Post on 25-Jul-2020
Middle-down Analysis of Monoclonal Antibody Middle using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerJie Qian,1 Keith A. Waddell,2 Zhiqi Hao2 1Thermo Fisher Scientific, Somerset, NJ, 2Thermo Fisher Scientific San Jose, CA
2 Middle-down Analysis of Monoclonal Antibody Middle using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass Spectrometer
Middle-down Analysis of Monoclonal Antibody using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerMiddle down Analysis of Monoclonal Antibody using Nano flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerJie Qian 1, Keith A. Waddell 2, Zhiqi Hao 2
Thermo Fisher Scientific, 1 Somerset, New Jersey, 2 San Jose, CaliforniaThermo Fisher Scientific, Somerset, New Jersey, San Jose, California
Overview Purpose: Sensitive analysis of monoclonal antibody using middle-down approach on a novel Thermo Scientific™ Orbitrap FusionTM TribridTM mass spectrometer
ResultsFull Mass Spectrum of Intact mAb
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC-MS. Data
Top Down Analysis of Proteolytic Fragmented MAb
Middle down analysis of mAb protein was performed by LC-MS analysis of the proteolytic fragments. Precursor ions at m/z 964.65 of light chain, m/z 1117.87 of Fd’ and m/z 902.20 of the G0F glycoform of Fc/2 were selected respectively for ETD and HCD
FIGURE 4. Full MS spectrum of mAb fragments at 240,000 resolution. (A) Full MS spectrum of Fc/2; (B) Full MS spectrum of light chain; (C) Full MS spectrum of Fd’.
100
935.7325z=27
1270 9351
NL: 1.39E7T: FTMS + p NSI Full
FIGURE 9. Comparison of experimental C57 ion of Fc/2+G0F chain to theoretically predicted isotope distribution. This ion is critical for identifying and localizing G0F on Asn61 residue.
719.3735719 2621Methods: A method combining nano-flow liquid chromatography and complementary higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) fragmentation on an Orbitrap Fusion Mass Spectrometer was developed and implemented
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd’
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC MS. Data shown below was acquired at 15,000 resolution on Orbitrap Fusion MS with source CID at 70. The application of 40-80 source CID is beneficial for most mAb proteins to help remove the adducts and thus promote a cleaner signal. The optimal SID setting is protein dependent.
g y p yMS/MS at 120,000 resolution. Reaction time of 3-5 ms was applied for ETD fragmentation. The normalized collision energy for HCD was 15-25 %. As shown in Figure 7, tandem spectra generated contain well resolved, multiply charged fragment ions. Interpretation of these ions based on the mAb protein sequence was performed using ProSightPC 3.0. The combined results of ETD and HCD suggest 50% sequence
20
40
60
80
100 1270.9351z=20
1329.0863z=19
pms [600.00-2000.00] (A) Fc/2
60
80
100
Abu
ndan
ce
z=9719.2621
z=9
719.4851z=9
719.1510z 9 719.5956
C57, Experimental<3ppm
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd , Fc/2 and light chain respectively. Intact masses of the monoclonal antibody and its proteolytic fragments were measured. The proteolytic fragments (Fd’, Fc/2 and light chains) were directly fragmented using HCD and ETD. An average 50% amino acid coverage was achieved and glycosylation site was unambiguously identified
FIGURE 1. Full MS spectrum of the intact mAb (candidate NIST RM 8670 mAblot #3F1b) obtained from LC-MS analysis.
coverage for light chain, 52% coverage for Fc/2 with G0F and 32% coverage for Fd’ respectively (Figure 8, 10, 11). N-terminal modification of pyroglutamate of Fd’chain was confirmed based on fragment ions from both ETD and HCD. Both intact and tandem spectra identified the Lys loss at C-terminus of Fc/2.
ETD is widely known for its advantage in preferentially fragmenting the peptide back bone
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
90
100 2965.00372906.8879
3025 5167
80
100
ndan
ce
0
20
1052.2509z=22 1218.2364
z=191361.4397
z=17890.4831
26
NL: 2.67E7T: FTMS + p NSI Full ms [600.00-2000.00] (B) Light chain
0
20
40
Rel
ativ
e A z=9 719.5956
z=9719.0392z=9 719.7081
z=9719.8182
z=9
719 2635719 3749
IntroductionMonoclonal antibodies (mAbs) are an increasingly important line of therapeutics for the
y g p y g g p pand keeping the labile modifications attached. It has been recognized as the method of choice for locating the sites of such labile PTMs including glycosylation. Multiple identified ETD fragments between Asn61 and Asn79 unambiguously located the glycan G0F on Asn61 of Fc/2 chain. In the highly complex tandem spectrum, the high resolution and accurate mass allows and is also necessary for confident identification of low abundance 30
40
50
60
70
80
90
Rel
ativ
e A
bund
ance
3025.5167
2851.0285
2797.26273154.1717
2695.5589 3222.7437
3294.30832647.47943369 25012601 0195
0
20
40
60
Rel
ativ
e A
bun z=17z=26
1928.2022z=121652.9607
z=14
1224.2858 NL: 1.75E7 60
80
100719.2635
z=9 719.3749z=9
719.4863z=9
719.1521z=9
719.5977z=9719.0407
C57, Theoretical
biopharmaceutical industry. The demand to better understand the biochemical and biophysical properties of mAbs has become critical. Recent developments in high resolution mass spectrometry (MS) with multiple dissociation techniques have clearly shown its distinctive power for characterization of intact proteins and in particular its subunits. The mass spectrometry–based study of mAbs in middle-down approach
f f f
glycan-containing fragment ions in the presence of interference. For example, shown in Figure 7 insert is the identification of the +9 charged C61 ion with G0F; Figure 9 presents the unambiguous identification of C57 without G0F. Although not all the isotopic peaks of this c ion were observed due to background interference, all seven isotopes identified were within 3 ppm mass error (external mass calibration). Both ETD ions, C61 and C57,
2400 2600 2800 3000 3200 3400 3600 3800 4000m/z
0
10
203369.25012601.0195
2558.9576 3447.50882430.4122 3615.6678 3805.1241 3912.0528
FIGURE 2. Expanded view of full MS spectrum of the intact mAb. Different glycoforms at charge +51 was shown 40
60
80
100 z=211428.1664
z=181511.9984
z=171976.9174
z=13
T: FTMS + p NSI Full ms [600.00-2000.00] (C) Fd’
0
20
40
719.0407z=9 719.7090
z=9719.8204
z=9718.9293z=9
provides a wealth of information to interpret its structural features. Here, we describe the use of an Orbitrap Fusion mass analyzer in combination with nano liquid chromatography (LC) for characterizing a mAb protein using different dissociation techniques.
have played critical roles in identification and localization of G0F on Asn61 residue.FIGURE 10. ETD (blue) and HCD (red) coverage of light chain.
FIGURE 5 Isotopically resolved mAb fragments at 240 000 resolution (A)
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
70
80
90
100
nce
2906.8879
2910.1479
glycoforms at charge +51 was shown.
1000 1500 2000m/z
0
20z 13
FIGURE 7. ETD spectrum of Fc/2 with G0F with precursor ion as m/z 902.20, charge +28. The insert is an expanded view of the spectrum covering C61 ion at charge +9 with 3ppm mass accuracy, which confirms the addition of G0F
719.0 719.2 719.4 719.6 719.8
MethodsSample Preparation
Th i t t Ab t i ti ll l d b l th hi i i t
FIGURE 5. Isotopically resolved mAb fragments at 240,000 resolution. (A) Fc/2+G1F, charge +28; (B) Light Chain, charge 21; (C) Fd’, charge 21.
60
70
80
90
100
unda
nce
939.2334z=9
939.1216z=9
939.3439z=9
939.5667z=9
90
100764.9279
z=2
656 8907
+9, C61 with G0F, <3ppm
0
10
20
30
40
50
60
Rel
ativ
e A
bund
an 2903.7472
2913.3612
2916.46952919.3575
2923.21022899.8132 2925.8599 2932.27852895.7333
T: FTMS + p NSI Full ms [600.00-2000.00]
100
908.0636z=28907.9929
z=28908.1366
z=28 908 2076907 9207 (A) F /2+G1F
glycan.
The intact mAb protein was enzymatically cleaved below the hinge region into a F(ab')2 fragment and two Fc fragments. Aliquot of candidate NIST RM 8670 mAb lot #3F1b was treated with FabRICATOR® (Genovis, Sweden) at 37 oC for 1 hour. The resulting F(ab’)2 and Fc fragments were further denatured and reduced in 50mM dithiothreitol (Sigma, Saint Louis, MO) at 56 oC for 1 hour. The proteolyticallyfragmented mAb was diluted to 1 5 µg/µL using 0 1% formic acid in water 0
10
20
30
40
50
Rel
ativ
e A
b
70
80
90
nce
656.8907z=2
Intact LC-MS Analysis of Proteolytic Fragmented mAb
A mixture including approximately 20 pmol of Fd’ Fc/2 and light chain respectively
2895 2900 2905 2910 2915 2920 2925 2930 2935m/z
0
907 7 907 8 907 9 908 0 908 1 908 2 908 3 908 4 908 5 908 60
20
40
60
80
100
Rel
ativ
e A
bund
ance
z 28 z=28 908.2076z=28
907.9207z=28
908.3156z=28907.7781
z=28
(A) Fc/2+G1F
fragmented mAb was diluted to 1.5 µg/µL using 0.1% formic acid in water.
