Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry

Kai Scheffler,1 Eugen Damoc,2 Mathias Müller,2 Martin Zeller, 2 Thomas Moehring2 Thermo Fisher Scientific, Dreieich1 and Bremen,2 Germany Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry Kai Scheffler1, Eugen Damoc2, Mathias Müller2, Martin Zeller2, Thomas Moehring2 Thermo Fisher Scientific, Dreieich1 and Bremen2, Germany

HUMIRA_heavy_av_SIM # 1 RT: 1556.88 AV: 1 NL: 3.30E3 Humira_heavy_av_SIM #1 RT: 1556.88 AV: 1 NL: 3.30E3 T: FTMS + p ESI SIM ms [1180.00-1190.00] T: FTMS + p ESI SIM ms [1180.00-1190.00] Monoisotopic mass (M) of the 50891.04317 z=43 1185.2190 100 Instrument 1185.2190 R=117700 1185.2921 Introduction Deconvolution intact heavy chain after R=117700 100 R=117704 80 100 1185.1287 zoom R=120804 60 deconvolution with Xtract 80 1185.3820 Recombinant monoclonal antibodies have gained significant importance in A Thermo Scientific™ Surveyor™ MS Pump Plus was coupled to an 90 1304.56287 R=118304 100 1185.4508 40 60 1185.4961 diagnostic and therapeutic applications over the past years. In order to verify Orbitrap Elite mass spectrometer that was equipped with ETD (Figure 3). z=18 80 R=122004 1235.95410 1183.9644 R=120004 20 90 (A) R=120204 40

Relative Abundance the correctness of the overall molecule to provide a reproducible, safe and 70 0 Square Quadrupole z=19 Electrospray source S-lens Octopole High Pressure Cell Low Pressure Cell Quadrupole Mass Filter HCD Collision Cell Transfer Multipole Reagent Ion Source 1182.5933 C-trap Abundance Relative

20 with Beam Blocker 1184.9670 50922.07492 80 z=17 60 R=116404 1183.5918 100 effective biological drug compound, the correct protein sequence, as well as R=121104 Isotope pattern and average R=122204 0 1381.19873 1182.2444 80 50 1185.2 1185.4 70 z=20 R=121404 mass of the intact heavy chain the presence and relative abundance of different glycoforms have to be z=16 m/z 1174.17102 1185.8925 60 1467.49915 40 R=109804 after deconvolution with Xtract confirmed. 40 60 1181.6860 1186.2635 z=15 Relative Abundance 30 R=117204 R=127904 20 Here we present an approach to analyze an intact monoclonal antibody in 1180.9857 50 1565.29358 z=21 20 R=133304 0 New z=12 non-reduced and reduced condition by LC-MS using the Thermo Scientific™ High-Field Orbitrap 1118.31189 50890 50900 50910 50920 50930 50940 Mass Analyzer z=14 10 m/z 40 1956.39819 z=11 Orbitrap Elite™ mass spectrometer. The intact antibody and the separated 1677.03210 0 Relative Abundance 2134.19409 Reagent 1 Reagent 2 30 z=13 1181 1182 1183 1184 1185 1186 Heated Inlet Heated Inlet light and heavy chains were analyzed in Full MS experiments as well as with z=22 1806.14465 m/z FIGURE 6: HUMIRA heavy chain acquired in SIM scan mode (z=43). 60 µscans were top-down experiments using in-source CID (SID), CID, HCD and ETD 20 1067.56909 z=10 averaged. Deconvoluted mass: Mr 50,891.04317 Da. The inserts on the right fragmentation techniques making use of the ultrahigh resolution of the mass FIGURE 3: Schematics of the Orbitrap Elite hybrid ion trap-Orbitrap mass 10 1021.25623 2347.60156 Conclusion demonstrate isotopic resolution of that charge state detected at m/z 1185 and masses spectrometer. For data evaluation ProSight software and Thermo Scientific™ spectrometer equipped with an ETD source. 2610.80981 0 obtained after deconvolution using Xtract. The analysis of intact and reduced antibodies on the Orbitrap Elite 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 Humira_CID_59_120210173005 #1 RT: 355.98 AV: 1 NL: 6.11E3 • Protein Deconvolution™ software version 1.0 packages were used. T: FTMS + p ESI Full ms2 [email protected] [325.00-4000.00] m/z 1395.24207 1507.50037 mass spectrometer provides the accurate molecular weight, as well as z=18 Samples were purified on a Thermo Scientific™ BioBasic™ C4 column (150 100 z=8 1321.86304 90 1594.27380 valuable information about the presence and abundance of glycoforms. x 1 mm, 5 µm particles), solvent A: 0.1 % FA, 2 % ACN in H O, solvent B: z=? z=8 2 80 CID 1629.42053 Physicochemical Characteris4cs R=124.100 70 Biological Characteris4cs 0.1 % FA in ACN. The LC gradient was 7 min 20–40 % B, 3 min 40–80 % B z=8 • Analysis of the reduced antibody provides isotopically resolved mass 60 1301.43152 NL: (A) 1217.55786 at a flow rate of 100 µL/min. 100 Δ=0.9ppm 50 z=? spectra for both light and heavy chain. 3.84E5 1076.95667 N-­‐terminal heterogeneity 40 551.31708 z=24 z=18 1301.26538 FS_tripleSIM_20uS_1#19 RT: z=1 Pyroglutamate forma.on 30 782.33112 1756.70886 Data analysis was done using Protein Deconvolution and ProSight software 1301.59814 Abundance Relative • The combination of multiple fragmentation techniques in top-down Other modifica.ons 80 (B) measured 7.66 AV: 1 T: FTMS + p ESI z=? 971.26849 z=7 SIM ms [1290.00-1320.00]20 452.24893 683.26221 z=? z=1 z=2 packages. 10 analysis (SID, CID, HCD and ETD) generates comprehensive Amino acid modifica4ons Fab 60 Deamidataion, oxida.on, 1301.70923 0 400 600 800 1000 1200 R=1110811400 1600 1800 2000 sequence coverage and enables fast localization of modifications with 100 glycosyla.on, isomeriza.on m/z R=105350 40 minimum sample preparation. -­‐S-­‐S-­‐ Results 90 Fragmenta4on -­‐S-­‐S-­‐ Cleavage in hinge region chain Heavy 80 7+ R=109093 Relative Abundance Relative 20 y • For measurements of intact light and heavy chain as well as for the S S The analysis of large proteins of the size of intact anti-bodies (~150 kDa) 91 R=104772 Oligosaccharides 70 2 detection of fragment ion spectra from top-down experiments ultra- Fucosyla.on, sialyla.on, galactosyla.on,... H R=108541 R=110104 C using Orbitrap mass spectrometers has been significantly improved over the 0 1301.43034 NL: 60 z = 6 S S Fc R=94282 high resolution as provided by the Orbitrap Elite mass spectrometer is 100 2.13E3(B) Disulfide Bonds past few years. Large molecules like mAbs show only very short transient 50 R=102177 Free thiols, disulfide shuffling, thioether 1301.20754 c h n o s +H: R=108646 essential. 3 1301.59741 1027 1606 282 332 6 8+ 8+ H life-times due to their relatively big cross section. Thus, the method of choice (C) simulated 40 c /c· R=108759 C 80 C 1027 H 1624 N 282 O 332 S 6 125 125 C-­‐terminal heterogeneity -­‐COO Relative Abundance p (gss, s /p:40) Chrg30 18 R=109703 Lysine processing, Proline amida.on for intact antibodies is to use the shortest transient duration (48 ms) R=101019 -­‐ R: 120000 Res .Pwr . @FWHM R=107362 60 1301.70878 R=97925 R=105375 1301.09615 20 R=112194 available on the Orbitrap Elite MS (Figure 4). R=103665 R=99113 R=82023 Abbreviations 10 R=86808 FIGURE 1: General structure of mAbs and their biological and 40 1301.82014 T: 0 ACN, acetonitrile; CID, collision-induced dissociation; C-trap, curved 2794.98103 physico-chemical characteristics. 100 2848.70886 1300.98475 1702.5 1703.0 1703.5 1704.0 1704.5 1705.0 1705.5 1706.0 1706.5 1707.0 2743.24976 20 m/z 90 +53 1301.98718 linear trap; DTT, dithiothreitol; ETD, electron transfer dissociation; FA, 80 +54 +52 intactHumira_ETD15_LP_HCD_240k #11-27 RT: 6.11-11.23 AV: 17 NL: 1.07E4 formic acid; HCD, higher energy collision-induced dissociation; mAb, 70 0 T: FTMS + p ESI Full ms2 [email protected] [200.00-4000.00] 2794.98103 100 100 Methods 60 1300.5 1301.0 1301.5 1302.0 1302.5 2693.38227 90 monoclonal antibody; µS, micro-scan; SID, in-source decay; SIM, single 50 m/z (C) (A) 80 ETD 90 2962.591 40 23407.63635 ion monitoring. 2749.10120 2800.91032 2854.81639 100 (D) 70 Sample Preparation 30 Relative Abundance Isotope pattern after 60 20

