A Complete Workflow Solution for Monoclonal
Antibody Glycoform Characterization Combining
a Novel Glycan Column Technology and
Bench-Top Orbitrap LC-MS/MS
Zhiqi Hao,1 Udayanath Aich, 2 Julian Saba,1 Rosa Viner,1 Xiaodong Liu, 2 Srinivasa Rao, 2 Chris Pohl, 2
Andreas Huhmer1 and Patrick Bennett1
1
Thermo Fisher Scientific, San Jose, CA, USA; 2Thermo Fisher Scientific, Sunnyvale, CA, USA
Thermo Fisher Scientific, San Jose, CA, USA, Thermo Fis
Overview
Purpose: To develop a complete workflow solution for monoclonal antibody (mAb)
glycoform characterization using a unique glycan column technology and a Thermo
Scientific™ bench-top Orbitrap™ LC-MS/MS.
Methods: Glycans are separated using a recently developed high-performance
HPLC/UHPLC column, a Thermo Scientific™ GlycanPac™ AXH-1 column. A datadependent high-energy collision dissociation (HCD) method was performed in negative
ion mode to analyze the glycans.
Results: The GlycanPac AXH-1 column separates glycans with unique selectivity
based on charge, size and polarity. A complete workflow solution was developed for
glycan profiling combining the unique column technology and a bench-top Orbitrap LCMS/MS (Figure 1). This workflow was applied to antibody glycoform characterization.
Confident identification and structural confirmation were achieved for released glycans
from a standard glycoprotein and a monoclonal antibody.
Data analysis
SimGlycan® software from PREMIER
structural elucidation2. SimGlycan so
Scientific mass spectrometers and el
database searching and scoring tech
Full MS spectra of mAb were analyze
Deconvolution™ 2.0 software. Mass
averaging spectra across the most ab
A minimum of at least 8 consecutive c
used to produce a deconvoluted peak
compared to the expected masses of
glycoforms
Figure 1. A complete LC-MS/MS wo
glycan profiling
PNGase F
digestion
Introduction
Native g
Because glycosylation is critical to the efficacy of antibody therapeutics, the FDA
requires that a consistent human-type glycosylation be maintained for recombinant
monoclonal antibodies (mAb), irrespective of the system in which they are produced.
The complex branching and isomeric nature of glycans pose significant analytical
challenges for their identification and characterization. Liquid chromatography (LC)
coupled to mass spectrometry (MS) has emerged as one of the most powerful tools for
the structural characterization of glycans.
The recently developed GlycanPac AXH-1 column is a high-performance
HPLC/UHPLC column specifically designed for structural, qualitative and quantitative
analysis of glycans. It has a unique selectivity for biologically relevant glycans
including glycans from antibodies, either labeled or native and is designed for highresolution, high-throughput analysis by LC-fluorescence or LC-MS methods. Because
glycans are very hydrophilic and polar, hydrophilic interaction liquid chromatography
(HILIC) columns based on amide, amine or zwitterionic packing materials are often
used for their analysis. HILIC columns separate glycans mainly by hydrogen bonding,
resulting in size and composition-based separation. Identification of the glycan charge
state is not possible by HILIC. The GlycanPac AXH-1 column overcomes these
limitations and can separate glycans based on charge, size and polarity configuration.
It provides both greater selectivity and higher resolution. In this study, we
characterized N-linked glycans released from a glycoprotein standard and a
monoclonal antibody by LC-MS/MS methods using the new column technology and
high-resolution Orbitrap mass spectrometry.