Liquid Chromatography
Fd' and Fc/2 fragments after reduction were chromatographically eluted from a Thermo Scientific™ PepSwift™ column, Monolithic easy spray column (200 µm x 25 cm, Thermo Fisher Scientific Amsterdam the Netherlands) One µL of the stock was
FIGURE 11. ETD (blue) and HCD (red) coverage of Fd’ chain.
939.0 939.2 939.4 939.6m/z
40
50
60
elat
ive
Abu
ndan
957.7858z=3
1203.0937z=4
924.4734z=6 1073 0261
A mixture including approximately 20 pmol of Fd , Fc/2 and light chain, respectively, was eluted at 800 nl/min and directly analyzed at 240,000 resolution on Orbitrap Fusion MS. The mAb fragments were separated by PepSwift monolithic column. For a 800 nl/min gradient of 5-60% in 32 minutes, different glycoforms of Fc/2 were first eluted at 20.67minute, followed by light chain at 22.34minute. Eluted last was Fd’ chain at 24.76min.
907.7 907.8 907.9 908.0 908.1 908.2 908.3 908.4 908.5 908.6m/z
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
ndan
ce
1102.3104z=21 1102.4048
z=211102.2147z=21
1102.4524z=211102.1195
21
(B) Light chain
Thermo Fisher Scientific, Amsterdam, the Netherlands). One µL of the stock was loaded per injection. Nano flow reverse phase chromatography was performed with a 800 nl/minute gradient of 5-60% in 32minutes using the Thermo Scientific™ EASY-nLC™ 1000 system. The proteins were directly detected by a standard Orbitrap fusion mass spectrometer. Liquid chromatography solvents used include the aqueous as 0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 10
20
30
Re z 6 1073.0261
z=6618.3590
z=1358.2070
z=1814.0227
z=51528.8462
z=11287.6293z=5
Full MS spectra acquired at 240,000 resolution provides base line resolution of the isotope distribution of the ~25,000Da mAb fragments. The isotopically resolved spectra were deconvoluted for monoisotopic masses. Figure 6 shows the deconvoluted monoisotopic mass of the light chain is 23113.3568Da, which suggests a mass accuracy of 2 2ppm comparing to its theoretical monoisotopic mass at
1101.8 1102.0 1102.2 1102.4 1102.6 1102.8m/z
0
20
40
60
Rel
ativ
e A
bun z=21 1102.5968
z=211102.0254z=21 1102.6899
z=21
RT: 18.71 - 27.44 SM: 7G20 67 NL:
0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 0.1% formic acid (Fisher Scientific, Fair Lawn, New Jersey) in acetronitirile (Fisher Scientific, Fair Lawn, New Jersey).
Mass Spectrometry
The Fd’, Fc/2 and light chain eluted were directly detected by a standard Orbitrap
200 400 600 800 1000 1200 1400m/z
0
10mass accuracy of 2.2ppm comparing to its theoretical monoisotopic mass at 23113.3041Da. FIGURE 3. Total Ion Current Chromatogram of MAb Fragments Eluted from a PepSwift Monolithic Nano Column.
Fc/2
T: FTMS + p NSI Full ms [600.00-2000.00]
40
60
80
100
ve A
bund
ance
1224.1915z=21
1224.2859z=21
1224.0952z=21
1224.4296z=21
1224.5247z=21
1224.0009z=21
1224.6193z=211223.9055
(C) Fd’
70
80
90
100
nce
20.67 NL:2.47E10TIC MS frg_89
ConclusionThe middle-down study indicated that complementary fragmentation mechanisms allow
g y y pFusion MS under both full scan mode and tandem scan mode using higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD). Full mass spectra of mAb fragments were acquired at 240,000 resolution at m/z 200 with mass range m/z 400-2000. AGC setting for full MS spectrum was at 1e5 with 100ms maximum injection. Ion transferring temperature was set at 300 oC. Full mass spectra
Li ht Ch i
1223.8 1224.0 1224.2 1224.4 1224.6 1224.8m/z
0
20
Rel
ativ z=21
z=211223.8147z=21
FIGURE 6. Deconvoluted monoisotopic mass of light chain.
FIGURE 8. ETD (blue) and HCD (red) coverage of Fc/2+G0F chain. Asn61 was highlighted for addition of G0F.
30
40
50
60
Rel
ativ
e A
bund
an
22.34
22.60 24.76
extensive sequence coverage as well as identification and localization of PTMs including labile glycosylation. The middle-down approach produced detailed structure information deep into the middle of the mAb chains especially in the epitope region.
Acknowledgements
of intact mAb were acquired at 15,000 resolution at m/z 200 with mass range m/z 1000-6000.
Data Analysis
The full mass spectra were analyzed with Thermo ScientificTM Protein DeconvolutionTM
3 0 ft f l l d t i ti Th t d t f Fd’ F /2
Light ChainFd’
Theoretical monoisotpic mass-23113.3041DaMass acccuracy with external calibration-2.2ppm
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
danc
e
23113.3568
19 20 21 22 23 24 25 26 27Time (min)
0
10
20AcknowledgementsWe would like to thank National Institute of Standards and Technology for providing the intact mAb protein sample (candidate NIST RM 8670 mAb lot #3F1b).
3.0 software for molecular mass determination . The tandem mass spectra of Fd’, Fc/2 and light chain were deconvoluted by Xtract. The deconvoluted spectra were processed by ProSightPC 3.0 software for middle-down data interpretation.
22700 22800 22900 23000 23100 23200m/z
0
20
40
60
Rel
ativ
e A
bund
23130.328223096.2937
FabRICATOR® is a registered trademark for recombinant IdeS. 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 intellectual property rights of others.
PO64147-EN 0614S
3Thermo Scientific Poster Note • PN-64147-ASMS-EN-0614S
Middle-down Analysis of Monoclonal Antibody using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerMiddle down Analysis of Monoclonal Antibody using Nano flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerJie Qian 1, Keith A. Waddell 2, Zhiqi Hao 2
Thermo Fisher Scientific, 1 Somerset, New Jersey, 2 San Jose, CaliforniaThermo Fisher Scientific, Somerset, New Jersey, San Jose, California
Overview Purpose: Sensitive analysis of monoclonal antibody using middle-down approach on a novel Thermo Scientific™ Orbitrap FusionTM TribridTM mass spectrometer
ResultsFull Mass Spectrum of Intact mAb
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC-MS. Data
Top Down Analysis of Proteolytic Fragmented MAb
Middle down analysis of mAb protein was performed by LC-MS analysis of the proteolytic fragments. Precursor ions at m/z 964.65 of light chain, m/z 1117.87 of Fd’ and m/z 902.20 of the G0F glycoform of Fc/2 were selected respectively for ETD and HCD
FIGURE 4. Full MS spectrum of mAb fragments at 240,000 resolution. (A) Full MS spectrum of Fc/2; (B) Full MS spectrum of light chain; (C) Full MS spectrum of Fd’.
100
935.7325z=27
1270 9351
NL: 1.39E7T: FTMS + p NSI Full
FIGURE 9. Comparison of experimental C57 ion of Fc/2+G0F chain to theoretically predicted isotope distribution. This ion is critical for identifying and localizing G0F on Asn61 residue.
719.3735719 2621Methods: A method combining nano-flow liquid chromatography and complementary higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) fragmentation on an Orbitrap Fusion Mass Spectrometer was developed and implemented
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd’
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC MS. Data shown below was acquired at 15,000 resolution on Orbitrap Fusion MS with source CID at 70. The application of 40-80 source CID is beneficial for most mAb proteins to help remove the adducts and thus promote a cleaner signal. The optimal SID setting is protein dependent.
g y p yMS/MS at 120,000 resolution. Reaction time of 3-5 ms was applied for ETD fragmentation. The normalized collision energy for HCD was 15-25 %. As shown in Figure 7, tandem spectra generated contain well resolved, multiply charged fragment ions. Interpretation of these ions based on the mAb protein sequence was performed using ProSightPC 3.0. The combined results of ETD and HCD suggest 50% sequence
20
40
60
80
100 1270.9351z=20
1329.0863z=19
pms [600.00-2000.00] (A) Fc/2
60
80
100
Abu
ndan
ce
z=9719.2621
z=9
719.4851z=9
719.1510z 9 719.5956
C57, Experimental<3ppm
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd , Fc/2 and light chain respectively. Intact masses of the monoclonal antibody and its proteolytic fragments were measured. The proteolytic fragments (Fd’, Fc/2 and light chains) were directly fragmented using HCD and ETD. An average 50% amino acid coverage was achieved and glycosylation site was unambiguously identified
FIGURE 1. Full MS spectrum of the intact mAb (candidate NIST RM 8670 mAblot #3F1b) obtained from LC-MS analysis.
coverage for light chain, 52% coverage for Fc/2 with G0F and 32% coverage for Fd’ respectively (Figure 8, 10, 11). N-terminal modification of pyroglutamate of Fd’chain was confirmed based on fragment ions from both ETD and HCD. Both intact and tandem spectra identified the Lys loss at C-terminus of Fc/2.