80 2787.42772 2840.04424 50 10 80 deconvolution with Xtract ™ 2767.72395 2820.90307 AbbVie™ HUMIRA (adalimumab, Figure 2) [1]: The intact antibody (144 2598.92826 40 0 References 2740 2760 2780 2800 2820 2840 2860 30 70 60 Abundance Relative kDa) was dissolved in 0.1 % FA to 1 µg/µL; 5 µg HUMIRA were loaded onto m/z

20 3085.98807 10 1. Bondarenko, P.V., Second, T.P., Zabrouskov, V., Makarov, A. & the column. 60 40 0 500 1000 1500 2000 2500 3000 3500 Zhang, Z. Mass measurement and top-down HPLC/MS analysis of 2510.86531 m/z Relative Abundance 20 23421.61756 50 23390.64710 23433.66736 intact monoclonal antibodies on a hybrid linear quadrupole ion trap- FIGURE 7: (A) CID spectrum and (C) ETD spectrum of intact HUMIRA 3151.64648 0 Orbitrap mass spectrometer. Journal of the American Society for 40 2428.59194 23393.62879 antibody. (B) Zoom in into the ETD fragment ion spectrum of intact Relative Abundance 3220.13151 Mass Spectrometry 20, 1415-24 (2009). 100 Monoisotopic mass (M) of the HUMIRA showing the need for highest resolution possible. 30 3291.67370 2351.52066 (E) Δm=2.4 ppm intact light chain after 3366.46848 80 2. Michalski, A. et al. Ultra high resolution linear ion trap Orbitrap mass 20 deconvolution with Xtract 2279.19708 3444.75592 spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and 60 2178.67441 3526.72930 10 versatile peptide fragmentation modes. Molecular & cellular 2029.53882 3703.00780 40 proteomics: MCP (2011).doi:10.1074/mcp.O111.013698 0 20 23435.63611 2000 2500 3000 3500 23407.58564 23419.59961 m/z 23428.60343 0 23380 23390 23400 23410 23420 23430 Acknowledgements m/z FIGURE 2: 3D structure of HUMIRA highlighting the attached glycans and (B) We would like to thank Paul Thomas from Northwestern University (USA) cystein residues forming inter- and intra-chain disulfide bridges. +Hex FIGURE 5. (A) Full MS spectrum of intact light chain of HUMIRA. (B) for processing the HUMIRA ETD data. For analyzing HUMIRA light chain (24 kDa) and heavy chain (51 kDa) +Hex Zoom into +18 charge state of intact light chain. (C) Simulation of isotope pattern of +18 charge state. (D) Isotope pattern of intact light chain after separately, 50 µg HUMIRA was reduced with DTT (20-fold molar excess, HUMIRA is a trademark of AbbVie. All other trademarks are the property of Thermo Fisher Scientific and 56°C for 1 h) and alkylated with iodoacetamide (50-fold molar excess, room deconvolution. (E) Monoisotopic mass (M) of the measured light chain of its subsidiaries. FIGURE 8: Summarized sequence coverage of the HUMIRA heavy chain temperature for 30 min in the dark). HUMIRA obtained after deconvolution with Xtract. This information is not intended to encourage use of these products in any manners that might infringe FIGURE 4: (A) Full MS spectrum of intact HUMIRA. The insert shows a using fragmentation techniques SID, CID, HCD, and ETD. Optimized the intellectual property rights of others. zoom into the three most abundant charge states z=52,53,54. (B) Spectrum conditions: trapping under high pressure settings. N: Putative For Research Use only, not for Use in Diagnostic Procedures. after deconvolution. glycosylation site.

2 Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry Kai Scheffler1, Eugen Damoc2, Mathias Müller2, Martin Zeller2, Thomas Moehring2 Thermo Fisher Scientific, Dreieich1 and Bremen2, Germany

HUMIRA_heavy_av_SIM # 1 RT: 1556.88 AV: 1 NL: 3.30E3 Humira_heavy_av_SIM #1 RT: 1556.88 AV: 1 NL: 3.30E3 T: FTMS + p ESI SIM ms [1180.00-1190.00] T: FTMS + p ESI SIM ms [1180.00-1190.00] Monoisotopic mass (M) of the 50891.04317 z=43 1185.2190 100 Instrument 1185.2190 R=117700 1185.2921 Introduction Deconvolution intact heavy chain after R=117700 100 R=117704 80 100 1185.1287 zoom R=120804 60 deconvolution with Xtract 80 1185.3820 Recombinant monoclonal antibodies have gained significant importance in A Thermo Scientific™ Surveyor™ MS Pump Plus was coupled to an 90 1304.56287 R=118304 100 1185.4508 40 60 1185.4961 diagnostic and therapeutic applications over the past years. In order to verify Orbitrap Elite mass spectrometer that was equipped with ETD (Figure 3). z=18 80 R=122004 1235.95410 1183.9644 R=120004 20 90 (A) R=120204 40

Relative Abundance the correctness of the overall molecule to provide a reproducible, safe and 70 0 Square Quadrupole z=19 Electrospray source S-lens Octopole High Pressure Cell Low Pressure Cell Quadrupole Mass Filter HCD Collision Cell Transfer Multipole Reagent Ion Source 1182.5933 C-trap Abundance Relative

