Monoclonal Antibody Characterization on Q Exactive and Oribtrap Elite Yi Zhang, Ph.D Senior Proteomic Marketing Specialist Oct. 12, 211
Outline Orbitrap Mass Spectrometer in mab Characterization Intact Protein Analysis on Q Exactive Using Protein Deconvolution Top-Down Analysis of mab Light Chain on Orbitrap Elite Conclusion 2
Mass Spectrometry in mab Characterization Monoclonal antibodies have become a major drug class. Highly specific Fewer side effects Quality control is challenging Big molecules (15kD) More things can go wrong. The role of mass spectrometry Mass measurement of intact mab Sequence confirmation Disulfide mapping PTM identification Glycan analysis 3
Challenges of Protein Detection in Orbitrap Analyzer Myoglobin (16.9 kd) z: +16 Fast Signal Decay: Collision of ions Fragmentation of ions De-phasing of ion packets Solutions: Higher Vacuum Hardware Innovation Software Innovation 1 2 3 t, ms Enolase (46kD) z: +24 1 2 3 t, ms Transferrin (78kD) z: +4 1 2 3 t, ms 4
Hardware Inovations in New Orbitrap Analyzer Improved Detector Hardware Reduces Delay in Transient Acquisition Relative Intensity 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5.3585.51 6.5629 6.4464 6.461 6.3446 6.3241 6.2629 6.2432 6.396 6.13 5.8363 5.7755 2.1482 4.9622 5.6327 2.67 4.5154 2.24 2.188 4.891 3.528 3.6833 3.2571 1.1884 8.955 9.5588 9.964 7.256 7.8633 8.8447 Relative Intensity.1758 1 8.9 ms <.6 ms Exactive 95 9 85 8.3435 75 7 65.46 6.5435 55.6147 1.611 2.9427 4.796 4.3266 4.799 5.5774 6.8835 7.3687 7.634 7.7499 8.8795 9.5881 1.3 5 45 4 35 3 Q Exactive & Orbitrap Elite 25 2 15 1 5 1 2 3 4 5 6 7 8 9 1 ms <1ms 1 2 3 4 ms 5 6 7 8 9 1 Efficient Capture of Initial Transient 5 Transferrin (78kD) z: +4 1 2 3 t, ms Stronger Signal Better Mass Accuracy See aslo ASMS Poster MP93 Oliver Lange et al.
Software Innovation Advanced Signal Processing Increases resolution by 2 fold for the same transient time Maximum Resolution: Q Exactive: 14K ( 2) 1K ( 4) Orbitrap Elite: 24K ( 4) 1 8 6 4 2 993.892 R=18377 993.82838 R=11331 993.9771 R=11221 994.8274 R=17785 994.2348 R=1323 Enolase (46.64 kda), 47+, Orbitrap Elite Isotopically Resolved LC & HC Top-Down Analysis on LC Time Scale 6
Outline Orbitrap Mass Spectrometer in mab Characterization Intact Protein Analysis on Q Exactive Using Protein Deconvolution Top Down Analysis of mab Light Chain on Orbitrap Elite Conclusion 7
Intact Protein Mass Measurement on Q Exactive Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 Protein: ~2kD R: 14K 1389.2991 z=13 154.8222 z=12 1575.62 z=12 1641.624 z=11 1717.9145 z=11 185.6858 1889.927 13 14 15 16 17 18 19 2 21 22 23 24 25 26.245 z=9 299.6662 z=9 2135.3361 z=9 Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 2256.9799 z=8 185.2842 185.4848 185.6858 185. 185.5 186. 186.5 187. 187.5 188. 2362.1277 z=8 185.8863 186.875 185.834 186.4884 2399.6221 z=8 Isotopically Resolved 187.2867 186.2882 186.9859 187.587 187.9868 Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 mab:~15kd R: 17.5K 2391.6288 2471.345 2556.4844 2745.772 3 25 2 15 1 5 16 18 2 22 24 26 28 3 32 34 36 38 4 2851.3544 297.2595 2965.3764 325.8632 Isotopically Unresolved 8
Protein Deconvolution 1. Workflow software for intact protein mass determination Includes 2 deconvolution algorithms: Xtract for isotopically resolved proteins ReSpect TM for isotopically unresolved proteins (e.g. IgG) As of today, the software is feature complete and undergoing testing Target release date: Late October, early November 9
