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Improving Intact Antibody Characterization by Orbitrap Mass

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Improving Intact Antibody Characterization by Orbitrap Mass 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] Monoisotopic mass (M) of the T: FTMS + p ESI SIM ms [1180.00-1190.00] 50891.04317 Instrument z=43 1185.2190 100 Introduction 1185.2190 R=117700 1185.2921 intact heavy chain after Humira_FullMS_LC #1 RT: 7.34 AV: 1 NL: 3.57E6 R=117700 100 R=117704 Deconvolution 80 T: FTMS + p ESI Full ms [400.00-4000.00] 100 1185.1287 TM zoom R=120804 60 deconvolution with Xtract 80 1185.3820 Recombinant monoclonal antibodies have gained significant importance in A Surveyor MS Pump Plus was coupled to an Orbitrap Elite ETD mass 1304.56287 90 100 R=118304 1185.4508 40 60 1185.4961 diagnostic and therapeutic applications over the past years. In order to verify spectrometer (all Thermo Fisher Scientific) (Figure 3) [2]. z=18 80 R=122004 1235.95410 1183.9644 R=120004 20 (A) R=120204 90 40 the correctness of the overall molecule to provide a reproducible, safe and 70 Relative Abundance 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 100 effective biological drug compound, the correct protein sequence as well as 1183.5918 R=121104 Isotope pattern and average 1381.19873 R=122204 0 1182.2444 80 the presence and relative abundance of different glycoforms have to be 70 z=20 50 R=121404 1185.2 1185.4 mass of the intact heavy chain z=16 m/z 1174.17102 1185.8925 60 1467.49915 40 R=109804 tive tive Abundance after deconvolution with Xtract confirmed. 40 dance 60 1181.6860 1186.2635 Relative Relative Ab 30 R=117204 z=15 R=127904 20 Here we present an approach to analyze an intact monoclonal antibody in 50 1565.29358 1180.9857 z=21 20 R=133304 0 New z=12 non-reduced and reduced condition by LC-MS using the Orbitrap Elite hybrid High-Field Orbitrap 1118.31189 50890 50900 50910 50920 50930 50940 Mass Analyzer z=14 10 m/z 40 1956.39819 z=11 mass spectrometer. The intact antibody respectively the separated light and 1677.03210 Relative Abundance 2134.19409 0 Reagent 1 Reagent 2 30 z=13 1181 1182 1183 1184 1185 1186 Heated Inlet Heated Inlet heavy chains were analyzed in Full MS experiments as well as with top-down z=22 1806.14465 m/z 20 FIGURE 6: Humira® heavy chain acquired in SIM scan mode (z=43). 60 experiments using in-source CID (SID), CID, HCD and ETD fragmentation 1067.56909 z=10 µscans were averaged. Deconvoluted mass: Mr 50,891.04317 Da. The inserts techniques making use of the ultrahigh resolution of the mass spectrometer. FIGURE 3: Schematics of the Orbitrap Elite hybrid mass spectrometer 10 1021.25623 2347.60156 Conclusion equipped with an ETD source. 2610.80981 on the right demonstrate isotopic resolution of that charge state detected at m/z For data evaluation ProSight PC 2.0 and Protein Deconvolution 1.0 software 0 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 1185Humira_CID_59_120210173005 and masses # obtained1 RT: 355.98 AV: 1 afterNL: 6.11E3 deconvolution using Xtract. • The analysis of intact and reduced antibodies on the Orbitrap Elite 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 BioBasic-C4 column(150 x 1 mm, 5 µm particles 100 z=8 1321.86304 90 1594.27380 valuable information about the presence and abundance of glycoforms. z=? z=8 Physicochemical Characteristics Biological Characteristics (Thermo Fisher Scientific), solvent A: 0.1 % FA, 2 % ACN in H2O, solvent B: 80 CID 1629.42053 R=124.100 70 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 ∆=0.9ppm (A) 1217.55786 spectra for both light and heavy chain. N-terminal heterogeneity at a flow rate of 100 µL/min. 100 ∆∆ 50 z=? 1076.95667 Pyroglutamate formation 40 551.31708 z=24 Other modifications z=18 1301.26538 z=1 PTM 30 782.33112 1756.70886 RelativeAbundance The combination of multiple fragmentation techniques in top-down Data analysis was done using Protein Deconvolution 1.0 and ProSight 80 (B) 1301.59814 z=7 • measured z=? 