20 with Beam Blocker 1184.9670 50922.07492 80 z=17 60 R=116404 1183.5918 100 effective biological drug compound, the correct protein sequence, as well as R=121104 Isotope pattern and average R=122204 0 1381.19873 1182.2444 80 50 1185.2 1185.4 70 z=20 R=121404 mass of the intact heavy chain the presence and relative abundance of different glycoforms have to be z=16 m/z 1174.17102 1185.8925 60 1467.49915 40 R=109804 after deconvolution with Xtract confirmed. 40 60 1181.6860 1186.2635 z=15 Relative Abundance 30 R=117204 R=127904 20 Here we present an approach to analyze an intact monoclonal antibody in 1180.9857 50 1565.29358 z=21 20 R=133304 0 New z=12 non-reduced and reduced condition by LC-MS using the Thermo Scientific™ High-Field Orbitrap 1118.31189 50890 50900 50910 50920 50930 50940 Mass Analyzer z=14 10 m/z 40 1956.39819 z=11 Orbitrap Elite™ mass spectrometer. The intact antibody and the separated 1677.03210 0 Relative Abundance 2134.19409 Reagent 1 Reagent 2 30 z=13 1181 1182 1183 1184 1185 1186 Heated Inlet Heated Inlet light and heavy chains were analyzed in Full MS experiments as well as with z=22 1806.14465 m/z FIGURE 6: HUMIRA heavy chain acquired in SIM scan mode (z=43). 60 µscans were top-down experiments using in-source CID (SID), CID, HCD and ETD 20 1067.56909 z=10 averaged. Deconvoluted mass: Mr 50,891.04317 Da. The inserts on the right fragmentation techniques making use of the ultrahigh resolution of the mass FIGURE 3: Schematics of the Orbitrap Elite hybrid ion trap-Orbitrap mass 10 1021.25623 2347.60156 Conclusion demonstrate isotopic resolution of that charge state detected at m/z 1185 and masses spectrometer. For data evaluation ProSight software and Thermo Scientific™ spectrometer equipped with an ETD source. 2610.80981 0 obtained after deconvolution using Xtract. The analysis of intact and reduced antibodies on the Orbitrap Elite 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 Humira_CID_59_120210173005 #1 RT: 355.98 AV: 1 NL: 6.11E3 • Protein Deconvolution™ software version 1.0 packages were used. T: FTMS + p ESI Full ms2 [email protected] [325.00-4000.00] m/z 1395.24207 1507.50037 mass spectrometer provides the accurate molecular weight, as well as z=18 Samples were purified on a Thermo Scientific™ BioBasic™ C4 column (150 100 z=8 1321.86304 90 1594.27380 valuable information about the presence and abundance of glycoforms. x 1 mm, 5 µm particles), solvent A: 0.1 % FA, 2 % ACN in H O, solvent B: z=? z=8 2 80 CID 1629.42053 Physicochemical Characteris4cs R=124.100 70 Biological Characteris4cs 0.1 % FA in ACN. The LC gradient was 7 min 20–40 % B, 3 min 40–80 % B z=8 • Analysis of the reduced antibody provides isotopically resolved mass 60 1301.43152 NL: (A) 1217.55786 at a flow rate of 100 µL/min. 100 Δ=0.9ppm 50 z=? spectra for both light and heavy chain. 3.84E5 1076.95667 N-­‐terminal heterogeneity 40 551.31708 z=24 z=18 1301.26538 FS_tripleSIM_20uS_1#19 RT: z=1 Pyroglutamate forma.on 30 782.33112 1756.70886 Data analysis was done using Protein Deconvolution and ProSight software 1301.59814 Abundance Relative • The combination of multiple fragmentation techniques in top-down Other modifica.ons 80 (B) measured 7.66 AV: 1 T: FTMS + p ESI z=? 971.26849 z=7 SIM ms [1290.00-1320.00]20 452.24893 683.26221 z=? z=1 z=2 packages. 10 analysis (SID, CID, HCD and ETD) generates comprehensive Amino acid modifica4ons Fab 60 Deamidataion, oxida.on, 1301.70923 0 400 600 800 1000 1200 R=1110811400 1600 1800 2000 sequence coverage and enables fast localization of modifications with 100 glycosyla.on, isomeriza.on m/z R=105350 40 minimum sample preparation. -­‐S-­‐S-­‐ Results 90 Fragmenta4on -­‐S-­‐S-­‐ Cleavage in hinge region chain Heavy 80 7+ R=109093 Relative Abundance Relative 20 y • For measurements of intact light and heavy chain as well as for the S S The analysis of large proteins of the size of intact anti-bodies (~150 kDa) 91 R=104772 Oligosaccharides 70 2 detection of fragment ion spectra from top-down experiments ultra- Fucosyla.on, sialyla.on, galactosyla.on,... H R=108541 R=110104 C using Orbitrap mass spectrometers has been significantly improved over the 0 1301.43034 NL: 60 z = 6 S S Fc R=94282 high resolution as provided by the Orbitrap Elite mass spectrometer is 100 2.13E3(B) Disulfide Bonds past few years. Large molecules like mAbs show only very short transient 50 R=102177 Free thiols, disulfide shuffling, thioether 1301.20754 c h n o s +H: R=108646 essential. 3 1301.59741 1027 1606 282 332 6 8+ 8+ H life-times due to their relatively big cross section. Thus, the method of choice (C) simulated 40 c /c· R=108759 C 80 C 1027 H 1624 N 282 O 332 S 6 125 125 C-­‐terminal heterogeneity -­‐COO Relative Abundance p (gss, s /p:40) Chrg30 18 R=109703 Lysine processing, Proline amida.on for intact antibodies is to use the shortest transient duration (48 ms) R=101019 -­‐ R: 120000 Res .Pwr . @FWHM R=107362 60 1301.70878 R=97925 R=105375 1301.09615 20 R=112194 available on the Orbitrap Elite MS (Figure 4). R=103665 R=99113 R=82023 Abbreviations 10 R=86808 FIGURE 1: General structure of mAbs and their biological and 40 1301.82014 T: 0 ACN, acetonitrile; CID, collision-induced dissociation; C-trap, curved 2794.98103 physico-chemical characteristics. 100 2848.70886 1300.98475 1702.5 1703.0 1703.5 1704.0 1704.5 1705.0 1705.5 1706.0 1706.5 1707.0 2743.24976 20 m/z 90 +53 1301.98718 linear trap; DTT, dithiothreitol; ETD, electron transfer dissociation; FA, 80 +54 +52 intactHumira_ETD15_LP_HCD_240k #11-27 RT: 6.11-11.23 AV: 17 NL: 1.07E4 formic acid; HCD, higher energy collision-induced dissociation; mAb, 70 0 T: FTMS + p ESI Full ms2 [email protected] [200.00-4000.00] 2794.98103 100 100 Methods 60 1300.5 1301.0 1301.5 1302.0 1302.5 2693.38227 90 monoclonal antibody; µS, micro-scan; SID, in-source decay; SIM, single 50 m/z (C) (A) 80 ETD 90 2962.591 40 23407.63635 ion monitoring. 2749.10120 2800.91032 2854.81639 100 (D) 70 Sample Preparation 30 Relative Abundance Isotope pattern after 60 20

80 2787.42772 2840.04424 50 10 80 deconvolution with Xtract ™ 2767.72395 2820.90307 AbbVie™ HUMIRA (adalimumab, Figure 2) [1]: The intact antibody (144 2598.92826 40 0 References 2740 2760 2780 2800 2820 2840 2860 30 70 60 Abundance Relative kDa) was dissolved in 0.1 % FA to 1 µg/µL; 5 µg HUMIRA were loaded onto m/z

20 3085.98807 10 1. Bondarenko, P.V., Second, T.P., Zabrouskov, V., Makarov, A. & the column. 60 40 0 500 1000 1500 2000 2500 3000 3500 Zhang, Z. Mass measurement and top-down HPLC/MS analysis of 2510.86531 m/z Relative Abundance 20 23421.61756 50 23390.64710 23433.66736 intact monoclonal antibodies on a hybrid linear quadrupole ion trap- FIGURE 7: (A) CID spectrum and (C) ETD spectrum of intact HUMIRA 3151.64648 0 Orbitrap mass spectrometer. Journal of the American Society for 40 2428.59194 23393.62879 antibody. (B) Zoom in into the ETD fragment ion spectrum of intact Relative Abundance 3220.13151 Mass Spectrometry 20, 1415-24 (2009). 100 Monoisotopic mass (M) of the HUMIRA showing the need for highest resolution possible. 30 3291.67370 2351.52066 (E) Δm=2.4 ppm intact light chain after 3366.46848 80 2. Michalski, A. et al. Ultra high resolution linear ion trap Orbitrap mass 20 deconvolution with Xtract 2279.19708 3444.75592 spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and 60 2178.67441 3526.72930 10 versatile peptide fragmentation modes. Molecular & cellular 2029.53882 3703.00780 40 proteomics: MCP (2011).doi:10.1074/mcp.O111.013698 0 20 23435.63611 2000 2500 3000 3500 23407.58564 23419.59961 m/z 23428.60343 0 23380 23390 23400 23410 23420 23430 Acknowledgements m/z FIGURE 2: 3D structure of HUMIRA highlighting the attached glycans and (B) We would like to thank Paul Thomas from Northwestern University (USA) cystein residues forming inter- and intra-chain disulfide bridges. +Hex FIGURE 5. (A) Full MS spectrum of intact light chain of HUMIRA. (B) for processing the HUMIRA ETD data. For analyzing HUMIRA light chain (24 kDa) and heavy chain (51 kDa) +Hex Zoom into +18 charge state of intact light chain. (C) Simulation of isotope pattern of +18 charge state. (D) Isotope pattern of intact light chain after separately, 50 µg HUMIRA was reduced with DTT (20-fold molar excess, HUMIRA is a trademark of AbbVie. All other trademarks are the property of Thermo Fisher Scientific and 56°C for 1 h) and alkylated with iodoacetamide (50-fold molar excess, room deconvolution. (E) Monoisotopic mass (M) of the measured light chain of its subsidiaries. FIGURE 8: Summarized sequence coverage of the HUMIRA heavy chain temperature for 30 min in the dark). HUMIRA obtained after deconvolution with Xtract. This information is not intended to encourage use of these products in any manners that might infringe FIGURE 4: (A) Full MS spectrum of intact HUMIRA. The insert shows a using fragmentation techniques SID, CID, HCD, and ETD. Optimized the intellectual property rights of others. zoom into the three most abundant charge states z=52,53,54. (B) Spectrum conditions: trapping under high pressure settings. N: Putative For Research Use only, not for Use in Diagnostic Procedures. after deconvolution. glycosylation site.

