± 0.53 1.13 ± 0.66
FA2G1S1 1897.6821 3085.1785* 3.10 37.93 ± 0.52 1.02 ± 0.48
A1G1 1257.4494 2446.9614 0.44 38.03 ± 0.48 0.78 ± 0.40
FA1G1S1 1694.6027 2884.1147 -3.15 34.39 ± 0.42 0.57 ± 0.03
A2G2 (G2) 1622.5816 2812.0936 -2.77 34.71 ± 0.39 0.55 ± 0.03
A1 1095.3966 2284.9086 0.57 41.57 ± 0.64 0.51 ± 0.32
FA2G2S2 2350.8303 3540.3423 1.77 35.11 ± 0.44 0.44 ± 0.05
A1G1S1 1548.5448 2738.0568 -2.81 37.90 ± 0.51 0.32 ± 0.15
FA2BG1 1809.6661 2999.1781 -3.00 35.09 ± 0.44 0.17 ± 0.01
M4A1G1S1 1710.5976 2900.1096 -1.62 34.83 ± 0.43 0.14 ± 0.01
M5A1 1419.5022 2609.0142 -1.92 37.95 ± 0.52 0.14 ±.0.07
A2G2S1 1913.677 3103.1890 -1.74 38.05 ± 0.57 0.06 ± 0.03
1Error was calculated as standard deviations from triplicate technical CESI-MS replicate runs.
2Relative abundances were calculated using peak areas of glycosylated peptides of the same sequence and charge state (+3).
*[M+H]+ mass has a -18.0106 Da loss due to water neutral loss.
p 10
For Research Use Only. Not for use in diagnostic procedures.
© 2014 AB SCIEX. SCIEX is part of AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte Ltd or their respective owners IEX™ is being used under license.
Publication number: 10400614-01