Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry Kai Scheffler, 1 Eugen Damoc, 2 Mathias Müller, 2 Martin Zeller, 2 Thomas Moehring 2 Thermo Fisher Scientific, Dreieich 1 and Bremen, 2 Germany
Introduction Recombinant monoclonal antibodies have gained significant importance in diagnostic and therapeutic applications over the past years. In order to verify the correctness of the overall molecule to provide a reproducible, safe and effective biological drug compound, the correct protein sequence, as well as the presence and relative abundance of different glycoforms have to be confirmed. Here we present an approach to analyze an intact monoclonal antibody in non-reduced and reduced condition by LC-MS using the Thermo Scientific Orbitrap Elite mass spectrometer. The intact antibody and the separated light and heavy chains were analyzed in Full MS experiments as well as with top-down experiments using in-source CID (SID), CID, HCD and ETD fragmentation techniques making use of the ultrahigh resolution of the mass spectrometer. For data evaluation ProSight software and Thermo Scientific Protein Deconvolution software version 1. packages were used. Electrospray source S-lens Square Quadrupole with Beam Blocker Octopole High Pressure Cell Low Press FIGURE 3: Schematics of the O spectrometer equipped with an E H Physicochemical Characteris4cs Biological Characteris4cs N- terminal heterogeneity Pyroglutamate forma.on Other modifica.ons Amino acid modifica4ons Deamidataion, oxida.on, glycosyla.on, isomeriza.on Fragmenta4on Cleavage in hinge region Oligosaccharides Fucosyla.on, sialyla.on, galactosyla.on,... Disulfide Bonds Free thiols, disulfide shuffling, thioether C- terminal heterogeneity Lysine processing, Proline amida.on S S S S Heavy chain - S- S- - S- S- - COO - C H 3 C H 2 Fab Fc Methods FIGURE 1: General structure of mabs and their biological and physico-chemical characteristics. Sample Preparation AbbVie HUMIRA (adalimumab, Figure 2) [1]: The intact antibody (144 kda) was dissolved in.1 % FA to 1 µg/µl; 5 µg HUMIRA were loaded onto the column. 1 9 8 7 6 5 4 3 2 1 2 25 279 2693.3822 2598.92826 251.86531 2428.59194 2351.5266 2279.1978 2178.67441 229.53882 FIGURE 2: 3D structure of HUMIRA highlighting the attached glycans and cystein residues forming inter- and intra-chain disulfide bridges. For analyzing HUMIRA light chain (24 kda) and heavy chain (51 kda) separately, 5 µg HUMIRA was reduced with DTT (2-fold molar excess, 56 C for 1 h) and alkylated with iodoacetamide (5-fold molar excess, room temperature for 3 min in the dark). FIGURE 4: Full MS spectru zoom into the three most abunda after deconvolution. 2 Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry
Instrument A Thermo Scientific Surveyor MS Pump Plus was coupled to an Orbitrap Elite mass spectrometer that was equipped with ETD (Figure 3). Electrospray source S-lens Square Quadrupole with Beam Blocker Octopole High Pressure Cell Low Pressure Cell Quadrupole Mass Filter C-trap HCD Collision Cell Transfer Multipole Reagent Ion Source New High-Field Orbitrap Mass Analyzer FIGURE 3: Schematics of the Orbitrap Elite hybrid ion trap-orbitrap mass spectrometer equipped with an ETD source. Reagent 1 Reagent 2 1 9 8 7 6 5 4 3 2 1 z=19 z=17 1381.19873 z=2 z=16 1174.1712 