Thermo Scientific Poster Note• BioPharma_PN63712_E 11/13S 3 Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry Kai Scheffler1, Eugen Damoc2, Mathias Müller2, Martin Zeller2, Thomas Moehring2 Thermo Fisher Scientific, Dreieich1 and Bremen2, Germany

HUMIRA_heavy_av_SIM # 1 RT: 1556.88 AV: 1 NL: 3.30E3 Humira_heavy_av_SIM #1 RT: 1556.88 AV: 1 NL: 3.30E3 T: FTMS + p ESI SIM ms [1180.00-1190.00] T: FTMS + p ESI SIM ms [1180.00-1190.00] Monoisotopic mass (M) of the 50891.04317 z=43 1185.2190 100 Instrument 1185.2190 R=117700 1185.2921 Introduction Deconvolution intact heavy chain after R=117700 100 R=117704 80 100 1185.1287 zoom R=120804 60 deconvolution with Xtract 80 1185.3820 Recombinant monoclonal antibodies have gained significant importance in A Thermo Scientific™ Surveyor™ MS Pump Plus was coupled to an 90 1304.56287 R=118304 100 1185.4508 40 60 1185.4961 diagnostic and therapeutic applications over the past years. In order to verify Orbitrap Elite mass spectrometer that was equipped with ETD (Figure 3). z=18 80 R=122004 1235.95410 1183.9644 R=120004 20 90 (A) R=120204 40

Relative Abundance the correctness of the overall molecule to provide a reproducible, safe and 70 0 Square Quadrupole z=19 Electrospray source S-lens Octopole High Pressure Cell Low Pressure Cell Quadrupole Mass Filter HCD Collision Cell Transfer Multipole Reagent Ion Source 1182.5933 C-trap Abundance Relative

20 with Beam Blocker 1184.9670 50922.07492 80 z=17 60 R=116404 1183.5918 100 effective biological drug compound, the correct protein sequence, as well as R=121104 Isotope pattern and average R=122204 0 1381.19873 1182.2444 80 50 1185.2 1185.4 70 z=20 R=121404 mass of the intact heavy chain the presence and relative abundance of different glycoforms have to be z=16 m/z 1174.17102 1185.8925 60 1467.49915 40 R=109804 after deconvolution with Xtract confirmed. 40 60 1181.6860 1186.2635 z=15 Relative Abundance 30 R=117204 R=127904 20 Here we present an approach to analyze an intact monoclonal antibody in 1180.9857 50 1565.29358 z=21 20 R=133304 0 New z=12 non-reduced and reduced condition by LC-MS using the Thermo Scientific™ High-Field Orbitrap 1118.31189 50890 50900 50910 50920 50930 50940 Mass Analyzer z=14 10 m/z 40 1956.39819 z=11 Orbitrap Elite™ mass spectrometer. The intact antibody and the separated 1677.03210 0 Relative Abundance 2134.19409 Reagent 1 Reagent 2 30 z=13 1181 1182 1183 1184 1185 1186 Heated Inlet Heated Inlet light and heavy chains were analyzed in Full MS experiments as well as with z=22 1806.14465 m/z FIGURE 6: HUMIRA heavy chain acquired in SIM scan mode (z=43). 60 µscans were top-down experiments using in-source CID (SID), CID, HCD and ETD 20 1067.56909 z=10 averaged. Deconvoluted mass: Mr 50,891.04317 Da. The inserts on the right fragmentation techniques making use of the ultrahigh resolution of the mass FIGURE 3: Schematics of the Orbitrap Elite hybrid ion trap-Orbitrap mass 10 1021.25623 2347.60156 Conclusion demonstrate isotopic resolution of that charge state detected at m/z 1185 and masses spectrometer. For data evaluation ProSight software and Thermo Scientific™ spectrometer equipped with an ETD source. 2610.80981 0 obtained after deconvolution using Xtract. The analysis of intact and reduced antibodies on the Orbitrap Elite 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 Humira_CID_59_120210173005 #1 RT: 355.98 AV: 1 NL: 6.11E3 • Protein Deconvolution™ software version 1.0 packages were used. T: FTMS + p ESI Full ms2 [email protected] [325.00-4000.00] m/z 1395.24207 1507.50037 mass spectrometer provides the accurate molecular weight, as well as z=18 Samples were purified on a Thermo Scientific™ BioBasic™ C4 column (150 100 z=8 1321.86304 90 1594.27380 valuable information about the presence and abundance of glycoforms. x 1 mm, 5 µm particles), solvent A: 0.1 % FA, 2 % ACN in H O, solvent B: z=? z=8 2 80 CID 1629.42053 Physicochemical Characteris4cs R=124.100 70 Biological Characteris4cs 0.1 % FA in ACN. The LC gradient was 7 min 20–40 % B, 3 min 40–80 % B z=8 • Analysis of the reduced antibody provides isotopically resolved mass 60 1301.43152 NL: (A) 1217.55786 at a flow rate of 100 µL/min. 100 Δ=0.9ppm 50 z=? spectra for both light and heavy chain. 3.84E5 1076.95667 N-­‐terminal heterogeneity 40 551.31708 z=24 z=18 1301.26538 FS_tripleSIM_20uS_1#19 RT: z=1 Pyroglutamate forma.on 30 782.33112 1756.70886 Data analysis was done using Protein Deconvolution and ProSight software 1301.59814 Abundance Relative • The combination of multiple fragmentation techniques in top-down Other modifica.ons 80 (B) measured 7.66 AV: 1 T: FTMS + p ESI z=? 971.26849 z=7 SIM ms [1290.00-1320.00]20 452.24893 683.26221 z=? z=1 z=2 packages. 10 analysis (SID, CID, HCD and ETD) generates comprehensive Amino acid modifica4ons Fab 60 Deamidataion, oxida.on, 1301.70923 0 400 600 800 1000 1200 R=1110811400 1600 1800 2000 sequence coverage and enables fast localization of modifications with 100 glycosyla.on, isomeriza.on m/z R=105350 40 minimum sample preparation. -­‐S-­‐S-­‐ Results 90 Fragmenta4on -­‐S-­‐S-­‐ Cleavage in hinge region chain Heavy 80 7+ R=109093 Relative Abundance Relative 20 y • For measurements of intact light and heavy chain as well as for the S S The analysis of large proteins of the size of intact anti-bodies (~150 kDa) 91 R=104772 Oligosaccharides 70 2 detection of fragment ion spectra from top-down experiments ultra- Fucosyla.on, sialyla.on, galactosyla.on,... H R=108541 R=110104 C using Orbitrap mass spectrometers has been significantly improved over the 0 1301.43034 NL: 60 z = 6 S S Fc R=94282 high resolution as provided by the Orbitrap Elite mass spectrometer is 100 2.13E3(B) Disulfide Bonds past few years. Large molecules like mAbs show only very short transient 50 R=102177 Free thiols, disulfide shuffling, thioether 1301.20754 c h n o s +H: R=108646 essential. 3 1301.59741 1027 1606 282 332 6 8+ 8+ H life-times due to their relatively big cross section. Thus, the method of choice (C) simulated 40 c /c· R=108759 C 80 C 1027 H 1624 N 282 O 332 S 6 125 125 C-­‐terminal heterogeneity -­‐COO Relative Abundance p (gss, s /p:40) Chrg30 18 R=109703 Lysine processing, Proline amida.on for intact antibodies is to use the shortest transient duration (48 ms) R=101019 -­‐ R: 120000 Res .Pwr . @FWHM R=107362 60 1301.70878 R=97925 R=105375 1301.09615 20 R=112194 available on the Orbitrap Elite MS (Figure 4). R=103665 R=99113 R=82023 Abbreviations 10 R=86808 FIGURE 1: General structure of mAbs and their biological and 40 1301.82014 T: 0 ACN, acetonitrile; CID, collision-induced dissociation; C-trap, curved 2794.98103 physico-chemical characteristics. 100 2848.70886 1300.98475 1702.5 1703.0 1703.5 1704.0 1704.5 1705.0 1705.5 1706.0 1706.5 1707.0 2743.24976 20 m/z 90 +53 1301.98718 linear trap; DTT, dithiothreitol; ETD, electron transfer dissociation; FA, 80 +54 +52 intactHumira_ETD15_LP_HCD_240k #11-27 RT: 6.11-11.23 AV: 17 NL: 1.07E4 formic acid; HCD, higher energy collision-induced dissociation; mAb, 70 0 T: FTMS + p ESI Full ms2 [email protected] [200.00-4000.00] 2794.98103 100 100 Methods 60 1300.5 1301.0 1301.5 1302.0 1302.5 2693.38227 90 monoclonal antibody; µS, micro-scan; SID, in-source decay; SIM, single 50 m/z (C) (A) 80 ETD 90 2962.591 40 23407.63635 ion monitoring. 2749.10120 2800.91032 2854.81639 100 (D) 70 Sample Preparation 30 Relative Abundance Isotope pattern after 60 20