Methods
Sample preparation
Native glycans are released from glycoproteins or mAb with PNGase F enzyme. The
released glycans are conjugated with 2-amino benzamide (2-AB) label group with
reported procedure of Bigge et. al.1
Liquid chromatography
All the glycans are separated using a recently developed high-performance
HPLC/UHPLC column, GlycanPac AXH-1, on a Thermo Scientific™ Dionex™ Ultimate
3000 UHPLC with either s fluorescence or MS detector.
For intact antibody, a Thermo Scientific™ ProSwift RP-10R monolithic column (1 x 50
mm) was used for desalting. LC solvents are 0.1% formic acid in H2O (Solvent A) and
0.1% formic acid in acetonitrile (Solvent B). Column was heated to 80 °C during
analysis. Flow rate was 60 µL/min. After injection of 1 µg mAb, a 15 min gradient was
used to elute mAbs from the column (0.0 min, 20%B; 1.0 min, 35%B; 3.0 min, 55%B;
4.0 min, 98%B; 7.0 min, 98% B; 7.1 min, 20%B; 15.0 min, 20%B).
Mass spectrometry
A data-dependent high-energy collision dissociation (HCD) method was performed in
negative ion mode to analyze the glycans. The following MS and MS/MS settings were
used: MS scan range 380-2000 m/z. FT-MS was acquired at 70,000 resolution at m/z
200 with AGC target of 1x106 and DDA MS2 acquired at 17,500 resolution at m/z 200
with AGC target of 2x105. Intact mAbs were analyzed by ESI-MS for intact molecular
mass. The spray voltage was 4kV. Sheath gas flow rate was set at 10. Auxiliary gas
flow rate was set at 5. Capillary temperature was 275 °C. S-lens level was set at 55.
In-source CID was set at 45 eV. For full MS, resolution was 17,500 for intact mAb. The
AGC target was set at 3x106. Maximum IT was set at 250 ms.
LC-MS
analysis
SimGlycan
software
The Q Exactiv
Results
Separation of Glycans Based on Ch
The GlycanPac AXH-1 column can be
analysis and characterization of uncha
proteins. The separation and elution of
glycans elute first, followed by the sepa
di-sialylated, tri-sialylated, tetra-sialylat
of each charge state are further separa
study, the structure of glycans present
resolution LC-MS/MS. As shown in Fig
obtained from the MS/MS data validate
separate labeled N-glycans based on c
different charge state glycans is comm
column as shown in Figure 3.
Figure 2. LC-MS analysis of 2-AB lab
GlycanPac AXH-1 (1.9 µm) column w
N-Acetyl-Glucosamine (GlcNAc),
N-Acetyl Neuraminic Acid (Neu5Ac),
Mannose (Man),
Galactose (Ga
N-Glycolyl-Nueraminic Acid (Ne
2 A Complete Workflow Solution for Monoclonal Antibody Glycoform Characterization Combininga Novel Glycan Column Technology and Bench-Top Orbitrap LC-MS/MS
olution for monoclonal antibody (mAb)
can column technology and a Thermo
ently developed high-performance
GlycanPac™ AXH-1 column. A data(HCD) method was performed in negative
arates glycans with unique selectivity
e workflow solution was developed for
technology and a bench-top Orbitrap LCto antibody glycoform characterization.
ation were achieved for released glycans
al antibody.
Data analysis
SimGlycan® software from PREMIER Biosoft was used for glycan identification and
structural elucidation2. SimGlycan software accepts raw data files from Thermo
Scientific mass spectrometers and elucidates the associated glycan structure by
database searching and scoring techniques.
Full MS spectra of mAb were analyzed using Thermo Scientific™ Protein
Deconvolution™ 2.0 software. Mass spectra for deconvolution were produced by
averaging spectra across the most abundant portion of the elution profile for the mAb.
A minimum of at least 8 consecutive charge states from the input m/z spectrum were
used to produce a deconvoluted peak. To identify glycoforms, the masses were
compared to the expected masses of various combinations of commonly found
glycoforms
Figure 1. A complete LC-MS/MS workflow solution for monoclonal antibody
glycan profiling
PNGase F
digestion
ciation (HCD) method was performed in
e following MS and MS/MS settings were
was acquired at 70,000 resolution at m/z
acquired at 17,500 resolution at m/z 200
nalyzed by ESI-MS for intact molecular
s flow rate was set at 10. Auxiliary gas
was 275 °C. S-lens level was set at 55.
esolution was 17,500 for intact mAb. The
as set at 250 ms.
