Application Note
Primary Characterization of a Monoclonal
Antibody Using Agilent HPLC-Chip
Accurate-Mass LC/MS Technology
Authors
Ravindra Gudihal
Sudha Ragalopalan
Ranjit Gulge
Andrew Gieschen
Christine Miller
Ning Tang
Keith Waddell
Agilent Technologies, Inc.,
Santa Clara, CA, USA
The characterization of a monoclonal antibody at low nanogram levels using
an Agilent HPLC-Chip system coupled to an Agilent Accurate-Mass Q-TOF MS
instrument is described. The superior sensitivity and mass accuracy of the
HPLC-Chip LC/MS platform, combined with the powerful data processing
capabilities of Agilent MassHunter and BioConfirm software, enabled easy and
rapid identification of antibody heterogeneity and cleaved fragments. Robust,
reliable, and easy to use, Agilent nanoflow LC/MS technology is ideally suited
for the routine analysis of biopharmaceuticals.
Introduction
Recombinant monoclonal antibodies (mAbs) represent an important class of biopharmaceutical products with a wide range of diagnostic and clinical applications.
The worldwide market for monoclonal antibodies is projected to reach $26 billion by
2010–2011. These therapeutic glycoproteins are usually produced from mammalian
cell lines (hybridomas), then purified, concentrated, and exchanged into an appropriate
formulation buffer prior to being sold. Although mAbs are relatively stable biomolecules, a number of chemical modifications and degradation reactions can occur
during manufacturing, formulation, and storage, thereby necessitating reliable and
sensitive methods for the characterization of protein purity and structural integrity.
Accurate mass measurements of intact proteins, whole subunits, or domains are
useful for the rapid verification of sequence composition and identification of posttranslational modifications and sample handling artifacts.
LC/MS technology is a powerful and sensitive technique for the characterization and
identification of proteins. Nanoflow LC/MS enables faster and more sensitive protein
identification while minimizing sample and solvent consumption. Agilent’s microfluidicbased HPLC-Chip integrates sample preparation, chromatographic separation, and
nanoelectrospray formation for efficient, high-sensitivity nanospray LC/MS. Agilent
Accurate-Mass LC/MS TOF and Q-TOF systems deliver exceptional mass resolution,
Application Note
mass accuracy, sensitivity, and data processing capabilities for
optimal MS characterization of proteins. In this note, we report
the rapid LC/MS characterization of low nanogram levels of
an intact mAb and its fragments using the Agilent HPLC-Chip
system coupled to an Accurate-Mass Q-TOF.
facturer. Briefly, 0.3 µL of 100 U/mL solution of PNGase was
added to 3 µg of antibody in 20 mM Tris-HCl buffer pH 8.2. The
mixture was incubated at 37°C for 24 hr. Samples were appropriately diluted in 3% acetonitrile/water with 0.1%
formic acid solution prior to LC/MS analysis.
Experimental
Papain digestion
The papain was activated using freshly prepared activation
buffer (papain activation/digestion buffer: 1 mM EDTA, 50 mM
sodium phosphate buffer and 10 mM cysteine, pH 7.0) for 15
min at 37°C. Activated papain solution was then added to the
mAb in papain digestion buffer. The mAb-to-protease ratio
was 60:1(w/w). The mixture was incubated at 37°C for 2 hr.
Samples were appropriately diluted in 3% acetonitrile/water
with 0.1% formic acid solution prior to LC/MS analysis.
Materials
L-cysteine, EDTA disodium, sodium phosphate dibasic anhydrous, sodium phosphate monobasic, sodium bicarbonate,
papain suspension (in 0.05 M sodium acetate), and DL-dithiothreitol (DTT) were purchased from Sigma. PNGase and the
Tris reaction buffer pH 8.2 were obtained from Prozyme.
Deglycosylation reaction
Deglycosylation of the mAb was performed using PNGase
in 20 mM Tris-HCl buffer pH 8.2 as described by the manu-
2
This item is intended for Research Use Only. Not for use in diagnostic procedures.