80 2787.42772 2840.04424 50 10 80 deconvolution with Xtract ™ 2767.72395 2820.90307 AbbVie™ HUMIRA (adalimumab, Figure 2) [1]: The intact antibody (144 2598.92826 40 0 References 2740 2760 2780 2800 2820 2840 2860 30 70 60 Abundance Relative kDa) was dissolved in 0.1 % FA to 1 µg/µL; 5 µg HUMIRA were loaded onto m/z

20 3085.98807 10 1. Bondarenko, P.V., Second, T.P., Zabrouskov, V., Makarov, A. & the column. 60 40 0 500 1000 1500 2000 2500 3000 3500 Zhang, Z. Mass measurement and top-down HPLC/MS analysis of 2510.86531 m/z Relative Abundance 20 23421.61756 50 23390.64710 23433.66736 intact monoclonal antibodies on a hybrid linear quadrupole ion trap- FIGURE 7: (A) CID spectrum and (C) ETD spectrum of intact HUMIRA 3151.64648 0 Orbitrap mass spectrometer. Journal of the American Society for 40 2428.59194 23393.62879 antibody. (B) Zoom in into the ETD fragment ion spectrum of intact Relative Abundance 3220.13151 Mass Spectrometry 20, 1415-24 (2009). 100 Monoisotopic mass (M) of the HUMIRA showing the need for highest resolution possible. 30 3291.67370 2351.52066 (E) Δm=2.4 ppm intact light chain after 3366.46848 80 2. Michalski, A. et al. Ultra high resolution linear ion trap Orbitrap mass 20 deconvolution with Xtract 2279.19708 3444.75592 spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and 60 2178.67441 3526.72930 10 versatile peptide fragmentation modes. Molecular & cellular 2029.53882 3703.00780 40 proteomics: MCP (2011).doi:10.1074/mcp.O111.013698 0 20 23435.63611 2000 2500 3000 3500 23407.58564 23419.59961 m/z 23428.60343 0 23380 23390 23400 23410 23420 23430 Acknowledgements m/z FIGURE 2: 3D structure of HUMIRA highlighting the attached glycans and (B) We would like to thank Paul Thomas from Northwestern University (USA) cystein residues forming inter- and intra-chain disulfide bridges. +Hex FIGURE 5. (A) Full MS spectrum of intact light chain of HUMIRA. (B) for processing the HUMIRA ETD data. For analyzing HUMIRA light chain (24 kDa) and heavy chain (51 kDa) +Hex Zoom into +18 charge state of intact light chain. (C) Simulation of isotope pattern of +18 charge state. (D) Isotope pattern of intact light chain after separately, 50 µg HUMIRA was reduced with DTT (20-fold molar excess, HUMIRA is a trademark of AbbVie. All other trademarks are the property of Thermo Fisher Scientific and 56°C for 1 h) and alkylated with iodoacetamide (50-fold molar excess, room deconvolution. (E) Monoisotopic mass (M) of the measured light chain of its subsidiaries. FIGURE 8: Summarized sequence coverage of the HUMIRA heavy chain temperature for 30 min in the dark). HUMIRA obtained after deconvolution with Xtract. This information is not intended to encourage use of these products in any manners that might infringe FIGURE 4: (A) Full MS spectrum of intact HUMIRA. The insert shows a using fragmentation techniques SID, CID, HCD, and ETD. Optimized the intellectual property rights of others. zoom into the three most abundant charge states z=52,53,54. (B) Spectrum conditions: trapping under high pressure settings. N: Putative For Research Use only, not for Use in Diagnostic Procedures. after deconvolution. glycosylation site.

4 Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry Kai Scheffler1, Eugen Damoc2, Mathias Müller2, Martin Zeller2, Thomas Moehring2 Thermo Fisher Scientific, Dreieich1 and Bremen2, Germany

HUMIRA_heavy_av_SIM # 1 RT: 1556.88 AV: 1 NL: 3.30E3 Humira_heavy_av_SIM #1 RT: 1556.88 AV: 1 NL: 3.30E3 T: FTMS + p ESI SIM ms [1180.00-1190.00] T: FTMS + p ESI SIM ms [1180.00-1190.00] Monoisotopic mass (M) of the 50891.04317 z=43 1185.2190 100 Instrument 1185.2190 R=117700 1185.2921 Introduction Deconvolution intact heavy chain after R=117700 100 R=117704 80 100 1185.1287 zoom R=120804 60 deconvolution with Xtract 80 1185.3820 Recombinant monoclonal antibodies have gained significant importance in A Thermo Scientific™ Surveyor™ MS Pump Plus was coupled to an 90 1304.56287 R=118304 100 1185.4508 40 60 1185.4961 diagnostic and therapeutic applications over the past years. In order to verify Orbitrap Elite mass spectrometer that was equipped with ETD (Figure 3). z=18 80 R=122004 1235.95410 1183.9644 R=120004 20 90 (A) R=120204 40

Relative Abundance the correctness of the overall molecule to provide a reproducible, safe and 70 0 Square Quadrupole z=19 Electrospray source S-lens Octopole High Pressure Cell Low Pressure Cell Quadrupole Mass Filter HCD Collision Cell Transfer Multipole Reagent Ion Source 1182.5933 C-trap Abundance Relative