ETD is widely known for its advantage in preferentially fragmenting the peptide back bone
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
90
100 2965.00372906.8879
3025 5167
80
100
ndan
ce
0
20
1052.2509z=22 1218.2364
z=191361.4397
z=17890.4831
26
NL: 2.67E7T: FTMS + p NSI Full ms [600.00-2000.00] (B) Light chain
0
20
40
Rel
ativ
e A z=9 719.5956
z=9719.0392z=9 719.7081
z=9719.8182
z=9
719 2635719 3749
IntroductionMonoclonal antibodies (mAbs) are an increasingly important line of therapeutics for the
y g p y g g p pand keeping the labile modifications attached. It has been recognized as the method of choice for locating the sites of such labile PTMs including glycosylation. Multiple identified ETD fragments between Asn61 and Asn79 unambiguously located the glycan G0F on Asn61 of Fc/2 chain. In the highly complex tandem spectrum, the high resolution and accurate mass allows and is also necessary for confident identification of low abundance 30
40
50
60
70
80
90
Rel
ativ
e A
bund
ance
3025.5167
2851.0285
2797.26273154.1717
2695.5589 3222.7437
3294.30832647.47943369 25012601 0195
0
20
40
60
Rel
ativ
e A
bun z=17z=26
1928.2022z=121652.9607
z=14
1224.2858 NL: 1.75E7 60
80
100719.2635
z=9 719.3749z=9
719.4863z=9
719.1521z=9
719.5977z=9719.0407
C57, Theoretical
biopharmaceutical industry. The demand to better understand the biochemical and biophysical properties of mAbs has become critical. Recent developments in high resolution mass spectrometry (MS) with multiple dissociation techniques have clearly shown its distinctive power for characterization of intact proteins and in particular its subunits. The mass spectrometry–based study of mAbs in middle-down approach
f f f
glycan-containing fragment ions in the presence of interference. For example, shown in Figure 7 insert is the identification of the +9 charged C61 ion with G0F; Figure 9 presents the unambiguous identification of C57 without G0F. Although not all the isotopic peaks of this c ion were observed due to background interference, all seven isotopes identified were within 3 ppm mass error (external mass calibration). Both ETD ions, C61 and C57,
2400 2600 2800 3000 3200 3400 3600 3800 4000m/z
0
10
203369.25012601.0195
2558.9576 3447.50882430.4122 3615.6678 3805.1241 3912.0528
FIGURE 2. Expanded view of full MS spectrum of the intact mAb. Different glycoforms at charge +51 was shown 40
60
80
100 z=211428.1664
z=181511.9984
z=171976.9174
z=13
T: FTMS + p NSI Full ms [600.00-2000.00] (C) Fd’
0
20
40
719.0407z=9 719.7090
z=9719.8204
z=9718.9293z=9
provides a wealth of information to interpret its structural features. Here, we describe the use of an Orbitrap Fusion mass analyzer in combination with nano liquid chromatography (LC) for characterizing a mAb protein using different dissociation techniques.
have played critical roles in identification and localization of G0F on Asn61 residue.FIGURE 10. ETD (blue) and HCD (red) coverage of light chain.
FIGURE 5 Isotopically resolved mAb fragments at 240 000 resolution (A)
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
70
80
90
100
nce
2906.8879
2910.1479
glycoforms at charge +51 was shown.
1000 1500 2000m/z
0
20z 13
FIGURE 7. ETD spectrum of Fc/2 with G0F with precursor ion as m/z 902.20, charge +28. The insert is an expanded view of the spectrum covering C61 ion at charge +9 with 3ppm mass accuracy, which confirms the addition of G0F
719.0 719.2 719.4 719.6 719.8
MethodsSample Preparation
Th i t t Ab t i ti ll l d b l th hi i i t
FIGURE 5. Isotopically resolved mAb fragments at 240,000 resolution. (A) Fc/2+G1F, charge +28; (B) Light Chain, charge 21; (C) Fd’, charge 21.
60
70
80
90
100
unda
nce
939.2334z=9
939.1216z=9
939.3439z=9
939.5667z=9
90
100764.9279
z=2
656 8907
+9, C61 with G0F, <3ppm
0
10
20
30
40
50
60
Rel
ativ
e A
bund
an 2903.7472
2913.3612
2916.46952919.3575
2923.21022899.8132 2925.8599 2932.27852895.7333
T: FTMS + p NSI Full ms [600.00-2000.00]
100
908.0636z=28907.9929
z=28908.1366
z=28 908 2076907 9207 (A) F /2+G1F
glycan.
The intact mAb protein was enzymatically cleaved below the hinge region into a F(ab')2 fragment and two Fc fragments. Aliquot of candidate NIST RM 8670 mAb lot #3F1b was treated with FabRICATOR® (Genovis, Sweden) at 37 oC for 1 hour. The resulting F(ab’)2 and Fc fragments were further denatured and reduced in 50mM dithiothreitol (Sigma, Saint Louis, MO) at 56 oC for 1 hour. The proteolyticallyfragmented mAb was diluted to 1 5 µg/µL using 0 1% formic acid in water 0
10
20
30
40
50
Rel
ativ
e A
b
70
80
90
nce
656.8907z=2
Intact LC-MS Analysis of Proteolytic Fragmented mAb
A mixture including approximately 20 pmol of Fd’ Fc/2 and light chain respectively
2895 2900 2905 2910 2915 2920 2925 2930 2935m/z
0
907 7 907 8 907 9 908 0 908 1 908 2 908 3 908 4 908 5 908 60
20
40
60
80
100
Rel
ativ
e A
bund
ance
z 28 z=28 908.2076z=28
907.9207z=28
908.3156z=28907.7781
z=28
(A) Fc/2+G1F
fragmented mAb was diluted to 1.5 µg/µL using 0.1% formic acid in water.
Liquid Chromatography
Fd' and Fc/2 fragments after reduction were chromatographically eluted from a Thermo Scientific™ PepSwift™ column, Monolithic easy spray column (200 µm x 25 cm, Thermo Fisher Scientific Amsterdam the Netherlands) One µL of the stock was
FIGURE 11. ETD (blue) and HCD (red) coverage of Fd’ chain.
939.0 939.2 939.4 939.6m/z
40
50
60
elat
ive
Abu
ndan
957.7858z=3
1203.0937z=4
924.4734z=6 1073 0261
A mixture including approximately 20 pmol of Fd , Fc/2 and light chain, respectively, was eluted at 800 nl/min and directly analyzed at 240,000 resolution on Orbitrap Fusion MS. The mAb fragments were separated by PepSwift monolithic column. For a 800 nl/min gradient of 5-60% in 32 minutes, different glycoforms of Fc/2 were first eluted at 20.67minute, followed by light chain at 22.34minute. Eluted last was Fd’ chain at 24.76min.
907.7 907.8 907.9 908.0 908.1 908.2 908.3 908.4 908.5 908.6m/z
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
ndan
ce
1102.3104z=21 1102.4048
z=211102.2147z=21
1102.4524z=211102.1195
21
(B) Light chain
Thermo Fisher Scientific, Amsterdam, the Netherlands). One µL of the stock was loaded per injection. Nano flow reverse phase chromatography was performed with a 800 nl/minute gradient of 5-60% in 32minutes using the Thermo Scientific™ EASY-nLC™ 1000 system. The proteins were directly detected by a standard Orbitrap fusion mass spectrometer. Liquid chromatography solvents used include the aqueous as 0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 10
20
30
Re z 6 1073.0261
z=6618.3590
z=1358.2070
z=1814.0227
z=51528.8462
z=11287.6293z=5
Full MS spectra acquired at 240,000 resolution provides base line resolution of the isotope distribution of the ~25,000Da mAb fragments. The isotopically resolved spectra were deconvoluted for monoisotopic masses. Figure 6 shows the deconvoluted monoisotopic mass of the light chain is 23113.3568Da, which suggests a mass accuracy of 2 2ppm comparing to its theoretical monoisotopic mass at
1101.8 1102.0 1102.2 1102.4 1102.6 1102.8m/z
0
20
40
60
Rel
ativ
e A
bun z=21 1102.5968
z=211102.0254z=21 1102.6899
z=21
RT: 18.71 - 27.44 SM: 7G20 67 NL:
0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 0.1% formic acid (Fisher Scientific, Fair Lawn, New Jersey) in acetronitirile (Fisher Scientific, Fair Lawn, New Jersey).
Mass Spectrometry
The Fd’, Fc/2 and light chain eluted were directly detected by a standard Orbitrap
200 400 600 800 1000 1200 1400m/z
0
10mass accuracy of 2.2ppm comparing to its theoretical monoisotopic mass at 23113.3041Da. FIGURE 3. Total Ion Current Chromatogram of MAb Fragments Eluted from a PepSwift Monolithic Nano Column.
Fc/2
T: FTMS + p NSI Full ms [600.00-2000.00]
40
60
80
100
ve A
bund
ance
1224.1915z=21
1224.2859z=21
1224.0952z=21
1224.4296z=21
1224.5247z=21
1224.0009z=21
1224.6193z=211223.9055
(C) Fd’
70
80
90
100
nce
20.67 NL:2.47E10TIC MS frg_89
ConclusionThe middle-down study indicated that complementary fragmentation mechanisms allow
g y y pFusion MS under both full scan mode and tandem scan mode using higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD). Full mass spectra of mAb fragments were acquired at 240,000 resolution at m/z 200 with mass range m/z 400-2000. AGC setting for full MS spectrum was at 1e5 with 100ms maximum injection. Ion transferring temperature was set at 300 oC. Full mass spectra
Li ht Ch i
1223.8 1224.0 1224.2 1224.4 1224.6 1224.8m/z
0
20
Rel
ativ z=21
z=211223.8147z=21
FIGURE 6. Deconvoluted monoisotopic mass of light chain.