Labeled glycans
N-Acetyl-Glucosamine (GlcNAc),
Mannose (Man),
N-Acetyl Neuraminic Acid (Neu5Ac),
GlycanPac AXH-1 solumn
LC-MS
analysis
SimGlycan
software
Ultimate 3000
UHPLC
Separation
of glycans
The Q Exactive MS
Galactose (Gal)
N-Glycolyl-Nueraminic Acid (Neu5Gc),
The GlycanPac AXH-1 column is also well su
separation and analysis of native glycans fro
unlabeled glycans not only eliminates the ex
cleanup methods during labeling, but also re
adding further ambiguity imposed by the lab
Monoclonal antibody (mAb) glycan profil
high resolution LC-MS/MS
Results
Separation of Glycans Based on Charge, Size and Polarity
The GlycanPac AXH-1 column can be used for qualitative, quantitative, structural
analysis and characterization of uncharged (neutral) and charged glycans present in
proteins. The separation and elution of glycans are based on charge; the neutral
glycans elute first, followed by the separation of acidic glycans from mono-sialylated,
di-sialylated, tri-sialylated, tetra-sialylated and finally penta-sialylated species. Glycans
of each charge state are further separated based on their size and polarity. In this
study, the structure of glycans present in each peak was determined using high
resolution LC-MS/MS. As shown in Figure 2, the detailed structural information
obtained from the MS/MS data validated the ability of GlycanPac AXH-1 column to
separate labeled N-glycans based on charge, size and polarity. However, co-elution of
different charge state glycans is common with other commercially available HILIC
column as shown in Figure 3.
Figure 2. LC-MS analysis of 2-AB labeled N-glycans from bovine fetuin by
GlycanPac AXH-1 (1.9 µm) column with MS detection.
Intact mass measurement of a monoclonal a
the combination of any two of the three N-gly
mass errors for some of the intact glycoform
(Figure 4A) which is larger than the <10 ppm
shown). Furthermore, the intact mass error f
was within 10 ppm (Figure 4B), suggesting t
molecule that were not detected at the intact
intact mass of the major glycoforms. To furth
glycans from this protein were separated usi
separation and elution of glycans from Glyca
with neutral glycans eluting first, followed by
each charge state are further separated bas
Figure 4. Observed molecular mass of gly
forms of a intact monoclonal antibody. So
glycoforms have an observed mass error
potentially double fucosylated peaks that
A Full MS spectrum of mAb
2797.59
90
60
50
20
10
3154.57
2647.78
3223.15
2601.35
40
2513.21
1899.12 2003.93 2149.04
2000
2246.72
2200
G0+G0F
3369.59
2430.84
2353.71
2400
G1F+G2F
3294.72
2556.54
A
0
1800
G0F+G2F
3088.89
2695.90
70
G0F+G0F
3025.87
2745.82
80
30
G0F+G1F
2907.25 2968.59
100
Relative Abundance
y developed high-performance
a Thermo Scientific™ Dionex™ Ultimate
S detector.
Swift RP-10R monolithic column (1 x 50
0.1% formic acid in H2O (Solvent A) and
olumn was heated to 80 °C during
tion of 1 µg mAb, a 15 min gradient was
, 20%B; 1.0 min, 35%B; 3.0 min, 55%B;
%B; 15.0 min, 20%B).
2AB
ns or mAb with PNGase F enzyme. The
o benzamide (2-AB) label group with
2AB/2AA labeling
Native glycans
y of antibody therapeutics, the FDA
ylation be maintained for recombinant
the system in which they are produced.
of glycans pose significant analytical
erization. Liquid chromatography (LC)
rged as one of the most powerful tools for
lumn is a high-performance
or structural, qualitative and quantitative
for biologically relevant glycans
led or native and is designed for highorescence or LC-MS methods. Because
philic interaction liquid chromatography
witterionic packing materials are often
ate glycans mainly by hydrogen bonding,
ration. Identification of the glycan charge
c AXH-1 column overcomes these
n charge, size and polarity configuration.
