Monoclonal Antibody Characterization on Q Exactive and Oribtrap Elite. Yi Zhang, Ph.D Senior Proteomic Marketing Specialist Oct.

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1 Monoclonal Antibody Characterization on Q Exactive and Oribtrap Elite Yi Zhang, Ph.D Senior Proteomic Marketing Specialist Oct. 12, 211

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 2

3 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

4 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 t, ms Enolase (46kD) z: t, ms Transferrin (78kD) z: t, ms 4

5 Hardware Inovations in New Orbitrap Analyzer Improved Detector Hardware Reduces Delay in Transient Acquisition Relative Intensity Relative Intensity ms <.6 ms Exactive Q Exactive & Orbitrap Elite ms <1ms ms Efficient Capture of Initial Transient 5 Transferrin (78kD) z: t, ms Stronger Signal Better Mass Accuracy See aslo ASMS Poster MP93 Oliver Lange et al.

6 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) R= R= R= R= R=1323 Enolase (46.64 kda), 47+, Orbitrap Elite Isotopically Resolved LC & HC Top-Down Analysis on LC Time Scale 6

7 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

8 Intact Protein Mass Measurement on Q Exactive Relative Abundance Protein: ~2kD R: 14K z= z= z= z= z= z= z= z=9 Relative Abundance z= z= z=8 Isotopically Resolved Relative Abundance mab:~15kd R: 17.5K Isotopically Unresolved 8

9 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

10 Protein Deconvolution 1. A Workflow Design Select Algorithm Load File Create/Select Method 1

11 Protein Deconvolution Method Parameters Straight-forward Parameter Setting Estimated Target Mass Automatic Resolution Detection 11

12 Protein Deconvolution Chromatogram Tab Averaged spectrum 12

13 Protein Deconvolution - ReSpect TM Deconvolution Fast Data Processing:1-2s Deconvoluted Spectrum Deconvolution Data 13

14 Protein Deconvolution - Report Comprehensive, Exportable Report 14

15 Isotopic Resolution for Accurate Mass Detection Relative Abundance Protein:~2kD R: 14K z= z= z= z= z= z=9 z= z= z= z=11 z= Relative Abundance Xtract Monoisotopic Mass (<5ppm) amu 1 Oxidation 15

16 Precise Mass Measurement of Intact Monoclonal Antibody Relative Abundance mab: ~15kD R: 17.5K ReSpect -.7 ppm GF+GF -7 ppm GF+G1F Relative Abundance ppm GF+G2F (or 2G1F) 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

17 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

18 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 /- 6.4 ppm mass tolerance for multiple measurements 18

19 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 CV 3.6% 1.7% N.A. 2.2% 4.9% GF+G1F GF+GF GF+G2F (or 2G1F) G+GF G1F+G2F 19

20 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

21 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

22 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

23 Step 1 Accurate MW Determination of mab Light Chain z= z=16 R: 24K z= z= z= z= z= z= z=18 Relative Abundance z= z= z=13 Relative Abundance z= z= z= z= z= z= z= z= Xtract Measured Mass = Target Mass = Dalton Mass Deviation 2 S-S? 23 Shiaw-Lin Wu, Barry Karger, Barnett Institute, Northeastern University

24 Step 2 - Top-Down Analysis with ETD at 12K Resolution Relative Abundance ETD R: 12K z= z= z= z= z= z= Relative Abundance z= z= z= z= z= z= z= z= z= z=? z= z=

25 Step 3 - Top-Down Analysis with HCD at 12K Resolution HCD R: 12K Relative Abundance [ ] x5 x5 x z= z= z= z= z= z= z= z= z= z= z= z=? z= z= z= z= z= z=? z= z=? z= z=

26 Step 4 ProSightPC Data Analysis 4 Dalton deviation is probably due to internal disulfides. 26

27 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

28 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 Relative Abundance Solutions RSLC nano/cap ProSwift RP columns Relative Abundance z=? R= R=6813 z=? z=? R=69867 z=? R=58977 z=? R=6784 z=? R= R=88597 z=? R=45346 z=? R=47666 R=43558 z=? z=? z=? Relative Abundance Solutions Orbitrap hybrid family Q Exactive Solutions ReSpect(unresolved spectra) Xtract (resolved spectra) 28

29 Conclusion Thermo Fisher provides complete LC/MS-based workflow solutions for intact protein analysis and comprehensive protein characterization. 29

30 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 31 Thank You!