1467.49915 z=15 z=21 1565.29358 1118.31189 z=1 1677.3 z=22 167.5699 121.25623 1235.9541 134.56287 8 1 12 14 16 1 f mabs and their biological and cs. Samples were purified on a Thermo Scientific BioBasic C4 column (15 x 1 mm, 5 µm particles), solvent A:.1 % FA, 2 % ACN in H 2 O, solvent B:.1 % FA in ACN. The LC gradient was 7 min 2 4 % B, 3 min 4 8 % B at a flow rate of 1 µl/min. Data analysis was done using Protein Deconvolution and ProSight software packages. Results The analysis of large proteins of the size of intact anti-bodies (~15 kda) using Orbitrap mass spectrometers has been significantly improved over the past few years. Large molecules like mabs show only very short transient life-times due to their relatively big cross section. Thus, the method of choice for intact antibodies is to use the shortest transient duration (48 ms) available on the Orbitrap Elite MS (Figure 4). 1 9 2693.38227 2794.9813 2962.591 T: 1 9 8 7 6 5 4 3 2743.24976 2749.112 2794.9813 2848.7886 +53 +54 +52 28.9132 2854.81639 1 8 6 4 2 1 8 6 4 2 1 (C) (D) 13.5 131. 131.26 131.275 131.9615 13.98475 2347.6 8 7 6 5 4 3 2 1 2598.92826 251.86531 2428.59194 2351.5266 2279.1978 2178.67441 229.53882 2 1 385.9887 2787.42772 284.4424 2767.72395 282.937 274 276 278 28 282 284 286 3151.64648 322.13151 3291.6737 3366.46848 3444.75592 3526.7293 373.78 8 6 4 2 1 8 6 4 (E) 2339.6471 23393.62879 Δm=2.4 ppm 2 25 3 35 +Hex +Hex FIGURE 4: Full MS spectrum of intact HUMIRA. The insert shows a zoom into the three most abundant charge states z=52,53,54. Spectrum after deconvolution. 2 2347.58 2338 2339 234 2 m FIGURE 5. Full MS spectrum Zoom into +18 charge state of intac pattern of +18 charge state. (D) Is deconvolution. (E) Monoisotopic m HUMIRA obtained after deconvolutio Thermo Scientific Poster Note BioPharma_PN63712_E 11/13S 3
HUMIRA_heavy_av_SIM # 1 RT: 1556.88 AV: 1 3.3E3 T: FTMS + p ESI SIM ms [118.-119.] uadrupole Mass Filter New itrap lyzer C-trap HCD Collision Cell Transfer Multipole Reagent Ion Source Reagent 1 Reagent 2 rap Elite hybrid ion trap-orbitrap mass source. 1 9 8 7 6 5 4 3 2 1 z=19 z=17 1381.19873 z=2 z=16 1174.1712 1467.49915 z=15 z=21 1565.29358 1118.31189 z=12 z=14 1956.39819 z=11 1677.321 z=13 2134.1949 z=22 186.14465 167.5699 121.25623 1235.9541 134.56287 z=1 2347.6156 261.8981 8 1 12 14 16 18 2 22 24 26 28 1 9 8 7 6 5 4 3 2 1 FIGURE 6: HUMIRA heavy chain acqu averaged. Deconvoluted mass: Mr 5 demonstrate isotopic resolution of that obtained after deconvolution using Xtrac Humira_CID_59_12211735 #1 RT: 355.98 AV: 1 6.11E3 T: FTMS + p ESI Full ms2 1185.@cid5. [325.-4.] 1 118.9857 R=13334 1181.686 R=11724 1182.5933 R=11644 1183.5918 R=12224 1182.2444 R=12144 1183.9644 R=1224 1181 1182 1183 1184 9 1 8 6 4 2 R=124.1 131.43152 Δ=.9ppm 131.26538 131.59814 131.7923 measured 6 5 3.84E5 4 551.3178 FS_tripleSIM_2uS_1#19 RT: z=1 3 7.66 AV: 1 T: FTMS + p ESI 2 SIM ms [129.-132.] 452.24893 z=1 1 8 7 683.26221 z=2 782.33112 971.26849 4 6 8 1 1 8 6 (C) 131.4334 131.2754 131.59741 131.9615 131.7878 simulated 2.13E3 c 127 h 166 n 282 o 332 s 6 +H: C 127 H 1624 N 282 O 332 S 6 p (gss, s /p:4) Chrg 18 R: 12 Res.Pwr. @FWHM 3 962.591 T: 1 9 8 7 6 5 4 3 2 1 385.9887 2743.24976 2749.112 2794.9813 2848.7886 +53 +54 +52 2767.72395 282.937 28.9132 2854.81639 2787.42772 284.4424 274 276 278 28 282 284 286 4 2 1 8 6 4 2 (D) 13.5 131. 