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

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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

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40

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60

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ativ

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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

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b

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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.

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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

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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

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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

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