FIGURE 8. ETD (blue) and HCD (red) coverage of Fc/2+G0F chain. Asn61 was highlighted for addition of G0F.
30
40
50
60
Rel
ativ
e A
bund
an
22.34
22.60 24.76
extensive sequence coverage as well as identification and localization of PTMs including labile glycosylation. The middle-down approach produced detailed structure information deep into the middle of the mAb chains especially in the epitope region.
Acknowledgements
of intact mAb were acquired at 15,000 resolution at m/z 200 with mass range m/z 1000-6000.
Data Analysis
The full mass spectra were analyzed with Thermo ScientificTM Protein DeconvolutionTM
3 0 ft f l l d t i ti Th t d t f Fd’ F /2
Light ChainFd’
Theoretical monoisotpic mass-23113.3041DaMass acccuracy with external calibration-2.2ppm
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
danc
e
23113.3568
19 20 21 22 23 24 25 26 27Time (min)
0
10
20AcknowledgementsWe would like to thank National Institute of Standards and Technology for providing the intact mAb protein sample (candidate NIST RM 8670 mAb lot #3F1b).
3.0 software for molecular mass determination . The tandem mass spectra of Fd’, Fc/2 and light chain were deconvoluted by Xtract. The deconvoluted spectra were processed by ProSightPC 3.0 software for middle-down data interpretation.
22700 22800 22900 23000 23100 23200m/z
0
20
40
60
Rel
ativ
e A
bund
23130.328223096.2937
FabRICATOR® is a registered trademark for recombinant IdeS. 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 intellectual property rights of others.
PO64147-EN 0614S
4 Middle-down Analysis of Monoclonal Antibody Middle using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass Spectrometer
Middle-down Analysis of Monoclonal Antibody using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerMiddle down Analysis of Monoclonal Antibody using Nano flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerJie Qian 1, Keith A. Waddell 2, Zhiqi Hao 2
Thermo Fisher Scientific, 1 Somerset, New Jersey, 2 San Jose, CaliforniaThermo Fisher Scientific, Somerset, New Jersey, San Jose, California
Overview Purpose: Sensitive analysis of monoclonal antibody using middle-down approach on a novel Thermo Scientific™ Orbitrap FusionTM TribridTM mass spectrometer
ResultsFull Mass Spectrum of Intact mAb
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC-MS. Data
Top Down Analysis of Proteolytic Fragmented MAb
Middle down analysis of mAb protein was performed by LC-MS analysis of the proteolytic fragments. Precursor ions at m/z 964.65 of light chain, m/z 1117.87 of Fd’ and m/z 902.20 of the G0F glycoform of Fc/2 were selected respectively for ETD and HCD
FIGURE 4. Full MS spectrum of mAb fragments at 240,000 resolution. (A) Full MS spectrum of Fc/2; (B) Full MS spectrum of light chain; (C) Full MS spectrum of Fd’.
100
935.7325z=27
1270 9351
NL: 1.39E7T: FTMS + p NSI Full
FIGURE 9. Comparison of experimental C57 ion of Fc/2+G0F chain to theoretically predicted isotope distribution. This ion is critical for identifying and localizing G0F on Asn61 residue.
719.3735719 2621Methods: A method combining nano-flow liquid chromatography and complementary higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) fragmentation on an Orbitrap Fusion Mass Spectrometer was developed and implemented
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd’
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC MS. Data shown below was acquired at 15,000 resolution on Orbitrap Fusion MS with source CID at 70. The application of 40-80 source CID is beneficial for most mAb proteins to help remove the adducts and thus promote a cleaner signal. The optimal SID setting is protein dependent.
g y p yMS/MS at 120,000 resolution. Reaction time of 3-5 ms was applied for ETD fragmentation. The normalized collision energy for HCD was 15-25 %. As shown in Figure 7, tandem spectra generated contain well resolved, multiply charged fragment ions. Interpretation of these ions based on the mAb protein sequence was performed using ProSightPC 3.0. The combined results of ETD and HCD suggest 50% sequence
20
40
60
80
100 1270.9351z=20
1329.0863z=19
pms [600.00-2000.00] (A) Fc/2
60
80
100
Abu
ndan
ce
z=9719.2621
z=9
719.4851z=9
719.1510z 9 719.5956
C57, Experimental<3ppm
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd , Fc/2 and light chain respectively. Intact masses of the monoclonal antibody and its proteolytic fragments were measured. The proteolytic fragments (Fd’, Fc/2 and light chains) were directly fragmented using HCD and ETD. An average 50% amino acid coverage was achieved and glycosylation site was unambiguously identified
FIGURE 1. Full MS spectrum of the intact mAb (candidate NIST RM 8670 mAblot #3F1b) obtained from LC-MS analysis.
coverage for light chain, 52% coverage for Fc/2 with G0F and 32% coverage for Fd’ respectively (Figure 8, 10, 11). N-terminal modification of pyroglutamate of Fd’chain was confirmed based on fragment ions from both ETD and HCD. Both intact and tandem spectra identified the Lys loss at C-terminus of Fc/2.
ETD is widely known for its advantage in preferentially fragmenting the peptide back bone
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
90
100 2965.00372906.8879
3025 5167
80
100nd
ance
0
20
1052.2509z=22 1218.2364
z=191361.4397
z=17890.4831
26
NL: 2.67E7T: FTMS + p NSI Full ms [600.00-2000.00] (B) Light chain
0
20
40
Rel
ativ
e A z=9 719.5956
z=9719.0392z=9 719.7081
z=9719.8182
z=9
719 2635719 3749
IntroductionMonoclonal antibodies (mAbs) are an increasingly important line of therapeutics for the
y g p y g g p pand keeping the labile modifications attached. It has been recognized as the method of choice for locating the sites of such labile PTMs including glycosylation. Multiple identified ETD fragments between Asn61 and Asn79 unambiguously located the glycan G0F on Asn61 of Fc/2 chain. In the highly complex tandem spectrum, the high resolution and accurate mass allows and is also necessary for confident identification of low abundance 30
40
50
60
70
80
90
Rel
ativ
e A
bund
ance
3025.5167
2851.0285
2797.26273154.1717
2695.5589 3222.7437
3294.30832647.47943369 25012601 0195
0
20
40
60
Rel
ativ
e A
bun z=17z=26
1928.2022z=121652.9607
z=14
1224.2858 NL: 1.75E7 60
80
100719.2635
z=9 719.3749z=9
719.4863z=9
719.1521z=9
719.5977z=9719.0407
C57, Theoretical
biopharmaceutical industry. The demand to better understand the biochemical and biophysical properties of mAbs has become critical. Recent developments in high resolution mass spectrometry (MS) with multiple dissociation techniques have clearly shown its distinctive power for characterization of intact proteins and in particular its subunits. The mass spectrometry–based study of mAbs in middle-down approach
f f f
glycan-containing fragment ions in the presence of interference. For example, shown in Figure 7 insert is the identification of the +9 charged C61 ion with G0F; Figure 9 presents the unambiguous identification of C57 without G0F. Although not all the isotopic peaks of this c ion were observed due to background interference, all seven isotopes identified were within 3 ppm mass error (external mass calibration). Both ETD ions, C61 and C57,
2400 2600 2800 3000 3200 3400 3600 3800 4000m/z
0
10
203369.25012601.0195
2558.9576 3447.50882430.4122 3615.6678 3805.1241 3912.0528
FIGURE 2. Expanded view of full MS spectrum of the intact mAb. Different glycoforms at charge +51 was shown 40
60
80
100 z=211428.1664
z=181511.9984
z=171976.9174
z=13
T: FTMS + p NSI Full ms [600.00-2000.00] (C) Fd’
0
20
40
719.0407z=9 719.7090
z=9719.8204
z=9718.9293z=9
provides a wealth of information to interpret its structural features. Here, we describe the use of an Orbitrap Fusion mass analyzer in combination with nano liquid chromatography (LC) for characterizing a mAb protein using different dissociation techniques.
have played critical roles in identification and localization of G0F on Asn61 residue.FIGURE 10. ETD (blue) and HCD (red) coverage of light chain.
FIGURE 5 Isotopically resolved mAb fragments at 240 000 resolution (A)
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
70
80
90
100
nce
2906.8879
2910.1479
glycoforms at charge +51 was shown.
1000 1500 2000m/z
0
20z 13
FIGURE 7. ETD spectrum of Fc/2 with G0F with precursor ion as m/z 902.20, charge +28. The insert is an expanded view of the spectrum covering C61 ion at charge +9 with 3ppm mass accuracy, which confirms the addition of G0F
719.0 719.2 719.4 719.6 719.8
MethodsSample Preparation
Th i t t Ab t i ti ll l d b l th hi i i t
FIGURE 5. Isotopically resolved mAb fragments at 240,000 resolution. (A) Fc/2+G1F, charge +28; (B) Light Chain, charge 21; (C) Fd’, charge 21.
60
70
80
90
100
unda
nce
939.2334z=9
939.1216z=9
939.3439z=9
939.5667z=9
90
100764.9279
z=2
656 8907
+9, C61 with G0F, <3ppm
0
10
20
30
40
50
60
Rel
ativ
e A
bund
an 2903.7472
2913.3612
2916.46952919.3575
2923.21022899.8132 2925.8599 2932.27852895.7333
T: FTMS + p NSI Full ms [600.00-2000.00]
100
908.0636z=28907.9929
z=28908.1366
z=28 908 2076907 9207 (A) F /2+G1F
glycan.