Primary Characterization of a Monoclonal Antibody Using
Agilent HPLC-Chip Accurate-Mass LC/MS Technology
Reduction reaction
The mAb (1.5 µg) was reduced with 25 mM DTT at pH 8.6 for 1
hr at 80°C. Samples were appropriately diluted in 3% acetonitrile/
water with 0.1% formic acid solution prior to LC/MS analysis.
chip positioning to the MS source. The Agilent 6520 Q-TOF LC/
MS system incorporates True High-Definition TOF technology
to provide optimal sensitivity, mass accuracy, mass resolution,
and wide dynamic range.
LC/MS Analysis
LC parameters
HPLC-Chip: 5 µm, ZORBAX 300SB-C8 (300Å), 40 nL enrichment
column, and a 75 µm x 43 mm analytical column.
Instrumentation
The Agilent 1200 HPLC-Chip system was coupled with the
Agilent 6520 Accurate-Mass Q-TOF LC/MS platform for LC/MS
analyses (Figure 1). The HPLC-Chip is a polymer microfluidicbased device that integrates sample preparation (enrichment
column), analytical separation (analytical column), and nanoelectrospray formation (emitter tip). The enrichment column
helps in efficient desalting and concentration of the sample,
reducing sample consumption. The HPLC-Chip Cube interface
enables automatic chip loading, sample and solvent delivery
to the chip, high pressure switching of flows, and automated
Flow rate: 3 µL/min from capillary pump to the enrichment
column and 300 nL/min from nano LC pump to the analytical
column.
Solvents: 0.1% formic acid in water (A); 90% acetonitrile in
water with 0.1% formic acid (B). Flush volume was set at 7 µL
for effective desalting of the mAb sample.
Sample load: 7.5-10 ng on-column for intact and deglycosylated mAb while 5 ng on-column was used for reduced and
papain-generated fragments of the mAb.
HPLC-Chip
HPLC-Chip Cube
6520 Q-TOF
Figure 1. Agilent’s HPLC-Chip technology coupled to a 6520 Accurate-Mass Q-TOF MS instrument.
www.proteomics-lab.com
3
Application Note
Sample loading: With cap pump at 3% B.
Vcap: 1750 V and drying gas flow of 5 L/min at 325°C was
used.
Sample analysis: Gradient with nano pump as shown below.
Fragmentor voltage: 420 V for intact antibody/deglycosylated
form and 350 V for the fragments.
MS parameters
Spectra were recorded in positive ion mode and in profile mode
on the Agilent 6520 Accurate-Mass Q-TOF MS instrument.
Time (minutes)
B (%)
Initial
3
1
25
4
60
8
80
12
95
15
5
Data were acquired in standard (20,000 m/z), 1 GHz, MS only
mode, range 500-4,000 m/z (for fragments of mAb); 1,000-4,000
m/z (for intact mAb).
Data analysis
AgilentMass Hunter Qualitative Analysis software and the
MassHunter BioConfirm software (version B.02.00) were used.
Protein and fragment masses were obtained using maximum
entropy deconvolution. Average masses for intact mAb, light
chain, heavy chain, Fab, and Fc were calculated from sequences
using the Sequence Editor feature found in the BioConfirm
software.
Stop time: 15 min
x10 4
x10 4
2
2
2706.7422
1.9
2658.4255
2706.7422
2756.7152
1.5
2658.4255
2808.7909
1.8
1.7
1
2756.7152
2611.8165
1.6
2680.4239
0.5
2730.0683
2781.5349
1.5
0
2660 2680 2700 2720 2740 2760 2780 2800 2820
1.4
2808.7909
2566.7952
Counts vs. Mass-to-Charge (m/z)
1.3
1.2
1.1
2862.7840
1
2523.3067
0.9
0.8
2481.2595
0.7
2919.0798
0.6
0.5
2440.5988
2977.3333
0.4
2401.2639
0.3
3038.0612
2363.1768
3101.3321
3167.3332
0.2
0.1
3308.0414
0
2200
2250
2300
2350
2400
2450
2500
2550
2600
2650
2700
2750
2800
2850
2900
2950
3000
3050
3100
3150
3200
3250
3300
3350
3400
Counts vs. Mass-to-Charge (m/z)
Figure 2. Mass spectrum of intact antibody with inset showing the expanded view of charge states of the antibody.