20 with Beam Blocker 1184.9670 50922.07492 80 z=17 60 R=116404 1183.5918 100 effective biological drug compound, the correct protein sequence, as well as R=121104 Isotope pattern and average R=122204 0 1381.19873 1182.2444 80 50 1185.2 1185.4 70 z=20 R=121404 mass of the intact heavy chain the presence and relative abundance of different glycoforms have to be z=16 m/z 1174.17102 1185.8925 60 1467.49915 40 R=109804 after deconvolution with Xtract confirmed. 40 60 1181.6860 1186.2635 z=15 Relative Abundance 30 R=117204 R=127904 20 Here we present an approach to analyze an intact monoclonal antibody in 1180.9857 50 1565.29358 z=21 20 R=133304 0 New z=12 non-reduced and reduced condition by LC-MS using the Thermo Scientific™ High-Field Orbitrap 1118.31189 50890 50900 50910 50920 50930 50940 Mass Analyzer z=14 10 m/z 40 1956.39819 z=11 Orbitrap Elite™ mass spectrometer. The intact antibody and the separated 1677.03210 0 Relative Abundance 2134.19409 Reagent 1 Reagent 2 30 z=13 1181 1182 1183 1184 1185 1186 Heated Inlet Heated Inlet light and heavy chains were analyzed in Full MS experiments as well as with z=22 1806.14465 m/z FIGURE 6: HUMIRA heavy chain acquired in SIM scan mode (z=43). 60 µscans were top-down experiments using in-source CID (SID), CID, HCD and ETD 20 1067.56909 z=10 averaged. Deconvoluted mass: Mr 50,891.04317 Da. The inserts on the right fragmentation techniques making use of the ultrahigh resolution of the mass FIGURE 3: Schematics of the Orbitrap Elite hybrid ion trap-Orbitrap mass 10 1021.25623 2347.60156 Conclusion demonstrate isotopic resolution of that charge state detected at m/z 1185 and masses spectrometer. For data evaluation ProSight software and Thermo Scientific™ spectrometer equipped with an ETD source. 2610.80981 0 obtained after deconvolution using Xtract. The analysis of intact and reduced antibodies on the Orbitrap Elite 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 Humira_CID_59_120210173005 #1 RT: 355.98 AV: 1 NL: 6.11E3 • Protein Deconvolution™ software version 1.0 packages were used. T: FTMS + p ESI Full ms2 [email protected] [325.00-4000.00] m/z 1395.24207 1507.50037 mass spectrometer provides the accurate molecular weight, as well as z=18 Samples were purified on a Thermo Scientific™ BioBasic™ C4 column (150 100 z=8 1321.86304 90 1594.27380 valuable information about the presence and abundance of glycoforms. x 1 mm, 5 µm particles), solvent A: 0.1 % FA, 2 % ACN in H O, solvent B: z=? z=8 2 80 CID 1629.42053 Physicochemical Characteris4cs R=124.100 70 Biological Characteris4cs 0.1 % FA in ACN. The LC gradient was 7 min 20–40 % B, 3 min 40–80 % B z=8 • Analysis of the reduced antibody provides isotopically resolved mass 60 1301.43152 NL: (A) 1217.55786 at a flow rate of 100 µL/min. 100 Δ=0.9ppm 50 z=? spectra for both light and heavy chain. 3.84E5 1076.95667 N-­‐terminal heterogeneity 40 551.31708 z=24 z=18 1301.26538 FS_tripleSIM_20uS_1#19 RT: z=1 Pyroglutamate forma.on 30 782.33112 1756.70886 Data analysis was done using Protein Deconvolution and ProSight software 1301.59814 Abundance Relative • The combination of multiple fragmentation techniques in top-down Other modifica.ons 80 (B) measured 7.66 AV: 1 T: FTMS + p ESI z=? 971.26849 z=7 SIM ms [1290.00-1320.00]20 452.24893 683.26221 z=? z=1 z=2 packages. 10 analysis (SID, CID, HCD and ETD) generates comprehensive Amino acid modifica4ons Fab 60 Deamidataion, oxida.on, 1301.70923 0 400 600 800 1000 1200 R=1110811400 1600 1800 2000 sequence coverage and enables fast localization of modifications with 100 glycosyla.on, isomeriza.on m/z R=105350 40 minimum sample preparation. -­‐S-­‐S-­‐ Results 90 Fragmenta4on -­‐S-­‐S-­‐ Cleavage in hinge region chain Heavy 80 7+ R=109093 Relative Abundance Relative 20 y • For measurements of intact light and heavy chain as well as for the S S The analysis of large proteins of the size of intact anti-bodies (~150 kDa) 91 R=104772 Oligosaccharides 70 2 detection of fragment ion spectra from top-down experiments ultra- Fucosyla.on, sialyla.on, galactosyla.on,... H R=108541 R=110104 C using Orbitrap mass spectrometers has been significantly improved over the 0 1301.43034 NL: 60 z = 6 S S Fc R=94282 high resolution as provided by the Orbitrap Elite mass spectrometer is 100 2.13E3(B) Disulfide Bonds past few years. Large molecules like mAbs show only very short transient 50 R=102177 Free thiols, disulfide shuffling, thioether 1301.20754 c h n o s +H: R=108646 essential. 3 1301.59741 1027 1606 282 332 6 8+ 8+ H life-times due to their relatively big cross section. Thus, the method of choice (C) simulated 40 c /c· R=108759 C 80 C 1027 H 1624 N 282 O 332 S 6 125 125 C-­‐terminal heterogeneity -­‐COO Relative Abundance p (gss, s /p:40) Chrg30 18 R=109703 Lysine processing, Proline amida.on for intact antibodies is to use the shortest transient duration (48 ms) R=101019 -­‐ R: 120000 Res .Pwr . @FWHM R=107362 60 1301.70878 R=97925 R=105375 1301.09615 20 R=112194 available on the Orbitrap Elite MS (Figure 4). R=103665 R=99113 R=82023 Abbreviations 10 R=86808 FIGURE 1: General structure of mAbs and their biological and 40 1301.82014 T: 0 ACN, acetonitrile; CID, collision-induced dissociation; C-trap, curved 2794.98103 physico-chemical characteristics. 100 2848.70886 1300.98475 1702.5 1703.0 1703.5 1704.0 1704.5 1705.0 1705.5 1706.0 1706.5 1707.0 2743.24976 20 m/z 90 +53 1301.98718 linear trap; DTT, dithiothreitol; ETD, electron transfer dissociation; FA, 80 +54 +52 intactHumira_ETD15_LP_HCD_240k #11-27 RT: 6.11-11.23 AV: 17 NL: 1.07E4 formic acid; HCD, higher energy collision-induced dissociation; mAb, 70 0 T: FTMS + p ESI Full ms2 [email protected] [200.00-4000.00] 2794.98103 100 100 Methods 60 1300.5 1301.0 1301.5 1302.0 1302.5 2693.38227 90 monoclonal antibody; µS, micro-scan; SID, in-source decay; SIM, single 50 m/z (C) (A) 80 ETD 90 2962.591 40 23407.63635 ion monitoring. 2749.10120 2800.91032 2854.81639 100 (D) 70 Sample Preparation 30 Relative Abundance Isotope pattern after 60 20

80 2787.42772 2840.04424 50 10 80 deconvolution with Xtract ™ 2767.72395 2820.90307 AbbVie™ HUMIRA (adalimumab, Figure 2) [1]: The intact antibody (144 2598.92826 40 0 References 2740 2760 2780 2800 2820 2840 2860 30 70 60 Abundance Relative kDa) was dissolved in 0.1 % FA to 1 µg/µL; 5 µg HUMIRA were loaded onto m/z