The intact mAb protein was enzymatically cleaved below the hinge region into a F(ab')2 fragment and two Fc fragments. Aliquot of candidate NIST RM 8670 mAb lot #3F1b was treated with FabRICATOR® (Genovis, Sweden) at 37 oC for 1 hour. The resulting F(ab’)2 and Fc fragments were further denatured and reduced in 50mM dithiothreitol (Sigma, Saint Louis, MO) at 56 oC for 1 hour. The proteolyticallyfragmented mAb was diluted to 1 5 µg/µL using 0 1% formic acid in water 0
10
20
30
40
50
Rel
ativ
e A
b
70
80
90
nce
656.8907z=2
Intact LC-MS Analysis of Proteolytic Fragmented mAb
A mixture including approximately 20 pmol of Fd’ Fc/2 and light chain respectively
2895 2900 2905 2910 2915 2920 2925 2930 2935m/z
0
907 7 907 8 907 9 908 0 908 1 908 2 908 3 908 4 908 5 908 60
20
40
60
80
100
Rel
ativ
e A
bund
ance
z 28 z=28 908.2076z=28
907.9207z=28
908.3156z=28907.7781
z=28
(A) Fc/2+G1F
fragmented mAb was diluted to 1.5 µg/µL using 0.1% formic acid in water.
Liquid Chromatography
Fd' and Fc/2 fragments after reduction were chromatographically eluted from a Thermo Scientific™ PepSwift™ column, Monolithic easy spray column (200 µm x 25 cm, Thermo Fisher Scientific Amsterdam the Netherlands) One µL of the stock was
FIGURE 11. ETD (blue) and HCD (red) coverage of Fd’ chain.
939.0 939.2 939.4 939.6m/z
40
50
60
elat
ive
Abu
ndan
957.7858z=3
1203.0937z=4
924.4734z=6 1073 0261
A mixture including approximately 20 pmol of Fd , Fc/2 and light chain, respectively, was eluted at 800 nl/min and directly analyzed at 240,000 resolution on Orbitrap Fusion MS. The mAb fragments were separated by PepSwift monolithic column. For a 800 nl/min gradient of 5-60% in 32 minutes, different glycoforms of Fc/2 were first eluted at 20.67minute, followed by light chain at 22.34minute. Eluted last was Fd’ chain at 24.76min.
907.7 907.8 907.9 908.0 908.1 908.2 908.3 908.4 908.5 908.6m/z
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
ndan
ce
1102.3104z=21 1102.4048
z=211102.2147z=21
1102.4524z=211102.1195
21
(B) Light chain
Thermo Fisher Scientific, Amsterdam, the Netherlands). One µL of the stock was loaded per injection. Nano flow reverse phase chromatography was performed with a 800 nl/minute gradient of 5-60% in 32minutes using the Thermo Scientific™ EASY-nLC™ 1000 system. The proteins were directly detected by a standard Orbitrap fusion mass spectrometer. Liquid chromatography solvents used include the aqueous as 0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 10
20
30R
e z 6 1073.0261z=6
618.3590z=1
358.2070z=1
814.0227z=5
1528.8462z=11287.6293
z=5
Full MS spectra acquired at 240,000 resolution provides base line resolution of the isotope distribution of the ~25,000Da mAb fragments. The isotopically resolved spectra were deconvoluted for monoisotopic masses. Figure 6 shows the deconvoluted monoisotopic mass of the light chain is 23113.3568Da, which suggests a mass accuracy of 2 2ppm comparing to its theoretical monoisotopic mass at
1101.8 1102.0 1102.2 1102.4 1102.6 1102.8m/z
0
20
40
60
Rel
ativ
e A
bun z=21 1102.5968
z=211102.0254z=21 1102.6899
z=21
RT: 18.71 - 27.44 SM: 7G20 67 NL:
0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 0.1% formic acid (Fisher Scientific, Fair Lawn, New Jersey) in acetronitirile (Fisher Scientific, Fair Lawn, New Jersey).
Mass Spectrometry
The Fd’, Fc/2 and light chain eluted were directly detected by a standard Orbitrap
200 400 600 800 1000 1200 1400m/z
0
10mass accuracy of 2.2ppm comparing to its theoretical monoisotopic mass at 23113.3041Da. FIGURE 3. Total Ion Current Chromatogram of MAb Fragments Eluted from a PepSwift Monolithic Nano Column.
Fc/2
T: FTMS + p NSI Full ms [600.00-2000.00]
40
60
80
100
ve A
bund
ance
1224.1915z=21
1224.2859z=21
1224.0952z=21
1224.4296z=21
1224.5247z=21
1224.0009z=21
1224.6193z=211223.9055
(C) Fd’
70
80
90
100
nce
20.67 NL:2.47E10TIC MS frg_89
ConclusionThe middle-down study indicated that complementary fragmentation mechanisms allow
g y y pFusion MS under both full scan mode and tandem scan mode using higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD). Full mass spectra of mAb fragments were acquired at 240,000 resolution at m/z 200 with mass range m/z 400-2000. AGC setting for full MS spectrum was at 1e5 with 100ms maximum injection. Ion transferring temperature was set at 300 oC. Full mass spectra
Li ht Ch i
1223.8 1224.0 1224.2 1224.4 1224.6 1224.8m/z
0
20
Rel
ativ z=21
z=211223.8147z=21
FIGURE 6. Deconvoluted monoisotopic mass of light chain.
FIGURE 8. ETD (blue) and HCD (red) coverage of Fc/2+G0F chain. Asn61 was highlighted for addition of G0F.
30
40
50
60
Rel
ativ
e A
bund
an
22.34
22.60 24.76
extensive sequence coverage as well as identification and localization of PTMs including labile glycosylation. The middle-down approach produced detailed structure information deep into the middle of the mAb chains especially in the epitope region.
Acknowledgements
of intact mAb were acquired at 15,000 resolution at m/z 200 with mass range m/z 1000-6000.
Data Analysis
The full mass spectra were analyzed with Thermo ScientificTM Protein DeconvolutionTM
3 0 ft f l l d t i ti Th t d t f Fd’ F /2
Light ChainFd’
Theoretical monoisotpic mass-23113.3041DaMass acccuracy with external calibration-2.2ppm
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
danc
e
23113.3568
19 20 21 22 23 24 25 26 27Time (min)
0
10
20AcknowledgementsWe would like to thank National Institute of Standards and Technology for providing the intact mAb protein sample (candidate NIST RM 8670 mAb lot #3F1b).
3.0 software for molecular mass determination . The tandem mass spectra of Fd’, Fc/2 and light chain were deconvoluted by Xtract. The deconvoluted spectra were processed by ProSightPC 3.0 software for middle-down data interpretation.
22700 22800 22900 23000 23100 23200m/z
0
20
40
60
Rel
ativ
e A
bund
23130.328223096.2937
FabRICATOR® is a registered trademark for recombinant IdeS. 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 intellectual property rights of others.
PO64147-EN 0614S
5Thermo Scientific Poster Note • PN-64147-ASMS-EN-0614S
Middle-down Analysis of Monoclonal Antibody using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerMiddle down Analysis of Monoclonal Antibody using Nano flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerJie Qian 1, Keith A. Waddell 2, Zhiqi Hao 2
Thermo Fisher Scientific, 1 Somerset, New Jersey, 2 San Jose, CaliforniaThermo Fisher Scientific, Somerset, New Jersey, San Jose, California
Overview Purpose: Sensitive analysis of monoclonal antibody using middle-down approach on a novel Thermo Scientific™ Orbitrap FusionTM TribridTM mass spectrometer
ResultsFull Mass Spectrum of Intact mAb
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC-MS. Data
Top Down Analysis of Proteolytic Fragmented MAb
Middle down analysis of mAb protein was performed by LC-MS analysis of the proteolytic fragments. Precursor ions at m/z 964.65 of light chain, m/z 1117.87 of Fd’ and m/z 902.20 of the G0F glycoform of Fc/2 were selected respectively for ETD and HCD
FIGURE 4. Full MS spectrum of mAb fragments at 240,000 resolution. (A) Full MS spectrum of Fc/2; (B) Full MS spectrum of light chain; (C) Full MS spectrum of Fd’.
100
935.7325z=27
1270 9351
NL: 1.39E7T: FTMS + p NSI Full
FIGURE 9. Comparison of experimental C57 ion of Fc/2+G0F chain to theoretically predicted isotope distribution. This ion is critical for identifying and localizing G0F on Asn61 residue.
719.3735719 2621Methods: A method combining nano-flow liquid chromatography and complementary higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) fragmentation on an Orbitrap Fusion Mass Spectrometer was developed and implemented
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd’
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC MS. Data shown below was acquired at 15,000 resolution on Orbitrap Fusion MS with source CID at 70. The application of 40-80 source CID is beneficial for most mAb proteins to help remove the adducts and thus promote a cleaner signal. The optimal SID setting is protein dependent.
g y p yMS/MS at 120,000 resolution. Reaction time of 3-5 ms was applied for ETD fragmentation. The normalized collision energy for HCD was 15-25 %. As shown in Figure 7, tandem spectra generated contain well resolved, multiply charged fragment ions. Interpretation of these ions based on the mAb protein sequence was performed using ProSightPC 3.0. The combined results of ETD and HCD suggest 50% sequence
20
40
60
80
100 1270.9351z=20
1329.0863z=19
pms [600.00-2000.00] (A) Fc/2
60
80
100
Abu
ndan
ce
z=9719.2621
z=9
719.4851z=9
719.1510z 9 719.5956
C57, Experimental<3ppm
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd , Fc/2 and light chain respectively. Intact masses of the monoclonal antibody and its proteolytic fragments were measured. The proteolytic fragments (Fd’, Fc/2 and light chains) were directly fragmented using HCD and ETD. An average 50% amino acid coverage was achieved and glycosylation site was unambiguously identified
FIGURE 1. Full MS spectrum of the intact mAb (candidate NIST RM 8670 mAblot #3F1b) obtained from LC-MS analysis.
coverage for light chain, 52% coverage for Fc/2 with G0F and 32% coverage for Fd’ respectively (Figure 8, 10, 11). N-terminal modification of pyroglutamate of Fd’chain was confirmed based on fragment ions from both ETD and HCD. Both intact and tandem spectra identified the Lys loss at C-terminus of Fc/2.