4
This item is intended for Research Use Only. Not for use in diagnostic procedures.
Primary Characterization of a Monoclonal Antibody Using
Agilent HPLC-Chip Accurate-Mass LC/MS Technology
Results and Discussion
Analysis of Intact Antibody
Figure 2 shows the averaged mass spectrum of the intact antibody after elution from the HPLC-Chip. A very well distributed
series of peaks is seen corresponding to the m/z of the many
different charge state species of the intact antibody. Small
satellite peaks within each charge state are also observed
(see inset), corresponding to either adducts or modifications
of the mAb. To gain more insight into the possible modification/adduct, deconvolution of the intact mass spectrum was
performed using a maximum entropy algorithm (Figure 3).
The deconvoluted spectrum shows three major mass peaks
at 148812.81 Da (calculated mass from sequence is 148811.95
Da; measured mass accuracy of 5.7 ppm), 147367.94 Da, and
145922.00 Da. The mass differences between the peaks are
marked in Figure 3. The observed mass difference of 1444.87
Da (calculated mass=1445.35 Da) is attributed to one unit of
the glycan (G0F) attached to the mAb. Table 1 lists some of
the commonly found N-linked glycans in the immunoglobulin.
The other observed mass difference of 2890.81 Da (calculated
mass=2890.7 Da) corresponds to two such units of G0F glycan,
indicating that the mAb sample has three species: intact mAb
with a pair of G0F glycans (148812.81 Da), mAb with one unit
x10 4
G0F/G0F
148812.81
6.5
6
x104
8
5.5
G0F/G0F
7
5
148812.81
6
5
4.5
4
162.16 hexose unit
4
3
3.5
148840.65
2
3
1
2890.81
145922.00
0
2.5
G0F/G1F
148916.37
148974.97
148765.43
148750
148800
148850
148900
148950
Counts vs. Deconvoluted Mass (amu)
2
1.5
147367.94
1
1444.87
146329.69
0.5
146816.21
147719.65
0
145500
146000
146500
147000
147500
148000
148500
149000
149500
150000
150500
151000
151500
Counts vs. Deconvoluted Mass (amu)
Figure 3. Deconvoluted spectrum of intact antibody with inset showing the expanded view of a small amount of G1F (addition of hexose unit to G0F, see Table 1)
of the mAb.
www.proteomics-lab.com
5
Application Note
of G0F glycan (147367.94 Da), and mAb devoid of any Nlinked glycan part (145922.00 Da). Another peak (Figure 3) at
148974.97 Da was assigned to the addition of a hexose unit to
the G0F glycan part of the antibody (observed mass increase
of 162.16 Da; calculated mass is 162.14 Da), denoted as G1F in
Figure 3 (see Table 1).
Code
Analysis of deglycosylated antibody
To confirm the assignment of the peak mass for the unglycosylated species at mass 145922.00 Da, a deglycosylation
reaction of the mAb was performed. The mass spectrum of
the deglycosylated mAb is shown in Figure 4. The spectrum
shows well-defined charge state species with minimal adducts
or satellite peaks. The deconvoluted spectrum (Figure 5) has
a single molecular species at 145924.41 Da (calculated mass
Oligosaccharide structure
Average mass
Comments
G2F
1769.64
G1F
1607.49
Small amount found
in this mAb
G0F
1445.35
Major form found
in this mAb
G0
1299.21
Galactose
Mannose
Fucose
N-acetylglucosamine
Table 1. Structures of N-linked glycans commonly found in IgG molecules [2] .
x104
2
2654.2106
1.9
2703.3116
2606.8056
1.8
1.7
1.6
2561.0999
2754.3171
1.5
1.4
1.3
1.2
2516.9783
2807.2538
1.1
1
0.9
2474.3228
0.8
2862.2881
0.7
2433.1203
0.6
0.5
0.4
2393.2569
2919.5091
2354.6563
2317.3069
2281.1008
0.3
2979.0715
0.2
3041.1043
3105.7939
3173.2776
0.1
0
1950 2000 2050 2100 2150 2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3100 3150 3200 3250 3300
Counts vs. Mass-to-Charge (m/z)
Figure 4. Mass spectrum of deglycosylated antibody.