resolution. In this study, we
a glycoprotein standard and a
using the new column technology and
Figure 3. LC-MS analysis of 2-AB labeled
commercial amide HILIC column (1.7 µm)
3447.92
3529.93
2600
2800
3000
m/z
3200
3400
3616.00
3600
3801.46 3901.52
3800
deconvolution
Mass error larger
than expected
-8.4 ppm
G1F/G1F (or G0F/G2F)
-6.8 ppm
G0F/G1F
-25.4 ppm
G1F/G2F
-57.3 ppm
-3.4 ppm
G2F/G2F
G0F/G0F
N-Acetyl-Glucosamine (GlcNAc),
N-Acetyl Neuraminic Acid (Neu5Ac),
Mannose (Man),
Galactose (Gal)
N-Glycolyl-Nueraminic Acid (Neu5Gc),
L-Fucose (L-Fuc)
Thermo Scientific Poster Note • PN ASMS13_WP24_ZHao_e 06/13S 3
?
?
sher Scientific, Sunnyvale, CA, USA
Figure 3. LC-MS analysis of 2-AB labeled N-glycans from bovine fetuin by a
commercial amide HILIC column (1.7 µm) with MS detection.
Figure 5. Separation of the major,
column
Extracted ion chroma
RT: 4.80 - 21.86
6.15
100
80
60
6.28
40
20
0
100
Relative Abundance
ER Biosoft was used for glycan identification and
oftware accepts raw data files from Thermo
elucidates the associated glycan structure by
hniques.
zed using Thermo Scientific™ Protein
s spectra for deconvolution were produced by
abundant portion of the elution profile for the mAb.
charge states from the input m/z spectrum were
ak. To identify glycoforms, the masses were
of various combinations of commonly found
workflow solution for monoclonal antibody
6.01
8.69
80
G0F
60
40
20
8.51
0
100
10.70
10.52
80
10.84
60
G1F
40
2AB/2AA labeling
20
2AB
glycans
0
100
N-Acetyl-Glucosamine (GlcNAc),
Mannose (Man),
N-Acetyl Neuraminic Acid (Neu5Ac),
GlycanPac AXH-1 solumn
Separation
of glycans
Ultimate 3000
UHPLC
60
Galactose (Gal)
N-Glycolyl-Nueraminic Acid (Neu5Gc),
40
L-Fucose (L-Fuc)
20
0
The GlycanPac AXH-1 column is also well suited for high performance LC/MS
separation and analysis of native glycans from proteins (data not shown). Analyzing
unlabeled glycans not only eliminates the extra reaction step and cumbersome
cleanup methods during labeling, but also retains the original glycan profile without
adding further ambiguity imposed by the labeling reaction.
Monoclonal antibody (mAb) glycan profiling using GlycanPac AXH-1 column and
high resolution LC-MS/MS
harge, Size and Polarity
e used for qualitative, quantitative, structural
arged (neutral) and charged glycans present in
of glycans are based on charge; the neutral
paration of acidic glycans from mono-sialylated,
ated and finally penta-sialylated species. Glycans
rated based on their size and polarity. In this
t in each peak was determined using high
gure 2, the detailed structural information
ted the ability of GlycanPac AXH-1 column to
charge, size and polarity. However, co-elution of
mon with other commercially available HILIC
abeled N-glycans from bovine fetuin by
with MS detection.
12.33
12.73
5.61
80
Labeled glycans
ive MS
Intact mass measurement of a monoclonal antibody identified glycoforms derived from
the combination of any two of the three N-glycans, G0F, G1F and G2F. However, the
mass errors for some of the intact glycoforms of this antibody ranged from 20-60 ppm
(Figure 4A) which is larger than the <10 ppm observed for other samples (data not
shown). Furthermore, the intact mass error for the deglycosylated form of this antibody
was within 10 ppm (Figure 4B), suggesting that some minor glycosylation forms of this
molecule that were not detected at the intact level had interfered with the observed
intact mass of the major glycoforms. To further characterize this antibody, released
glycans from this protein were separated using the GlycanPac AXH-1column. The
separation and elution of glycans from GlycanPac AXH-1 column are based on charge
with neutral glycans eluting first, followed by the acidic sialylated species. Glycans of
each charge state are further separated based on their size and polarity (Figure 5).