ETD is widely known for its advantage in preferentially fragmenting the peptide back bone
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
90
100 2965.00372906.8879
3025 5167
80
100
ndan
ce
0
20
1052.2509z=22 1218.2364
z=191361.4397
z=17890.4831
26
NL: 2.67E7T: FTMS + p NSI Full ms [600.00-2000.00] (B) Light chain
0
20
40
Rel
ativ
e A z=9 719.5956z=9719.0392
z=9 719.7081z=9
719.8182z=9
719 2635719 3749
IntroductionMonoclonal antibodies (mAbs) are an increasingly important line of therapeutics for the
y g p y g g p pand keeping the labile modifications attached. It has been recognized as the method of choice for locating the sites of such labile PTMs including glycosylation. Multiple identified ETD fragments between Asn61 and Asn79 unambiguously located the glycan G0F on Asn61 of Fc/2 chain. In the highly complex tandem spectrum, the high resolution and accurate mass allows and is also necessary for confident identification of low abundance 30
40
50
60
70
80
90
Rel
ativ
e A
bund
ance
3025.5167
2851.0285
2797.26273154.1717
2695.5589 3222.7437
3294.30832647.47943369 25012601 0195
0
20
40
60
Rel
ativ
e A
bun z=17z=26
1928.2022z=121652.9607
z=14
1224.2858 NL: 1.75E7 60
80
100719.2635
z=9 719.3749z=9
719.4863z=9
719.1521z=9
719.5977z=9719.0407
C57, Theoretical
biopharmaceutical industry. The demand to better understand the biochemical and biophysical properties of mAbs has become critical. Recent developments in high resolution mass spectrometry (MS) with multiple dissociation techniques have clearly shown its distinctive power for characterization of intact proteins and in particular its subunits. The mass spectrometry–based study of mAbs in middle-down approach
f f f
glycan-containing fragment ions in the presence of interference. For example, shown in Figure 7 insert is the identification of the +9 charged C61 ion with G0F; Figure 9 presents the unambiguous identification of C57 without G0F. Although not all the isotopic peaks of this c ion were observed due to background interference, all seven isotopes identified were within 3 ppm mass error (external mass calibration). Both ETD ions, C61 and C57,
2400 2600 2800 3000 3200 3400 3600 3800 4000m/z
0
10
203369.25012601.0195
2558.9576 3447.50882430.4122 3615.6678 3805.1241 3912.0528
FIGURE 2. Expanded view of full MS spectrum of the intact mAb. Different glycoforms at charge +51 was shown 40
60
80
100 z=211428.1664
z=181511.9984
z=171976.9174
z=13
T: FTMS + p NSI Full ms [600.00-2000.00] (C) Fd’
0
20
40
719.0407z=9 719.7090
z=9719.8204
z=9718.9293z=9
provides a wealth of information to interpret its structural features. Here, we describe the use of an Orbitrap Fusion mass analyzer in combination with nano liquid chromatography (LC) for characterizing a mAb protein using different dissociation techniques.
have played critical roles in identification and localization of G0F on Asn61 residue.FIGURE 10. ETD (blue) and HCD (red) coverage of light chain.
FIGURE 5 Isotopically resolved mAb fragments at 240 000 resolution (A)
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
70
80
90
100
nce
2906.8879
2910.1479
glycoforms at charge +51 was shown.
1000 1500 2000m/z
0
20z 13
FIGURE 7. ETD spectrum of Fc/2 with G0F with precursor ion as m/z 902.20, charge +28. The insert is an expanded view of the spectrum covering C61 ion at charge +9 with 3ppm mass accuracy, which confirms the addition of G0F
719.0 719.2 719.4 719.6 719.8
MethodsSample Preparation
Th i t t Ab t i ti ll l d b l th hi i i t
FIGURE 5. Isotopically resolved mAb fragments at 240,000 resolution. (A) Fc/2+G1F, charge +28; (B) Light Chain, charge 21; (C) Fd’, charge 21.
60
70
80
90
100
unda
nce
939.2334z=9
939.1216z=9
939.3439z=9
939.5667z=9
90
100764.9279
z=2
656 8907
+9, C61 with G0F, <3ppm
0
10
20
30
40
50
60
Rel
ativ
e A
bund
an 2903.7472
2913.3612
2916.46952919.3575
2923.21022899.8132 2925.8599 2932.27852895.7333
T: FTMS + p NSI Full ms [600.00-2000.00]
100
908.0636z=28907.9929
z=28908.1366
z=28 908 2076907 9207 (A) F /2+G1F
glycan.
The intact mAb protein was enzymatically cleaved below the hinge region into a F(ab')2 fragment and two Fc fragments. Aliquot of candidate NIST RM 8670 mAb lot #3F1b was treated with FabRICATOR® (Genovis, Sweden) at 37 oC for 1 hour. The resulting F(ab’)2 and Fc fragments were further denatured and reduced in 50mM dithiothreitol (Sigma, Saint Louis, MO) at 56 oC for 1 hour. The proteolyticallyfragmented mAb was diluted to 1 5 µg/µL using 0 1% formic acid in water 0
10
20
30
40
50
Rel
ativ
e A
b
70
80
90
nce
656.8907z=2
Intact LC-MS Analysis of Proteolytic Fragmented mAb
A mixture including approximately 20 pmol of Fd’ Fc/2 and light chain respectively
2895 2900 2905 2910 2915 2920 2925 2930 2935m/z
0
907 7 907 8 907 9 908 0 908 1 908 2 908 3 908 4 908 5 908 60
20
40
60
80
100
Rel
ativ
e A
bund
ance
z 28 z=28 908.2076z=28
907.9207z=28
908.3156z=28907.7781
z=28
(A) Fc/2+G1F
fragmented mAb was diluted to 1.5 µg/µL using 0.1% formic acid in water.
Liquid Chromatography
Fd' and Fc/2 fragments after reduction were chromatographically eluted from a Thermo Scientific™ PepSwift™ column, Monolithic easy spray column (200 µm x 25 cm, Thermo Fisher Scientific Amsterdam the Netherlands) One µL of the stock was
FIGURE 11. ETD (blue) and HCD (red) coverage of Fd’ chain.
939.0 939.2 939.4 939.6m/z
40
50
60
elat
ive
Abu
ndan
957.7858z=3
1203.0937z=4
924.4734z=6 1073 0261
A mixture including approximately 20 pmol of Fd , Fc/2 and light chain, respectively, was eluted at 800 nl/min and directly analyzed at 240,000 resolution on Orbitrap Fusion MS. The mAb fragments were separated by PepSwift monolithic column. For a 800 nl/min gradient of 5-60% in 32 minutes, different glycoforms of Fc/2 were first eluted at 20.67minute, followed by light chain at 22.34minute. Eluted last was Fd’ chain at 24.76min.
907.7 907.8 907.9 908.0 908.1 908.2 908.3 908.4 908.5 908.6m/z
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
ndan
ce
1102.3104z=21 1102.4048
z=211102.2147z=21
1102.4524z=211102.1195
21
(B) Light chain
Thermo Fisher Scientific, Amsterdam, the Netherlands). One µL of the stock was loaded per injection. Nano flow reverse phase chromatography was performed with a 800 nl/minute gradient of 5-60% in 32minutes using the Thermo Scientific™ EASY-nLC™ 1000 system. The proteins were directly detected by a standard Orbitrap fusion mass spectrometer. Liquid chromatography solvents used include the aqueous as 0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 10
20
30
Re z 6 1073.0261
z=6618.3590
z=1358.2070
z=1814.0227
z=51528.8462
z=11287.6293z=5
Full MS spectra acquired at 240,000 resolution provides base line resolution of the isotope distribution of the ~25,000Da mAb fragments. The isotopically resolved spectra were deconvoluted for monoisotopic masses. Figure 6 shows the deconvoluted monoisotopic mass of the light chain is 23113.3568Da, which suggests a mass accuracy of 2 2ppm comparing to its theoretical monoisotopic mass at
1101.8 1102.0 1102.2 1102.4 1102.6 1102.8m/z
0
20
40
60
Rel
ativ
e A
bun z=21 1102.5968
z=211102.0254z=21 1102.6899
z=21
RT: 18.71 - 27.44 SM: 7G20 67 NL:
0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 0.1% formic acid (Fisher Scientific, Fair Lawn, New Jersey) in acetronitirile (Fisher Scientific, Fair Lawn, New Jersey).
Mass Spectrometry
The Fd’, Fc/2 and light chain eluted were directly detected by a standard Orbitrap
200 400 600 800 1000 1200 1400m/z
0
10mass accuracy of 2.2ppm comparing to its theoretical monoisotopic mass at 23113.3041Da. FIGURE 3. Total Ion Current Chromatogram of MAb Fragments Eluted from a PepSwift Monolithic Nano Column.