6
This item is intended for Research Use Only. Not for use in diagnostic procedures.
Primary Characterization of a Monoclonal Antibody Using
Agilent HPLC-Chip Accurate-Mass LC/MS Technology
Analysis of reduced antibody
To obtain more insight into the structure of the mAb, complete
reduction using DTT was performed. The deconvoluted spectra
of the light and heavy chains of the mAb are shown in Figure 6.
The light chain has a measured mass of 23746.5 Da (calculated
mass from sequence is 23746.63 Da; mass accuracy obtained
is -5.45 ppm) and the heavy chain has a measured mass of
50675.58 Da (calculated mass from sequence is 50675.68 Da;
mass accuracy obtained is 1.9 ppm).
from sequence, and accounting for N to D conversion which
occurs during deglycation reaction, is 145923.24 Da; mass
accuracy obtained is 8 ppm), indicating removal of all glycan
moieties from the mAb. The mass obtained is also in close
agreement with the mass assignment for the unglycosylated
form (145922.00 Da) of the mAb in Figure 3.
x10 4
145924.41
8
7
6
5
4
3
No glycan attached species
2
146272.08
1
0
144500
145000
145500
146000
146500
147000
147500
148000
148500
149000
149500
Counts vs. Deconvoluted Mass (amu)
Figure 5. Deconvoluted spectrum of deglycosylated antibody.
www.proteomics-lab.com
7
Application Note
Analysis of Fc and Fab fragments generated by
papain digestion
Papain is used to generate Fab and Fc fragments from a mAb
for various biotechnological/therapeutic applications. These
fragments are also useful for additional structural characterization of a mAb. The mass spectra and deconvoluted spectra for
Fc and Fab fragments are shown in Figures 7 and 8, respectively. Figure 7B further validates glycan attachment for the
intact mAb.
The Fc fragment has a measured mass of 52755.64 Da (calculated mass from sequence is 52755.62 Da; mass accuracy ob-
x10 5
23746.50
5
4
A
3
2
1
23727.82
23762.96
0
23540 23560 23580 23600 23620 23640 23660 23680 23700 23720 23740 23760 23780 23800 23820 23840 23860 23880 23900 23920 23940 23960 23980
Counts vs. Deonvoluted Mass (amu)
x10 4
50675.58
5
4
B
3
2
1446.6
49228.96
50838.33
162.7
1
49435.14
51023.43
50121.16
49920.85
49009.52
0
48600
48800
49000
49200
49400
49600
49800
50000
50200
50400
50600
50800
51000
51200
Counts vs. Deconvoluted Mass (amu)
Figure 6.
8
A) Deconvoluted spectrum of light chain of mAb (shown as blue bar).
B) Deconvoluted spectrum of heavy chain of mAb (shown as red bar with different sugar attachment).
This item is intended for Research Use Only. Not for use in diagnostic procedures.
Primary Characterization of a Monoclonal Antibody Using
Agilent HPLC-Chip Accurate-Mass LC/MS Technology
significantly less sample compared to other LC methods, as
shown in Table 2, making it well suited for the analysis of small
amounts of biotherapeutics during the R&D stage or during
product release.
tained is 0.38 ppm) and the Fab fragment has a measured mass
of 48046.09 Da (calculated mass from sequence is 48046.18
Da; mass accuracy obtained is -1.9 ppm).