12.39
6
8
10
B
G0F+G1F
HC
Full MS spectrum of deglycosylated mAb
5
100
2671.11
95
90
2907.25 2968.59
100
2797.59
Relative Abundance
80
50
30
20
10
3223.15
2601.35
40
70
3154.57
2647.78
A
0
1800
2513.21
2000
2246.72
2200
G0+G0F
3369.59
2430.84
2353.71
2400
G1F+G2F
3294.72
2556.54
1899.12 2003.93 2149.04
2530.57
2800
3000
m/z
3200
3400
55
2943.60
50
2486.98
45
40
3004.84
35
2444.78
3600
10
3801.46 3901.52
5
0
3800
3135.42
2326.63 2404.34
2289.67
15
3616.00
3068.71
25
20
3529.93
2884.72
60
30
3447.92
2600
3205.20
3278.05 3354.23
2254.13
2200
2300
3434.14
2400
2500
2600
deconvolution
Mass error larger
than expected
-8.4 ppm
-6.8 ppm
G0F/G1F
-0.7 ppm
-7.0 ppm
-25.4 ppm
G1F/G2F
2800
m/z
2900
3000
3100
3200
3300
3400
3497.41
3500
Mass error as expected
Fragment ion type
Single glycosidic
Glycosidic/glycosidic
Single cross ring
-57.3 ppm
G0F/G0F
2700
deconvolution
G1F/G1F (or G0F/G2F)
-3.4 ppm
2828.15
2575.73
75
65
2695.90
60
G0F+G2F
3088.89
2745.82
70
G0F+G0F
3025.87
80
Relative Abundance
90
85
G2F/G2F
?
?
Tim
Figure 6. Identification and struct
resolution HCD MS/MS
Figure 4. Observed molecular mass of glycosylated (A) and deglycosylated (B)
forms of a intact monoclonal antibody. Some of the intact antibody major
glycoforms have an observed mass error larger than expected. There are also two
potentially double fucosylated peaks that need to be confirmed.
A Full MS spectrum of mAb
12
Characterization of glycans in each
dependent MS/MS using HCD. The
that were generated from both cross
Three different types of glycans wer
of glycans identified were neutral, in
major glycoforms identified at the in
identified were less abundant, non-f
mono-sialylated and di-sialylated sp
double fucosylated species that wer
Cross ring/glycosidic
Gal)
Neu5Gc),
12.31 1
L-Fucose (L-Fuc)
4 A Complete Workflow Solution for Monoclonal Antibody Glycoform Characterization Combininga Novel Glycan Column Technology and Bench-Top Orbitrap LC-MS/MS
glycans from bovine fetuin by a
th MS detection.
Figure 7. Identified glycans from monocl
Figure 5. Separation of the major, neutral N-glycans on GlycanPac AXH-1
column
Glycans identified
only in released form
Extracted ion chromatogram of the neutral glycans
Gl
pro
RT: 4.80 - 21.86
6.15
100
80
60
6.28
40
20
Relative Abundance
0
100
6.01
8.69
80
G0F
60
40
20
8.51
0
100
10.70
10.52
12.31 13.28
80
10.84
60
14.58 15.15 16.65
17.92
19.81 20.79
G1F
40
20
0
100
12.33
12.73
5.61
80
14.51
15.98 16.54 17.91
19.79 20.68
G2F
60
40
ose (L-Fuc)
20
0
d for high performance LC/MS
proteins (data not shown). Analyzing
reaction step and cumbersome
ns the original glycan profile without
g reaction.
using GlycanPac AXH-1 column and
body identified glycoforms derived from
ns, G0F, G1F and G2F. However, the
this antibody ranged from 20-60 ppm
served for other samples (data not
he deglycosylated form of this antibody
some minor glycosylation forms of this
el had interfered with the observed
characterize this antibody, released
the GlycanPac AXH-1column. The
ac AXH-1 column are based on charge
acidic sialylated species. Glycans of
on their size and polarity (Figure 5).