Protein Deconvolution 1. A Workflow Design Select Algorithm Load File Create/Select Method 1
Protein Deconvolution Method Parameters Straight-forward Parameter Setting Estimated Target Mass Automatic Resolution Detection 11
Protein Deconvolution Chromatogram Tab Averaged spectrum 12
Protein Deconvolution - ReSpect TM Deconvolution Fast Data Processing:1-2s Deconvoluted Spectrum Deconvolution Data 13
Protein Deconvolution - Report Comprehensive, Exportable Report 14
Isotopic Resolution for Accurate Mass Detection Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 Protein:~2kD R: 14K 1389.2991 z=13 154.8222 z=12 1641.624 z=11 185.6858 1889.927 26.245 z=9 299.6662 z=9 2135.3361 2362.1277 z=9 z=8 2256.9799 z=8 2399.6221 z=8 1717.9145 5 1575.62 z=11 z=12 185. 185.5 186. 186.5 187. 187.5 188. 13 14 15 16 17 18 19 2 21 22 23 24 25 Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 185.2842 185.4848 185.6858 185.8863 186.875 185.834 186.4884 187.2867 186.2882 186.9859 187.587 Xtract 187.9868 Monoisotopic Mass (<5ppm) 15.96 amu 1 Oxidation 15
Precise Mass Measurement of Intact Monoclonal Antibody Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 mab: ~15kD R: 17.5K 2391.6288 2471.345 2556.4844 2745.772 2851.3544 297.2595 2965.3764 325.8632 1 5 16 18 2 22 24 26 28 3 32 34 36 38 4 ReSpect -.7 ppm GF+GF -7 ppm GF+G1F Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1-8.5 ppm 5 2695.8919 268 27 272 274 276 278 28 282 GF+G2F (or 2G1F) 2745.772 2797.5697 5. ppm G+GF 2xMan5 G+G -.9 ppm G1F+G2F G2F+G2F G1F+G2F+SA 16 Shiaw-Lin Wu, Barry Karger, Barnett Institute, Northeastern University
Accurate Mass Measurement from a Single Charge State GF/G1F Δ= 7. ppm 52+ GF/GF Δ=.1 ppm G1F/G1F or GF/G2F Δ = 1 ppm G/GF Δ= 1 ppm G1F/G2F Δ = 8 ppm 2835 284 2845 285 2855 286 2865 287 17
Reproducibility of Accurate MW Determination ReSpect TM Deconvolution of Q Exactive IgG Data ppm mass measurement errors RAW file Q Exactive G+GF GF+GF GF+G1F GF+G2F G1F+G2F 1 1-1.5.7-1.5-13.8-18. 2 1-11.6-1.1-8.8-11.2-12. 3 1 5.1-5. -2.6 5.1 5.6 4 2-14.3 3. -6.9-5.4-5.9 5 2-8.6-2.2-12.2-12.5-12.9 6 2-14.3-6.6-12.3-14.8-1.1-6.9 +/- 6.4 ppm mass tolerance for multiple measurements 18
Reproducibility of Relative Abundance Measurement ReSpect TM Deconvolution of Q Exactive IgG Data Relative Abundances RAW file Q Exactive G+GF GF+GF GF+G1F GF+G2F G1F+G2F 1 1 12.9 74.1 1. 67. 23.4 2 1 12. 72.8 1. 66.2 22. 3 1 12.2 75. 1. 67. 23.6 4 2 12.7 75.7 1. 63.6 21.6 5 2 13.2 75.4 1. 64.8 21. 6 2 12.9 76.6 1. 64.7 21.6 CV 3.6% 1.7% N.A. 2.2% 4.9% GF+G1F GF+GF GF+G2F (or 2G1F) G+GF G1F+G2F 19
Summary of Q Exactive and Protein Deconvolution 1. Q Exactive accurately and reproducibly measures the mass of intact antibody to ~7ppm. The new Protein Deconvolution software enables fast and accurate calculation of the intact mass of mabs. Q Exactive is also a powerful HR/AM mass spectrometer for routine peptide identification and peptide mapping. This LC/MS-based workflow solution provides a robust QC solution for biopharmaceutical products. 2
Outline Orbitrap Mass Spectrometer in mab Characterization Intact Protein Analysis on Q Exactive Using Protein Deconvolution Top-Down Analysis of mab Light Chain on Orbitrap Elite Conclusion 21
Orbitrap Elite: Velos Pro Iontrap + High Field Orbitrap Key Features for Intact Protein Analysis: Higher Resolution (24, at 1Hz) Faster Scan Speed 4X more scans at same resolution Multiple Fragmentation Mechanism CID HCD ETD Case Study: Top-Down Analysis of mab Light Chain 22