Low sample consumption
In our analyses, the amount of sample consumed was in the
low nanogram level. The HPLC-Chip LC/MS method consumes
x10 4
1319.9174
2.4
2.3
1287.7236
2.2
1257.0902
2.1
2
1.9
1.8
1.7
1227.8910
1.6
1353.7175
1.5
1389.3139
1.4
A
1.3
1.2
1426.7975
1199.9867
1521.9735
1.1
1466.4584
1
0.9
1552.6289
1147.8771
0.8
0.7
0.6
1599.6583
1123.4876
0.5
1100.0744
0.4
0.3
0.2
0.1
0
1075
1100
1125
1150
1175
1200
1225
1250
1275
1300
1325
1350
1375
1400
1425
1450
1475
1500
1525
1550
1575
1600
1625
1650
Counts vs. Mass-to-Charge (m/z)
x105
x10 5
52755.64
1.8
1.9
1.6
1.8
1.4
1.7
1.2
1
1.6
162.47
0.8
1.5
B
52755.64
2
2
0.6
1.4
0.4
1.3
0.2
1.2
0
52918.11
52553.59
52200
1.1
52400
52600
53081.26
52800
53000
53200
53400
53600
Counts vs. Deconvoluted Mass (amu)
1
0.9
49864.71
2890.93
0.8
0.7
0.6
0.5
0.4
51309.87
0.3
47485.72
0.2
1445.8
50272.22
0.1
0
44000
44500
45000
45500
46000
46500
47000
47500
48000
48500
49000
49500
50000
50500
51000
51500
52000
52500
53000
53500
54000
54500
55000
Counts vs. Deconvoluted Mass (amu)
Figure 7.
A) Mass spectrum of Fc fragment of mAb.
B) Deconvoluted mass of Fc fragment of mAb; inset shows the expanded view of small peak.
www.proteomics-lab.com
9
Application Note
x10 5
1265.3752
1299.5473
1335.6141
1.5
1232.9565
1373.7449
1.25
1202.1551
1
1414.1268
1172.8591
A
0.75
1456.9391
1144.9538
1502.4368
0.5
1118.3572
1550.8752
1602.5404
0.25
1092.9598
1521.9702
0
1075
1100
1125
1150
1175
1200
1225
1250
1275
1300
1325
1350
1375
1400
1425
1450
1475
1500
1525
1550
1575
1600
1625
1650
Counts vs. Mass-to-Charge (m/z)
x10 6
48046.09
2
Fab fragments
1.5
B
1
0.5
52755.52
0
44000
45000
46000
47000
48000
49000
50000
51000
52000
53000
54000
55000
Counts vs. Deconvoluted Mass (amu)
Figure 8.
A) Mass spectrum of Fab fragment of mAb.
B) Deconvoluted mass of Fab fragment of mAb.
Column(dimension)
Amount of sample injected onto column
Reference
HPLC-Chip (43 × 0.075 mm)
5-10 ng
This note and [4]
Cap LC,C8 (50 × 1 mm)
~ 4 µg
[3]
Poroshell C8 (75 × 2.1 mm)
~200 ng
[4]
Table 2. Sample load on different columns.
10
This item is intended for Research Use Only. Not for use in diagnostic procedures.
Primary Characterization of a Monoclonal Antibody Using
Agilent HPLC-Chip Accurate-Mass LC/MS Technology
Conclusions
Rapid and sensitive characterization of an intact mAb and its
fragments at low nanogram levels has been demonstrated
using Agilent’s microfluidic HPLC-Chip/MS system coupled to
the Agilent 6520 Accurate-Mass Q-TOF MS platform. The high
sensitivity and sub-10 ppm mass accuracy of the nanoflow LC/
MS system enabled identification of mAb heterogeneity, confirmation of mAb deglycosylation, and identification of Fab and
Fc fragments. This approach is ideal for the routine analysis of
small amounts of expensive and valuable biopharmaceuticals.
www.proteomics-lab.com
References
1. www.drugresearcher.com.
2. Gadgil HS, Pipes GD, Dillon TM, Treuheit MJ, Bondarenko PV. J Am Soc
Mass Spectrom. 2006 Jun; 17(6):867-72.
3. Gadgil HS, Bondarenko PV, Pipes GD, Dillon TM, Banks D, Abel J,
Kleemann GR, Treuheit MJ. Anal Biochem. 2006 Aug; 355(2):165-74.
4. Dayin Lin and Hongbin Liu, Protein Society 2008 Poster 236, HPLC-Chip
QTOF Analysis of MAb.
11
Application Note
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© Agilent Technologies, Inc. 2008
Published in the U.S.A. December 16, 2008
5990-3445EN