20 3085.98807 10 1. Bondarenko, P.V., Second, T.P., Zabrouskov, V., Makarov, A. & the column. 60 40 0 500 1000 1500 2000 2500 3000 3500 Zhang, Z. Mass measurement and top-down HPLC/MS analysis of 2510.86531 m/z Relative Abundance 20 23421.61756 50 23390.64710 23433.66736 intact monoclonal antibodies on a hybrid linear quadrupole ion trap- FIGURE 7: (A) CID spectrum and (C) ETD spectrum of intact HUMIRA 3151.64648 0 Orbitrap mass spectrometer. Journal of the American Society for 40 2428.59194 23393.62879 antibody. (B) Zoom in into the ETD fragment ion spectrum of intact Relative Abundance 3220.13151 Mass Spectrometry 20, 1415-24 (2009). 100 Monoisotopic mass (M) of the HUMIRA showing the need for highest resolution possible. 30 3291.67370 2351.52066 (E) Δm=2.4 ppm intact light chain after 3366.46848 80 2. Michalski, A. et al. Ultra high resolution linear ion trap Orbitrap mass 20 deconvolution with Xtract 2279.19708 3444.75592 spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and 60 2178.67441 3526.72930 10 versatile peptide fragmentation modes. Molecular & cellular 2029.53882 3703.00780 40 proteomics: MCP (2011).doi:10.1074/mcp.O111.013698 0 20 23435.63611 2000 2500 3000 3500 23407.58564 23419.59961 m/z 23428.60343 0 23380 23390 23400 23410 23420 23430 Acknowledgements m/z FIGURE 2: 3D structure of HUMIRA highlighting the attached glycans and (B) We would like to thank Paul Thomas from Northwestern University (USA) cystein residues forming inter- and intra-chain disulfide bridges. +Hex FIGURE 5. (A) Full MS spectrum of intact light chain of HUMIRA. (B) for processing the HUMIRA ETD data. For analyzing HUMIRA light chain (24 kDa) and heavy chain (51 kDa) +Hex Zoom into +18 charge state of intact light chain. (C) Simulation of isotope pattern of +18 charge state. (D) Isotope pattern of intact light chain after separately, 50 µg HUMIRA was reduced with DTT (20-fold molar excess, HUMIRA is a trademark of AbbVie. All other trademarks are the property of Thermo Fisher Scientific and 56°C for 1 h) and alkylated with iodoacetamide (50-fold molar excess, room deconvolution. (E) Monoisotopic mass (M) of the measured light chain of its subsidiaries. FIGURE 8: Summarized sequence coverage of the HUMIRA heavy chain temperature for 30 min in the dark). HUMIRA obtained after deconvolution with Xtract. This information is not intended to encourage use of these products in any manners that might infringe FIGURE 4: (A) Full MS spectrum of intact HUMIRA. The insert shows a using fragmentation techniques SID, CID, HCD, and ETD. Optimized the intellectual property rights of others. zoom into the three most abundant charge states z=52,53,54. (B) Spectrum conditions: trapping under high pressure settings. N: Putative For Research Use only, not for Use in Diagnostic Procedures. after deconvolution. glycosylation site.

Thermo Scientific Poster Note• BioPharma_PN63712_E 11/13S 5 Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry Kai Scheffler1, Eugen Damoc2, Mathias Müller2, Martin Zeller2, Thomas Moehring2 Thermo Fisher Scientific, Dreieich1 and Bremen2, Germany

HUMIRA_heavy_av_SIM # 1 RT: 1556.88 AV: 1 NL: 3.30E3 Humira_heavy_av_SIM #1 RT: 1556.88 AV: 1 NL: 3.30E3 T: FTMS + p ESI SIM ms [1180.00-1190.00] T: FTMS + p ESI SIM ms [1180.00-1190.00] Monoisotopic mass (M) of the 50891.04317 z=43 1185.2190 100 Instrument 1185.2190 R=117700 1185.2921 Introduction Deconvolution intact heavy chain after R=117700 100 R=117704 80 100 1185.1287 zoom R=120804 60 deconvolution with Xtract 80 1185.3820 Recombinant monoclonal antibodies have gained significant importance in A Thermo Scientific™ Surveyor™ MS Pump Plus was coupled to an 90 1304.56287 R=118304 100 1185.4508 40 60 1185.4961 diagnostic and therapeutic applications over the past years. In order to verify Orbitrap Elite mass spectrometer that was equipped with ETD (Figure 3). z=18 80 R=122004 1235.95410 1183.9644 R=120004 20 90 (A) R=120204 40

Relative Abundance the correctness of the overall molecule to provide a reproducible, safe and 70 0 Square Quadrupole z=19 Electrospray source S-lens Octopole High Pressure Cell Low Pressure Cell Quadrupole Mass Filter HCD Collision Cell Transfer Multipole Reagent Ion Source 1182.5933 C-trap Abundance Relative