12.39
6
8
10
12
14.05 14.65
14
Time (min)
16
15.84 17.26 18.76
18
20.01 21.10
20
Characterization of glycans in each peak was performed by Full MS and data
dependent MS/MS using HCD. The information-rich HCD spectra contain fragment ions
that were generated from both cross-ring and glycosidic bond fragmentations (Figure 6).
Three different types of glycans were found from this monoclonal antibody, the majority
of glycans identified were neutral, including G0F, G1F and G2F which were also the
major glycoforms identified at the intact protein level for this antibody (Figure 4A). Also
identified were less abundant, non-fucosylated forms of G1 and G2, minor amounts of
mono-sialylated and di-sialylated species with and without fucosylation, as well as
double fucosylated species that were not identified at the intact protein level (Figure 7).
G0F/G1F
HCD spectrum of G2F
 The GlycanPac AXH-1 columns are compa
or FT-MS/MS analysis of both native and la
antibodies were carried out successfully us
 Confident identification and structural confir
using high-resolution HCD MS/MS which pr
containing glycosidic and cross ring fragme
2671.11
2828.15
2575.73
 A complete workflow solution was develope
unique GlycanPac AXH-1 column technolog
70
2530.57
65
2884.72
55
2943.60
50
2486.98
45
40
3004.84
35
2444.78
30
3068.71
25
20
15
10
5
0
3135.42
2326.63 2404.34
2289.67
3205.20
3278.05 3354.23
2254.13
2200
2300
3434.14
2400
2500
2600
2700
2800
m/z
2900
3000
3100
3200
3300
3400
3497.41
3500
deconvolution
-0.7 ppm
-7.0 ppm
G0F/G0F
 GlycanPac AXH-1 column separates glycan
charge, size and polarity.
75
60
G1F
Conclusion
90
80
-25.4
-3.4 ppm
Figure 6. Identification and structural confirmation of released glycan using high
resolution HCD MS/MS
5
85
-8.4 ppm
-6.8 ppm
Full MS spectrum of deglycosylated mAb
95
Figure 8 Annotated glycoforms of a mon
G1F/G1F (or G0F/G2F)
sylated (A) and deglycosylated (B)
e of the intact antibody major
ger than expected. There are also two
ed to be confirmed.
100
These results explain that the unexpected mas
interfering minor glycoforms that have a molec
deconvoluted MS spectrum, the base of the an
mass range of about 40 Da due to the distribut
large protein of this size. Therefore any interfe
difference would cause a mass shift of the maj
separate peak. For example, in this case, the r
Neu5Ac, which would have a mass difference
shift observed in this study, especially when th
abundance (Figure 8). Results in this study in
glycan profiling can be achieved using GlycanP
LC-MS/MS.
Mass error as expected
Fragment ion type
Percentage match (%) of theoretical fragments
Single glycosidic
32.14
Glycosidic/glycosidic
30.95
Single cross ring
20.21
Cross ring/glycosidic
14.95
 This workflow was applied to characterize a
Confident identification and structural confir
glycans from the monoclonal antibody.
References
1.
2.
Bigge, J. C. et al., Non-selective and effic
2-amino benzamide and anthranilic acid.
238.
Apte, A and Meitei, N.S., Bioinformatics in
mass spectrometric data using SimGlycan
SimGlycan is a registered trademark of PREMIER Biosoft In
Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these pr
intellectual property rights of others.