131.5 132. 132.5 2347.63635 2339.6471 13.98475 131.8214 131.98718 Isotope pattern after 23421.61756 23433.66736 3151.64648 322.13151 3291.6737 3366.46848 3444.75592 3526.7293 1 8 6 (E) 23393.62879 Δm=2.4 ppm Monoisotopic mass (M) of the intact light chain after 373.78 4 3 35 /z 2 2347.58564 23419.59961 23428.6343 2338 2339 234 2341 2342 2343 23435.63611 ex +Hex f intact HUMIRA. The insert shows a harge states z=52,53,54. Spectrum FIGURE 5. Full MS spectrum of intact light chain of HUMIRA. 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 deconvolution. (E) Monoisotopic mass (M) of the measured light chain of HUMIRA obtained after. 4 Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry
z=12 1956.39819 z=11 13 2134.1949 4465 24.1.43152 Δ=.9ppm 131.59814 131.7923 z=1 2347.6156 261.8981 2 22 24 26 28 measured HUMIRA_heavy_av_SIM # 1 RT: 1556.88 AV: 1 3.3E3 T: FTMS + p ESI SIM ms [118.-119.] 1 9 8 7 6 5 4 3 2 1 FIGURE 6: HUMIRA heavy chain acquired in SIM scan mode (z=43). 6 µscans were averaged. Deconvoluted mass: Mr 5,891.4317 Da. The inserts on the right demonstrate isotopic resolution of that charge state detected at 1185 and masses obtained after deconvolution using Xtract. Humira_CID_59_12211735 #1 RT: 355.98 AV: 1 6.11E3 T: FTMS + p ESI Full ms2 1185.@cid5. [325.-4.] 1 1 9 8 7 118.9857 R=13334 CID 6 5 3.84E5 4 551.3178 FS_tripleSIM_2uS_1#19 RT: z=1 3 7.66 AV: 1 T: FTMS + p ESI 2 SIM ms [129.-132.] 452.24893 z=1 1 1181 1182 1183 1184 1185 1186 683.26221 z=2 1183.9644 R=1224 1182.5933 R=11644 1184.967 1183.5918 R=12114 R=12224 1182.2444 R=12144 782.33112 971.26849 176.95667 z=24 1185.219 R=1177 1185.458 R=1224 1217.55786 zoom 1185.8925 R=1984 1181.686 1186.2635 R=11724 R=12794 1321.8634 1395.2427 157.537 1594.2738 1629.4253 1756.7886 z=7 4 6 8 1 12 R=11181 14 R=1535 16 18 2 Humira_heavy_av_SIM #1 RT: 1556.88 AV: 1 3.3E3 T: FTMS + p ESI SIM ms [118.-119.] z=43 1185.219 R=1177 1185.2921 1 R=11774 1185.1287 8 R=1284 1185.382 R=11834 6 1185.4961 R=124 4 2 1185.2 1185.4 Deconvolution 1 8 6 4 2 1 8 6 4 2 5891.4317 5922.7492 589 59 591 592 5.4334 131.59741 131.7878 simulated 9 8 7 y 91 7+ z = 6 6 2.13E3 5 c 127 h 166 n 282 o 332 s 6 +H: 4 c 8+ 8+ C 127 H 1624 N 282 O 332 S 6 125 /c 125 p (gss, s /p:4) Chrg 3 18 R: 12 Res.Pwr. @FWHM 2 R=94282 R=12177 R=18759 R=1993 R=14772 R=18541 R=1114 R=18646 R=1973 R=1119 R=17362 R=97925 R=15375 R=13665 R=112194 R=99113 131.8214 131.98718 131.5 132. 132.5 Isotope pattern after (C) R=8223 1 R=8688 172.5 173. 173.5 174. 174.5 175. 175.5 176. 176.5 177. intacthumira_etd15_lp_hcd_24k #11-27 RT: 6.11-11.23 AV: 17 1.7E4 T: FTMS + p ESI Full ms2 28.@etd15. [2.-4.] 1 9 8 7 6 5 4 ETD 3 2 1 23421.61756 23433.66736 Monoisotopic mass (M) of the intact light chain after 5 1 15 2 25 3 35 FIGURE 7: CID spectrum and (C) ETD spectrum of intact HUMIRA antibody. Zoom in into the ETD fragment ion spectrum of intact HUMIRA showing the need for highest resolution possible. 23419.59961 23428.6343 23435.63611 2342 2343 tact light chain of HUMIRA. ht chain. (C) Simulation of isotope e pattern of intact light chain after (M) of the measured light chain of ith Xtract. FIGURE 8: Summarized sequence coverage of the HUMIRA heavy chain using fragmentation techniques SID, CID, HCD, and ETD. Optimized conditions: trapping under high pressure settings. N: Putative glycosylation site. Thermo Scientific Poster Note BioPharma_PN63712_E 11/13S 5