20 with Beam Blocker 1184.9670 50922.07492 80 z=17 60 R=116404 1183.5918 100 effective biological drug compound, the correct protein sequence, as well as R=121104 Isotope pattern and average R=122204 0 1381.19873 1182.2444 80 50 1185.2 1185.4 70 z=20 R=121404 mass of the intact heavy chain the presence and relative abundance of different glycoforms have to be z=16 m/z 1174.17102 1185.8925 60 1467.49915 40 R=109804 after deconvolution with Xtract confirmed. 40 60 1181.6860 1186.2635 z=15 Relative Abundance 30 R=117204 R=127904 20 Here we present an approach to analyze an intact monoclonal antibody in 1180.9857 50 1565.29358 z=21 20 R=133304 0 New z=12 non-reduced and reduced condition by LC-MS using the Thermo Scientific™ High-Field Orbitrap 1118.31189 50890 50900 50910 50920 50930 50940 Mass Analyzer z=14 10 m/z 40 1956.39819 z=11 Orbitrap Elite™ mass spectrometer. The intact antibody and the separated 1677.03210 0 Relative Abundance 2134.19409 Reagent 1 Reagent 2 30 z=13 1181 1182 1183 1184 1185 1186 Heated Inlet Heated Inlet light and heavy chains were analyzed in Full MS experiments as well as with z=22 1806.14465 m/z FIGURE 6: HUMIRA heavy chain acquired in SIM scan mode (z=43). 60 µscans were top-down experiments using in-source CID (SID), CID, HCD and ETD 20 1067.56909 z=10 averaged. Deconvoluted mass: Mr 50,891.04317 Da. The inserts on the right fragmentation techniques making use of the ultrahigh resolution of the mass FIGURE 3: Schematics of the Orbitrap Elite hybrid ion trap-Orbitrap mass 10 1021.25623 2347.60156 Conclusion demonstrate isotopic resolution of that charge state detected at m/z 1185 and masses spectrometer. For data evaluation ProSight software and Thermo Scientific™ spectrometer equipped with an ETD source. 2610.80981 0 obtained after deconvolution using Xtract. The analysis of intact and reduced antibodies on the Orbitrap Elite 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 Humira_CID_59_120210173005 #1 RT: 355.98 AV: 1 NL: 6.11E3 • Protein Deconvolution™ software version 1.0 packages were used. T: FTMS + p ESI Full ms2 [email protected] [325.00-4000.00] m/z 1395.24207 1507.50037 mass spectrometer provides the accurate molecular weight, as well as z=18 Samples were purified on a Thermo Scientific™ BioBasic™ C4 column (150 100 z=8 1321.86304 90 1594.27380 valuable information about the presence and abundance of glycoforms. x 1 mm, 5 µm particles), solvent A: 0.1 % FA, 2 % ACN in H O, solvent B: z=? z=8 2 80 CID 1629.42053 Physicochemical Characteris4cs R=124.100 70 Biological Characteris4cs 0.1 % FA in ACN. The LC gradient was 7 min 20–40 % B, 3 min 40–80 % B z=8 • Analysis of the reduced antibody provides isotopically resolved mass 60 1301.43152 NL: (A) 1217.55786 at a flow rate of 100 µL/min. 100 Δ=0.9ppm 50 z=? spectra for both light and heavy chain. 3.84E5 1076.95667 N-­‐terminal heterogeneity 40 551.31708 z=24 z=18 1301.26538 FS_tripleSIM_20uS_1#19 RT: z=1 Pyroglutamate forma.on 30 782.33112 1756.70886 Data analysis was done using Protein Deconvolution and ProSight software 1301.59814 Abundance Relative • The combination of multiple fragmentation techniques in top-down Other modifica.ons 80 (B) measured 7.66 AV: 1 T: FTMS + p ESI z=? 971.26849 z=7 SIM ms [1290.00-1320.00]20 452.24893 683.26221 z=? z=1 z=2 packages. 10 analysis (SID, CID, HCD and ETD) generates comprehensive Amino acid modifica4ons Fab 60 Deamidataion, oxida.on, 1301.70923 0 400 600 800 1000 1200 R=1110811400 1600 1800 2000 sequence coverage and enables fast localization of modifications with 100 glycosyla.on, isomeriza.on m/z R=105350 40 minimum sample preparation. -­‐S-­‐S-­‐ Results 90 Fragmenta4on -­‐S-­‐S-­‐ Cleavage in hinge region chain Heavy 80 7+ R=109093 Relative Abundance Relative 20 y • For measurements of intact light and heavy chain as well as for the S S The analysis of large proteins of the size of intact anti-bodies (~150 kDa) 91 R=104772 Oligosaccharides 70 2 detection of fragment ion spectra from top-down experiments ultra- Fucosyla.on, sialyla.on, galactosyla.on,... H R=108541 R=110104 C using Orbitrap mass spectrometers has been significantly improved over the 0 1301.43034 NL: 60 z = 6 S S Fc R=94282 high resolution as provided by the Orbitrap Elite mass spectrometer is 100 2.13E3(B) Disulfide Bonds past few years. Large molecules like mAbs show only very short transient 50 R=102177 Free thiols, disulfide shuffling, thioether 1301.20754 c h n o s +H: R=108646 essential. 3 1301.59741 1027 1606 282 332 6 8+ 8+ H life-times due to their relatively big cross section. Thus, the method of choice (C) simulated 40 c /c· R=108759 C 80 C 1027 H 1624 N 282 O 332 S 6 125 125 C-­‐terminal heterogeneity -­‐COO Relative Abundance p (gss, s /p:40) Chrg30 18 R=109703 Lysine processing, Proline amida.on for intact antibodies is to use the shortest transient duration (48 ms) R=101019 -­‐ R: 120000 Res .Pwr . @FWHM R=107362 60 1301.70878 R=97925 R=105375 1301.09615 20 R=112194 available on the Orbitrap Elite MS (Figure 4). R=103665 R=99113 R=82023 Abbreviations 10 R=86808 FIGURE 1: General structure of mAbs and their biological and 40 1301.82014 T: 0 ACN, acetonitrile; CID, collision-induced dissociation; C-trap, curved 2794.98103 physico-chemical characteristics. 100 2848.70886 1300.98475 1702.5 1703.0 1703.5 1704.0 1704.5 1705.0 1705.5 1706.0 1706.5 1707.0 2743.24976 20 m/z 90 +53 1301.98718 linear trap; DTT, dithiothreitol; ETD, electron transfer dissociation; FA, 80 +54 +52 intactHumira_ETD15_LP_HCD_240k #11-27 RT: 6.11-11.23 AV: 17 NL: 1.07E4 formic acid; HCD, higher energy collision-induced dissociation; mAb, 70 0 T: FTMS + p ESI Full ms2 [email protected] [200.00-4000.00] 2794.98103 100 100 Methods 60 1300.5 1301.0 1301.5 1302.0 1302.5 2693.38227 90 monoclonal antibody; µS, micro-scan; SID, in-source decay; SIM, single 50 m/z (C) (A) 80 ETD 90 2962.591 40 23407.63635 ion monitoring. 2749.10120 2800.91032 2854.81639 100 (D) 70 Sample Preparation 30 Relative Abundance Isotope pattern after 60 20

80 2787.42772 2840.04424 50 10 80 deconvolution with Xtract ™ 2767.72395 2820.90307 AbbVie™ HUMIRA (adalimumab, Figure 2) [1]: The intact antibody (144 2598.92826 40 0 References 2740 2760 2780 2800 2820 2840 2860 30 70 60 Abundance Relative kDa) was dissolved in 0.1 % FA to 1 µg/µL; 5 µg HUMIRA were loaded onto m/z

20 3085.98807 10 1. Bondarenko, P.V., Second, T.P., Zabrouskov, V., Makarov, A. & the column. 60 40 0 500 1000 1500 2000 2500 3000 3500 Zhang, Z. Mass measurement and top-down HPLC/MS analysis of 2510.86531 m/z Relative Abundance 20 23421.61756 50 23390.64710 23433.66736 intact monoclonal antibodies on a hybrid linear quadrupole ion trap- FIGURE 7: (A) CID spectrum and (C) ETD spectrum of intact HUMIRA 3151.64648 0 Orbitrap mass spectrometer. Journal of the American Society for 40 2428.59194 23393.62879 antibody. (B) Zoom in into the ETD fragment ion spectrum of intact Relative Abundance 3220.13151 Mass Spectrometry 20, 1415-24 (2009). 100 Monoisotopic mass (M) of the HUMIRA showing the need for highest resolution possible. 30 3291.67370 2351.52066 (E) Δm=2.4 ppm intact light chain after 3366.46848 80 2. Michalski, A. et al. Ultra high resolution linear ion trap Orbitrap mass 20 deconvolution with Xtract 2279.19708 3444.75592 spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and 60 2178.67441 3526.72930 10 versatile peptide fragmentation modes. Molecular & cellular 2029.53882 3703.00780 40 proteomics: MCP (2011).doi:10.1074/mcp.O111.013698 0 20 23435.63611 2000 2500 3000 3500 23407.58564 23419.59961 m/z 23428.60343 0 23380 23390 23400 23410 23420 23430 Acknowledgements m/z FIGURE 2: 3D structure of HUMIRA highlighting the attached glycans and (B) We would like to thank Paul Thomas from Northwestern University (USA) cystein residues forming inter- and intra-chain disulfide bridges. +Hex FIGURE 5. (A) Full MS spectrum of intact light chain of HUMIRA. (B) for processing the HUMIRA ETD data. For analyzing HUMIRA light chain (24 kDa) and heavy chain (51 kDa) +Hex Zoom into +18 charge state of intact light chain. (C) Simulation of isotope pattern of +18 charge state. (D) Isotope pattern of intact light chain after separately, 50 µg HUMIRA was reduced with DTT (20-fold molar excess, HUMIRA is a trademark of AbbVie. All other trademarks are the property of Thermo Fisher Scientific and 56°C for 1 h) and alkylated with iodoacetamide (50-fold molar excess, room deconvolution. (E) Monoisotopic mass (M) of the measured light chain of its subsidiaries. FIGURE 8: Summarized sequence coverage of the HUMIRA heavy chain temperature for 30 min in the dark). HUMIRA obtained after deconvolution with Xtract. This information is not intended to encourage use of these products in any manners that might infringe FIGURE 4: (A) Full MS spectrum of intact HUMIRA. The insert shows a using fragmentation techniques SID, CID, HCD, and ETD. Optimized the intellectual property rights of others. zoom into the three most abundant charge states z=52,53,54. (B) Spectrum conditions: trapping under high pressure settings. N: Putative For Research Use only, not for Use in Diagnostic Procedures. after deconvolution. glycosylation site. www.thermoscientific.com Thermo Fisher Scientific, ©2013 Thermo Fisher Scientific Inc. All rights reserved. HUMIRA is a trademark of AbbVie. ISO is a trademark of the International Standards San Jose, CA USA is Organization. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Specifications, terms and pricing are ISO 9001:2008 Certified. subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

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