Thermo Scientific Poster Note • PN ASMS13_WP24_ZHao_e 06/13S 5
Figure 7. Identified glycans from monoclonal antibody
, neutral N-glycans on GlycanPac AXH-1
Glycans identified
only in released form
atogram of the neutral glycans
13.28
14.58 15.15 16.65
17.92
Glycans identified both at intact
protein level and in released form
19.81 20.79
F
3
3
14.51
15.98 16.54 17.91
19.79 20.68
G2F
14.05 14.65
14
me (min)
15.84 17.26 18.76
16
18
20.01 21.10
20
peak was performed by Full MS and data
information-rich HCD spectra contain fragment ions
s-ring and glycosidic bond fragmentations (Figure 6).
re found from this monoclonal antibody, the majority
ncluding G0F, G1F and G2F which were also the
ntact protein level for this antibody (Figure 4A). Also
fucosylated forms of G1 and G2, minor amounts of
pecies with and without fucosylation, as well as
re not identified at the intact protein level (Figure 7).
These results explain that the unexpected mass error observed previously is due to the
interfering minor glycoforms that have a molecular mass close to the major ones. In the
deconvoluted MS spectrum, the base of the antibody major glycoform peaks covers a
mass range of about 40 Da due to the distribution of the unresolved isotopic peaks of a
large protein of this size. Therefore any interfering species within 20 Da of mass
difference would cause a mass shift of the major glycoform peaks, rather than forming a
separate peak. For example, in this case, the replacement of a Fuc and a Gal by
Neu5Ac, which would have a mass difference of -17Da, could cause the negative mass
shift observed in this study, especially when the interfering species is relatively low in
abundance (Figure 8). Results in this study indicate that rapid and sensitive antibody
glycan profiling can be achieved using GlycanPac AXH-1 column and HR/AM Orbitrap
LC-MS/MS.
Figure 8 Annotated glycoforms of a monoclonal antibody
-8.4 ppm
Mass error caused by
G1F/G1F (or G0F/G2F)
interfering minor glycoforms
-25.4 ppm
containing sialic acid
-6.8 ppm
G0F/G1F
G1F/G2F
-57.3 ppm
-3.4 ppm
tural confirmation of released glycan using high
CD spectrum of G2F
G0F/G0F
G2F/G2F
-24.7 ppm
G2F/G2F2
?
-36.8 ppm
?
G2F2/G2F2
Conclusion
 GlycanPac AXH-1 column separates glycans with unique selectivity based on
charge, size and polarity.
 The GlycanPac AXH-1 columns are compatible with MS instruments. LC-ESI-FTMS
or FT-MS/MS analysis of both native and labeled glycans from proteins and
antibodies were carried out successfully using GlycanPac AXH-1 columns.
 Confident identification and structural confirmation of glycans can be achieved
using high-resolution HCD MS/MS which produces an informative spectrum
containing glycosidic and cross ring fragment ions.
 A complete workflow solution was developed for glycan profiling combining the
unique GlycanPac AXH-1 column technology and a bench-top Orbitrap LC-MS/MS.
Percentage match (%) of theoretical fragments
32.14
30.95
 This workflow was applied to characterize a monoclonal antibody glycoforms.
Confident identification and structural confirmation was achieved for released
glycans from the monoclonal antibody.
References
1.
20.21
14.95
2.
Bigge, J. C. et al., Non-selective and efficient fluroscent labeling of glycans using
2-amino benzamide and anthranilic acid. Analytical Biochemistry, 1995, 230, 229238.
Apte, A and Meitei, N.S., Bioinformatics in glycomics: glycan characterization with
mass spectrometric data using SimGlycan, 2010, 600, 269-81.
SimGlycan is a registered trademark of PREMIER Biosoft International. 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 manners that might infringe the
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6 A Complete Workflow Solution for Monoclonal Antibody Glycoform Characterization Combininga Novel Glycan Column Technology and Bench-Top Orbitrap LC-MS/MS
www.thermoscientific.com
©2013 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization.
SimGlycan is a registered trademark of PREMIER Biosoft International. All other trademarks are the property of Thermo Fisher
Scientific and its subsidiaries. All other trademarks are the property of Thermo Fisher Scientific, Inc. and its subsidiaries.
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