Fc/2
T: FTMS + p NSI Full ms [600.00-2000.00]
40
60
80
100
ve A
bund
ance
1224.1915z=21
1224.2859z=21
1224.0952z=21
1224.4296z=21
1224.5247z=21
1224.0009z=21
1224.6193z=211223.9055
(C) Fd’
70
80
90
100
nce
20.67 NL:2.47E10TIC MS frg_89
ConclusionThe middle-down study indicated that complementary fragmentation mechanisms allow
g y y pFusion MS under both full scan mode and tandem scan mode using higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD). Full mass spectra of mAb fragments were acquired at 240,000 resolution at m/z 200 with mass range m/z 400-2000. AGC setting for full MS spectrum was at 1e5 with 100ms maximum injection. Ion transferring temperature was set at 300 oC. Full mass spectra
Li ht Ch i
1223.8 1224.0 1224.2 1224.4 1224.6 1224.8m/z
0
20
Rel
ativ z=21
z=211223.8147z=21
FIGURE 6. Deconvoluted monoisotopic mass of light chain.
FIGURE 8. ETD (blue) and HCD (red) coverage of Fc/2+G0F chain. Asn61 was highlighted for addition of G0F.
30
40
50
60
Rel
ativ
e A
bund
an
22.34
22.60 24.76
extensive sequence coverage as well as identification and localization of PTMs including labile glycosylation. The middle-down approach produced detailed structure information deep into the middle of the mAb chains especially in the epitope region.
Acknowledgements
of intact mAb were acquired at 15,000 resolution at m/z 200 with mass range m/z 1000-6000.
Data Analysis
The full mass spectra were analyzed with Thermo ScientificTM Protein DeconvolutionTM
3 0 ft f l l d t i ti Th t d t f Fd’ F /2
Light ChainFd’
Theoretical monoisotpic mass-23113.3041DaMass acccuracy with external calibration-2.2ppm
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
danc
e
23113.3568
19 20 21 22 23 24 25 26 27Time (min)
0
10
20AcknowledgementsWe would like to thank National Institute of Standards and Technology for providing the intact mAb protein sample (candidate NIST RM 8670 mAb lot #3F1b).
3.0 software for molecular mass determination . The tandem mass spectra of Fd’, Fc/2 and light chain were deconvoluted by Xtract. The deconvoluted spectra were processed by ProSightPC 3.0 software for middle-down data interpretation.
22700 22800 22900 23000 23100 23200m/z
0
20
40
60
Rel
ativ
e A
bund
23130.328223096.2937
FabRICATOR® is a registered trademark for recombinant IdeS. 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 intellectual property rights of others.
PO64147-EN 0614S
6 Middle-down Analysis of Monoclonal Antibody Middle using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass Spectrometer
Middle-down Analysis of Monoclonal Antibody using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerMiddle down Analysis of Monoclonal Antibody using Nano flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass SpectrometerJie Qian 1, Keith A. Waddell 2, Zhiqi Hao 2
Thermo Fisher Scientific, 1 Somerset, New Jersey, 2 San Jose, CaliforniaThermo Fisher Scientific, Somerset, New Jersey, San Jose, California
Overview Purpose: Sensitive analysis of monoclonal antibody using middle-down approach on a novel Thermo Scientific™ Orbitrap FusionTM TribridTM mass spectrometer
ResultsFull Mass Spectrum of Intact mAb
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC-MS. Data
Top Down Analysis of Proteolytic Fragmented MAb
Middle down analysis of mAb protein was performed by LC-MS analysis of the proteolytic fragments. Precursor ions at m/z 964.65 of light chain, m/z 1117.87 of Fd’ and m/z 902.20 of the G0F glycoform of Fc/2 were selected respectively for ETD and HCD
FIGURE 4. Full MS spectrum of mAb fragments at 240,000 resolution. (A) Full MS spectrum of Fc/2; (B) Full MS spectrum of light chain; (C) Full MS spectrum of Fd’.
100
935.7325z=27
1270 9351
NL: 1.39E7T: FTMS + p NSI Full
FIGURE 9. Comparison of experimental C57 ion of Fc/2+G0F chain to theoretically predicted isotope distribution. This ion is critical for identifying and localizing G0F on Asn61 residue.
719.3735719 2621Methods: A method combining nano-flow liquid chromatography and complementary higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) fragmentation on an Orbitrap Fusion Mass Spectrometer was developed and implemented
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd’
Intact mAb (candidate NIST RM 8670 mAb lot #3F1b) was surveyed by LC MS. Data shown below was acquired at 15,000 resolution on Orbitrap Fusion MS with source CID at 70. The application of 40-80 source CID is beneficial for most mAb proteins to help remove the adducts and thus promote a cleaner signal. The optimal SID setting is protein dependent.
g y p yMS/MS at 120,000 resolution. Reaction time of 3-5 ms was applied for ETD fragmentation. The normalized collision energy for HCD was 15-25 %. As shown in Figure 7, tandem spectra generated contain well resolved, multiply charged fragment ions. Interpretation of these ions based on the mAb protein sequence was performed using ProSightPC 3.0. The combined results of ETD and HCD suggest 50% sequence
20
40
60
80
100 1270.9351z=20
1329.0863z=19
pms [600.00-2000.00] (A) Fc/2
60
80
100
Abu
ndan
ce
z=9719.2621
z=9
719.4851z=9
719.1510z 9 719.5956
C57, Experimental<3ppm
Results: A monoclonal antibody was enzymatically and chemically cleaved into Fd , Fc/2 and light chain respectively. Intact masses of the monoclonal antibody and its proteolytic fragments were measured. The proteolytic fragments (Fd’, Fc/2 and light chains) were directly fragmented using HCD and ETD. An average 50% amino acid coverage was achieved and glycosylation site was unambiguously identified
FIGURE 1. Full MS spectrum of the intact mAb (candidate NIST RM 8670 mAblot #3F1b) obtained from LC-MS analysis.
coverage for light chain, 52% coverage for Fc/2 with G0F and 32% coverage for Fd’ respectively (Figure 8, 10, 11). N-terminal modification of pyroglutamate of Fd’chain was confirmed based on fragment ions from both ETD and HCD. Both intact and tandem spectra identified the Lys loss at C-terminus of Fc/2.
ETD is widely known for its advantage in preferentially fragmenting the peptide back bone
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
90
100 2965.00372906.8879
3025 5167
80
100
ndan
ce
0
20
1052.2509z=22 1218.2364
z=191361.4397
z=17890.4831
26
NL: 2.67E7T: FTMS + p NSI Full ms [600.00-2000.00] (B) Light chain
0
20
40
Rel
ativ
e A z=9 719.5956
z=9719.0392z=9 719.7081
z=9719.8182
z=9
719 2635719 3749
IntroductionMonoclonal antibodies (mAbs) are an increasingly important line of therapeutics for the
y g p y g g p pand keeping the labile modifications attached. It has been recognized as the method of choice for locating the sites of such labile PTMs including glycosylation. Multiple identified ETD fragments between Asn61 and Asn79 unambiguously located the glycan G0F on Asn61 of Fc/2 chain. In the highly complex tandem spectrum, the high resolution and accurate mass allows and is also necessary for confident identification of low abundance 30
40
50
60
70
80
90
Rel
ativ
e A
bund
ance
3025.5167
2851.0285
2797.26273154.1717
2695.5589 3222.7437
3294.30832647.47943369 25012601 0195
0
20
40
60
Rel
ativ
e A
bun z=17z=26
1928.2022z=121652.9607
z=14
1224.2858 NL: 1.75E7 60
80
100719.2635
z=9 719.3749z=9
719.4863z=9
719.1521z=9
719.5977z=9719.0407
C57, Theoretical
biopharmaceutical industry. The demand to better understand the biochemical and biophysical properties of mAbs has become critical. Recent developments in high resolution mass spectrometry (MS) with multiple dissociation techniques have clearly shown its distinctive power for characterization of intact proteins and in particular its subunits. The mass spectrometry–based study of mAbs in middle-down approach
f f f
glycan-containing fragment ions in the presence of interference. For example, shown in Figure 7 insert is the identification of the +9 charged C61 ion with G0F; Figure 9 presents the unambiguous identification of C57 without G0F. Although not all the isotopic peaks of this c ion were observed due to background interference, all seven isotopes identified were within 3 ppm mass error (external mass calibration). Both ETD ions, C61 and C57,
2400 2600 2800 3000 3200 3400 3600 3800 4000m/z
0
10
203369.25012601.0195
2558.9576 3447.50882430.4122 3615.6678 3805.1241 3912.0528
FIGURE 2. Expanded view of full MS spectrum of the intact mAb. Different glycoforms at charge +51 was shown 40
60
80
100 z=211428.1664
z=181511.9984
z=171976.9174
z=13
T: FTMS + p NSI Full ms [600.00-2000.00] (C) Fd’
0
20
40
719.0407z=9 719.7090
z=9719.8204
z=9718.9293z=9
provides a wealth of information to interpret its structural features. Here, we describe the use of an Orbitrap Fusion mass analyzer in combination with nano liquid chromatography (LC) for characterizing a mAb protein using different dissociation techniques.
have played critical roles in identification and localization of G0F on Asn61 residue.FIGURE 10. ETD (blue) and HCD (red) coverage of light chain.