Step 1 Accurate MW Determination of mab Light Chain 1 133.917 z=18 95 9 85 8 75 1465.9155 z=16 R: 24K 1 95 9 85 133.917 z=18 133.224 z=18 133.364 z=18 7 65 1234.6137 z=19 1563.576 z=15 8 75 7 132.985 z=18 133.3139 z=18 Relative Abundance 6 55 5 45 4 35 3 1172.8326 z=2 1675.1174 z=14 183.8945 z=13 Relative Abundance 65 6 55 5 45 4 35 3 25 2 15 132.9253 z=18 132.8691 z=18 132.7578 z=18 133.4251 z=18 133.536 z=18 25 2 15 1 1117.1264 z=21 1954.1323 z=12 2131.7785 z=11 1 5 132.6 132.8 133. 133.2 133.4 133.6 133.8 134. 5 12 14 16 18 2 22 24 Xtract Measured Mass = 23424.4845 Target Mass = 23428.416 4 Dalton Mass Deviation 2 S-S? 23 Shiaw-Lin Wu, Barry Karger, Barnett Institute, Northeastern University
Step 2 - Top-Down Analysis with ETD at 12K Resolution Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 ETD R: 12K 1416.4512 z=8 1545.427 z=7 1675.4727 z=14 182.8888 z=13 1954.555 z=12 2132.2379 z=11 2345.6667 1 12 14 16 18 2 22 24 26 28 3 Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 1819.8239 z=13 1824.591 z=6 1828.3897 z=6 1833.3963 z=6 1843.693 z=6 185.455 z=6 1859.5754 z=6 1871.923 z=6 1888.963 z=6 1898.973 z=? 194.925 z=5 199.9393 z=6 182 183 184 185 186 187 188 189 19 191 24
Step 3 - Top-Down Analysis with HCD at 12K Resolution HCD R: 12K Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 p @ [ ] x5 x5 x5 1954.22 z=12 397.17 z=1 571.25 z=1 589.26 z=1 654.27 z=1 84.36 z=1 812.34 z=1 895.45 z=1 141.48 z=1 1237.6 z=1 1763.19 z=6 1411.66 1697. z=? z=7 1511.45 z=7 2131.78 z=11 2197.7 z=5 242.42 z=4 2344.76 z=? 262.1 z=4 2573.89 z=? 2644.27 z=4 2681.29 z=4 5 1 15 2 25 3 25
Step 4 ProSightPC Data Analysis 4 Dalton deviation is probably due to internal disulfides. 26
Summary of Top-Down Analysis on Orbitrap Elite Orbitrap Elite enables mab subunit analysis at isotopic resolution, allows accurate mass measurement (<5ppm), and quick identification of minor modifications. The combination of multiple fragmentation mechanisms in top-down analysis generates comprehensive sequence coverage, enables fast localization of modification with minimum sample preparation. Orbitrap Elite enables comprehensive characterization of intact antibody with its superior performance and versatility. 27
HR/AM Intact Mass Measurement Workflow Exact Mass Determination Solutions On-line Desalting: HPLC Separation Sample Analysis: Orbitrap Elite or Q Exactive High RP for resolution of proteins ~5k low RP for larger proteins Data Analysis: Protein Deconvolution 1. with ReSpector Xtract to zero charge accurate mass 1 2997.31777 9 292.44812 8 2664.4983 7 Relative Abundance 6 5 4 2178.4685 3 2 1 3535.7427 3619.4765 2 25 3 35 Solutions RSLC nano/cap ProSwift RP columns Relative Abundance z=? 1 1311.87212 1311.54737 R=7792 9 R=6813 z=? z=? 8 7 6 1311.98174 R=69867 z=? 5 4 1311.43967 R=58977 z=? 1312.9147 3 R=6784 z=? 2 1312.287 1311.31799 R=56981 131.98824 R=88597 z=? 1312.42258 1 1312.64646 R=45346 z=? R=47666 R=43558 z=? z=? z=? 1311. 1311.5 1312. 1312.5 Relative Abundance 1 9 8 7 6 5 4 3 2 1 23578.58636 2358.66451 23565 2357 23575 2358 23585 2359 Solutions Orbitrap hybrid family Q Exactive Solutions ReSpect(unresolved spectra) Xtract (resolved spectra) 28
Conclusion Thermo Fisher provides complete LC/MS-based workflow solutions for intact protein analysis and comprehensive protein characterization. 29
Acknowledgement Q Exactive R&D Team Orbitrap Elite R&D Team San Jose Proteomics Marketing Team San Jose Demo Lab Barnett Institute, Northeastern University Shiaw-Lin Wu Barry Karger 3
31 Thank You!