zoom 1185.458 R=1224 Humira_heavy_av_SIM #1 RT: 1556.88 AV: 1 3.3E3 T: FTMS + p ESI SIM ms [118.-119.] z=43 1185.219 R=1177 1185.2921 1 R=11774 1185.1287 8 R=1284 1185.382 R=11834 6 1185.4961 R=124 4 2 1185.2 1185.4 1185.8925 R=1984 1186.2635 R=12794 Deconvolution 1 8 6 4 2 1 8 6 4 2 5891.4317 5922.7492 Monoisotopic mass (M) of the intact heavy chain after Isotope pattern and average mass of the intact heavy chain after 589 59 591 592 593 594 IM scan mode (z=43). 6 µscans were 317 Da. The inserts on the right tate detected at 1185 and masses 17.55786 1186 1321.8634 1395.2427 157.537 1594.2738 1629.4253 1756.7886 z=7 2 14 16 18 2 Conclusion The analysis of intact and reduced antibodies on the Orbitrap Elite mass spectrometer provides the accurate molecular weight, as well as valuable information about the presence and abundance of glycoforms. Analysis of the reduced antibody provides isotopically resolved mass spectra for both light and heavy chain. The combination of multiple fragmentation techniques in top-down analysis (SID, CID, HCD and ETD) generates comprehensive sequence coverage and enables fast localization of modifications with minimum sample preparation. For measurements of intact light and heavy chain as well as for the detection of fragment ion spectra from top-down experiments ultrahigh resolution as provided by the Orbitrap Elite mass spectrometer is essential. Abbreviations ACN, acetonitrile; CID, collision-induced dissociation; C-trap, curved linear trap; DTT, dithiothreitol; ETD, electron transfer dissociation; FA, formic acid; HCD, higher energy collision-induced dissociation; mab, monoclonal antibody; µs, micro-scan; SID, in-source decay; SIM, single ion monitoring. References 1. Bondarenko, P.V., Second, T.P., Zabrouskov, V., Makarov, A. & Zhang, Z. Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap- Orbitrap mass spectrometer. Journal of the American Society for Mass Spectrometry 2, 1415-24 (29). 2. Michalski, A. et al. Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes. Molecular & cellular proteomics : MCP (211).doi:1.174/mcp.O111.13698 Acknowledgements We would like to thank Paul Thomas from Northwestern University (USA) for processing the HUMIRA ETD data. www.thermoscientific.com 213 Thermo Fisher Scientific Inc. All rights reserved. HUMIRA is a trademark of AbbVie. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Thermo Fisher Scientific, San Jose, CA USA is ISO 91:28 Certified. Africa +43 1 333 5 34 Australia +61 3 9757 43 Austria +43 81 282 26 Belgium +32 53 73 42 41 Canada +1 8 53 8447 China 8 81 5118 (free call domestic) 4 65 5118 Denmark +45 7 23 62 6 Europe-Other +43 1 333 5 34 Finland +358 9 3291 2 France +33 1 6 92 48 Germany +49 613 48 114 India +91 22 6742 9494 Italy +39 2 95 591 Japan +81 45 453 91 Latin America +1 561 688 87 Middle East +43 1 333 5 34 Netherlands +31 76 579 55 55 New Zealand +64 9 98 67 Norway +46 8 556 468 Russia/CIS +43 1 333 5 34 Singapore +65 6289 119 Spain +34 914 845 965 Sweden +46 8 556 468 Switzerland +41 61 716 77 UK +44 1442 233555 USA +1 8 532 4752 BioPharma 213_PN63712_E_11/13S