FIGURE 5 Isotopically resolved mAb fragments at 240 000 resolution (A)
T: FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00]
70
80
90
100
nce
2906.8879
2910.1479
glycoforms at charge +51 was shown.
1000 1500 2000m/z
0
20z 13
FIGURE 7. ETD spectrum of Fc/2 with G0F with precursor ion as m/z 902.20, charge +28. The insert is an expanded view of the spectrum covering C61 ion at charge +9 with 3ppm mass accuracy, which confirms the addition of G0F
719.0 719.2 719.4 719.6 719.8
MethodsSample Preparation
Th i t t Ab t i ti ll l d b l th hi i i t
FIGURE 5. Isotopically resolved mAb fragments at 240,000 resolution. (A) Fc/2+G1F, charge +28; (B) Light Chain, charge 21; (C) Fd’, charge 21.
60
70
80
90
100
unda
nce
939.2334z=9
939.1216z=9
939.3439z=9
939.5667z=9
90
100764.9279
z=2
656 8907
+9, C61 with G0F, <3ppm
0
10
20
30
40
50
60
Rel
ativ
e A
bund
an 2903.7472
2913.3612
2916.46952919.3575
2923.21022899.8132 2925.8599 2932.27852895.7333
T: FTMS + p NSI Full ms [600.00-2000.00]
100
908.0636z=28907.9929
z=28908.1366
z=28 908 2076907 9207 (A) F /2+G1F
glycan.
The intact mAb protein was enzymatically cleaved below the hinge region into a F(ab')2 fragment and two Fc fragments. Aliquot of candidate NIST RM 8670 mAb lot #3F1b was treated with FabRICATOR® (Genovis, Sweden) at 37 oC for 1 hour. The resulting F(ab’)2 and Fc fragments were further denatured and reduced in 50mM dithiothreitol (Sigma, Saint Louis, MO) at 56 oC for 1 hour. The proteolyticallyfragmented mAb was diluted to 1 5 µg/µL using 0 1% formic acid in water 0
10
20
30
40
50
Rel
ativ
e A
b
70
80
90
nce
656.8907z=2
Intact LC-MS Analysis of Proteolytic Fragmented mAb
A mixture including approximately 20 pmol of Fd’ Fc/2 and light chain respectively
2895 2900 2905 2910 2915 2920 2925 2930 2935m/z
0
907 7 907 8 907 9 908 0 908 1 908 2 908 3 908 4 908 5 908 60
20
40
60
80
100
Rel
ativ
e A
bund
ance
z 28 z=28 908.2076z=28
907.9207z=28
908.3156z=28907.7781
z=28
(A) Fc/2+G1F
fragmented mAb was diluted to 1.5 µg/µL using 0.1% formic acid in water.
Liquid Chromatography
Fd' and Fc/2 fragments after reduction were chromatographically eluted from a Thermo Scientific™ PepSwift™ column, Monolithic easy spray column (200 µm x 25 cm, Thermo Fisher Scientific Amsterdam the Netherlands) One µL of the stock was
FIGURE 11. ETD (blue) and HCD (red) coverage of Fd’ chain.
939.0 939.2 939.4 939.6m/z
40
50
60
elat
ive
Abu
ndan
957.7858z=3
1203.0937z=4
924.4734z=6 1073 0261
A mixture including approximately 20 pmol of Fd , Fc/2 and light chain, respectively, was eluted at 800 nl/min and directly analyzed at 240,000 resolution on Orbitrap Fusion MS. The mAb fragments were separated by PepSwift monolithic column. For a 800 nl/min gradient of 5-60% in 32 minutes, different glycoforms of Fc/2 were first eluted at 20.67minute, followed by light chain at 22.34minute. Eluted last was Fd’ chain at 24.76min.
907.7 907.8 907.9 908.0 908.1 908.2 908.3 908.4 908.5 908.6m/z
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
ndan
ce
1102.3104z=21 1102.4048
z=211102.2147z=21
1102.4524z=211102.1195
21
(B) Light chain
Thermo Fisher Scientific, Amsterdam, the Netherlands). One µL of the stock was loaded per injection. Nano flow reverse phase chromatography was performed with a 800 nl/minute gradient of 5-60% in 32minutes using the Thermo Scientific™ EASY-nLC™ 1000 system. The proteins were directly detected by a standard Orbitrap fusion mass spectrometer. Liquid chromatography solvents used include the aqueous as 0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 10
20
30
Re z 6 1073.0261
z=6618.3590
z=1358.2070
z=1814.0227
z=51528.8462
z=11287.6293z=5
Full MS spectra acquired at 240,000 resolution provides base line resolution of the isotope distribution of the ~25,000Da mAb fragments. The isotopically resolved spectra were deconvoluted for monoisotopic masses. Figure 6 shows the deconvoluted monoisotopic mass of the light chain is 23113.3568Da, which suggests a mass accuracy of 2 2ppm comparing to its theoretical monoisotopic mass at
1101.8 1102.0 1102.2 1102.4 1102.6 1102.8m/z
0
20
40
60
Rel
ativ
e A
bun z=21 1102.5968
z=211102.0254z=21 1102.6899
z=21
RT: 18.71 - 27.44 SM: 7G20 67 NL:
0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as 0.1% formic acid (Fisher Scientific, Fair Lawn, New Jersey) in acetronitirile (Fisher Scientific, Fair Lawn, New Jersey).
Mass Spectrometry
The Fd’, Fc/2 and light chain eluted were directly detected by a standard Orbitrap
200 400 600 800 1000 1200 1400m/z
0
10mass accuracy of 2.2ppm comparing to its theoretical monoisotopic mass at 23113.3041Da. FIGURE 3. Total Ion Current Chromatogram of MAb Fragments Eluted from a PepSwift Monolithic Nano Column.
Fc/2
T: FTMS + p NSI Full ms [600.00-2000.00]
40
60
80
100
ve A
bund
ance
1224.1915z=21
1224.2859z=21
1224.0952z=21
1224.4296z=21
1224.5247z=21
1224.0009z=21
1224.6193z=211223.9055
(C) Fd’
70
80
90
100
nce
20.67 NL:2.47E10TIC MS frg_89
ConclusionThe middle-down study indicated that complementary fragmentation mechanisms allow
g y y pFusion MS under both full scan mode and tandem scan mode using higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD). Full mass spectra of mAb fragments were acquired at 240,000 resolution at m/z 200 with mass range m/z 400-2000. AGC setting for full MS spectrum was at 1e5 with 100ms maximum injection. Ion transferring temperature was set at 300 oC. Full mass spectra
Li ht Ch i
1223.8 1224.0 1224.2 1224.4 1224.6 1224.8m/z
0
20
Rel
ativ z=21
z=211223.8147z=21
FIGURE 6. Deconvoluted monoisotopic mass of light chain.
FIGURE 8. ETD (blue) and HCD (red) coverage of Fc/2+G0F chain. Asn61 was highlighted for addition of G0F.
30
40
50
60
Rel
ativ
e A
bund
an
22.34
22.60 24.76
extensive sequence coverage as well as identification and localization of PTMs including labile glycosylation. The middle-down approach produced detailed structure information deep into the middle of the mAb chains especially in the epitope region.
Acknowledgements
of intact mAb were acquired at 15,000 resolution at m/z 200 with mass range m/z 1000-6000.
Data Analysis
The full mass spectra were analyzed with Thermo ScientificTM Protein DeconvolutionTM
3 0 ft f l l d t i ti Th t d t f Fd’ F /2
Light ChainFd’
Theoretical monoisotpic mass-23113.3041DaMass acccuracy with external calibration-2.2ppm
T: FTMS + p NSI Full ms [600.00-2000.00]
80
100
danc
e
23113.3568
19 20 21 22 23 24 25 26 27Time (min)
0
10
20AcknowledgementsWe would like to thank National Institute of Standards and Technology for providing the intact mAb protein sample (candidate NIST RM 8670 mAb lot #3F1b).
3.0 software for molecular mass determination . The tandem mass spectra of Fd’, Fc/2 and light chain were deconvoluted by Xtract. The deconvoluted spectra were processed by ProSightPC 3.0 software for middle-down data interpretation.
22700 22800 22900 23000 23100 23200m/z
0
20
40
60
Rel
ativ
e A
bund
23130.328223096.2937
FabRICATOR® is a registered trademark for recombinant IdeS. 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 intellectual property rights of others.
PO64147-EN 0614S
www.thermofisher.com©2016 Thermo Fisher Scientific Inc. All rights reserved. FabRICATOR® is a registered trademark for recombinant IdeS. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented 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 might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
PN-64147-EN-0716S
Africa +43 1 333 50 34 0Australia +61 3 9757 4300Austria +43 810 282 206Belgium +32 53 73 42 41Canada +1 800 530 8447China 800 810 5118 (free call domestic)
400 650 5118
Denmark +45 70 23 62 60Europe-Other +43 1 333 50 34 0Finland +358 9 3291 0200France +33 1 60 92 48 00Germany +49 6103 408 1014India +91 22 6742 9494Italy +39 02 950 591
Japan +81 45 453 9100Latin America +1 561 688 8700Middle East +43 1 333 50 34 0Netherlands +31 76 579 55 55New Zealand +64 9 980 6700Norway +46 8 556 468 00Russia/CIS +43 1 333 50 34 0
Singapore +65 6289 1190Spain +34 914 845 965Sweden +46 8 556 468 00Switzerland +41 61 716 77 00UK +44 1442 233555USA +1 800 532 4752