971.26849 Amino acid modifications 20 452.24893 683.26221 z=? Fab z=1 z=2 analysis (SID, CID, HCD and ETD) generates comprehensive Deamidataion, oxidation, 2.0. 10 60 glycosylation, isomerization 1301.70923 0 sequence coverage and enables fast localization of modifications with 400 600 800 1000 1200 R=1110811400 1600 1800 2000 100 R=105350 Fragmentation -S-S- m/z -S-S- 40 minimum sample preparation. Cleavage in hinge region chain Heavy Results 90 S S S 80 7+ R=109093 Oligosaccharides Abundance Relative 20 y R=104772 • For measurements of intact light and heavy chain as well as for the 2 91 H Fucosylation, sialylation, galactosylation,... ain The analysis of large proteins of the size of intact anti-bodies (~150 kDa) C 70 R=108541 detection of fragment ion spectra from top-down experiments ultra- S S S Fc R=110104 Disulfide Bonds using the Orbitrap detector has been significantly improved over the past 0 60 1301.43034 dance z = 6 Free thiols, disulfide shuffling, thioether 100 (B) R=94282 high resolution as provided by the Orbitrap Elite mass spectrometer 3 years. Large molecules like mAbs show only very short transient life-times 50 R=102177 H 1301.20754 C is essential. C-terminal heterogeneity -COO 1301.59741 8+ 8+ R=108646 (C) 40 c /c R=108759 Lysine processing, Proline amidation due to their relatively big cross section. Thus the method of choice for intact 80 simulated 125 125 Relative Abundance Relative - 30 R=109703 antibodies is to use the shortest transient duration (48 ms) available on the R=101019 R=107362 60 1301.70878 R=97925 R=105375 1301.09615 20 R=112194 Orbitrap Elite (Figure 4). R=103665 R=99113 Abbreviations FIGURE 1: General structure of mAbs and their biological and R=82023 10 R=86808 40 physico-chemical characteristics. humira_FullMS #1 RT: 4.84 AV: 1 NL: 3.92E5 1301.82014 T: FTMS + p ESI Full ms [1000.00-4000.00] 0 ACN, acetonitrile; CID, collision-induced dissociation; C-trap, curved 2794.98103 2848.70886 1702.5 1703.0 1703.5 1704.0 1704.5 1705.0 1705.5 1706.0 1706.5 1707.0 100 2743.24976 1300.98475 20 m/z humira_FullMS #1 RT: 4.84 AV: 1 NL: 3.92E5 90 +53 1301.98718 linear trap; DTT, dithiothreitol; ETD, electron transfer dissociation; FA, T: FTMS + p ESI Full ms [1000.00-4000.00] 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, 2794.98103 70 0 T: FTMS + p ESI Full ms2 [email protected] [200.00-4000.00] 100 100 Methods 60 1300.5 1301.0 1301.5 1302.0 1302.5 monoclonal antibody; µS, micro-scan; SID, in-source decay; SIM, single 2693.38227 90 50 m/z (C) (A) 80 ETD 90 2962.5915240 23407.63635 ion monitoring. ive Abundance ive 2749.10120 2800.91032 2854.81639 100 (D) 70 Sample Preparation 30 Relative Ab Relative Isotope pattern after 60 20 80 2787.42772 2840.04424 50 10 80 deconvolution with Xtract ® 2767.72395 2820.90307 Humira (adalimumab, Figure 2) [1]: The intact antibody (144 kDa) was 2598.92826 40 0 References ® 2740 2760 2780 2800 2820 2840 2860 30 70 60 RelativeAbundance dissolved in 0.1 % FA to 1 µg/µL; 5 µg Humira were loaded onto the column. m/z 20 3085.98807 60 40 10 1. Bondarenko, P.V., Second, T.P., Zabrouskov, V., Makarov, A. & 0 500 1000 1500 2000 2500 3000 3500 Zhang, Z. Mass measurement and top-down HPLC/MS analysis of 2510.86531 m/z 50 Abundance Relative 20 23421.61756 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 40 Orbitrap mass spectrometer. Journal of the American Society for 2428.59194 antibody. (B) Zoom in into the ETD fragment ion spectrum of intact Relative Abundance 23393.62879 3220.13151 100 Monoisotopic mass (M) of the ® Mass Spectrometry 20, 1415-24 (2009). 30 3291.67370 Humira showing the need for highest resolution possible. 2351.52066 (E) ∆∆∆m=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 60 spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and 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 the North-Western Univ.
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