Advances in Protein Therapeutics Characterization Using LC/MS

This presentation by Steve Madden, MassHunter Software Product Manager at Agilent Technologies, highlights the tools and techniques for protein characterization using LC/MS. The agenda covers topics like intact proteins, peptides, PTMs, glycan profiling, and more, emphasizing the importance of sample preparation, separation, and data analysis. The Agilent BioPharma Workflow Platform offers a comprehensive solution for conducting multiple experiments efficiently. The stepwise process involves sample prep, separation, detection, and analysis using advanced tools like Agilent AssayMAP Bravo and AdvanceBio LC/Q-TOF. The presentation aims to address various aspects of protein characterization, from intact mass to glycan profiles, in a research setting.

• Protein Therapeutics
• LC/MS
• Agilent Technologies
• BioPharma Workflow
• Protein Characterization

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1. Advances in the Characterization of Protein Therapeutics by LC/MS Steve Madden MassHunter Software Product Manager Agilent Technologies, Inc. 22 September 2017 For Research Use Only. Not for use in diagnostic procedures.

2. Agenda The Right Tools for the Job Tackling Intact Proteins Break it Down Peptides and PTMs Sweet Success Released Glycan Profiling Conclusion For Research Use Only. Not for use in diagnostic procedures.

3. Agenda The Right Tools for the Job Tackling Intact Proteins Break it Down Peptides and PTMs Sweet Success Released Glycan Profiling Conclusion For Research Use Only. Not for use in diagnostic procedures.

4. Protein Characterization Poses Many Questions Intact Mass? Relative Glycoforms? ADC DAR Calculation? Peptide Sequence Mapping? Post Translational Modifications? Disulfide Bonds? Host Cell Proteins? Glycan Profile? For Research Use Only. Not for use in diagnostic procedures.

5. Multiple Experiments One Set of Tools Sample Preparation Separation Data Processing Detection For Research Use Only. Not for use in diagnostic procedures.

6. Agilent BioPharma Workflow Platform for LC/MS Sample Prep Separation Detection Data Analysis Agilent AssayMAP Bravo InfinityLab LC Series and AdvanceBio Columns 6545XT MassHunter BioConfirm AdvanceBio LC/Q-TOF For Research Use Only. Not for use in diagnostic procedures.

7. Step 1 Sample Prep Separation Detection Analysis Agilent AssayMAP Bravo Bravo Liquid Handling Robot Micro- chromatography cartridges Graphical Software (no programming language needed!) For Research Use Only. Not for use in diagnostic procedures.

8. Step 2 Separation Sample Prep Detection Analysis Agilent AdvanceBio LC Columns LC/MS compatible columns designed for: 7100 CE 2D LC Intact Proteins, Heavy/Light Chains, Fab/Fc Regions Peptide Mapping, PTM separations Glycan Profiling Agilent 1290 Infinity II UHPLC For Research Use Only. Not for use in diagnostic procedures.

9. Sample Preparation Sample Prep Separation Detection Analysis Intact Protein Industry leading data quality and sensitivity at the intact level Peptide Mapping and PTMs Ready for routine confirmation or digging deeper with IterativeMS/MS K A N L H V E V T F D SGT R S T S YRVV Glycan Profiling Full released glycan workflow with BioConfirm B.09 New New 6545XT AdvanceBio LC/Q-TOF For Research Use Only. Not for use in diagnostic procedures.

10. Separation Detection Step 4 Analysis Sample Prep MassHunter BioConfirm One Stop Protein Analysis Software Input Output Protein Sequence Deconvolved mass confirmation Glycoform results Peptide Sequence Coverage Glycan Profile Experimental Conditions For Research Use Only. Not for use in diagnostic procedures.

11. Agenda The Right Tools for the Job Tackling Intact Proteins Break it Down Peptides and PTMs Sweet Success Released Glycan Profiling Conclusion For Research Use Only. Not for use in diagnostic procedures.

12. Intact Protein Characterization Workflow Sample Prep Separation Detection Analysis Intact mAb HC & LC Deglycosylated mAb Deglycosylated HC & LC ProZyme InstantPC glycan tag or 2-AB tag Fc F(ab) 2 Fd & LC For Research Use Only. Not for use in diagnostic procedures.

13. Intact Protein Characterization Sample Prep Separation Detection Analysis PLRP-S 1000 AdvanceBio RP mAb Zorbax SB 300 RRHD Poroshell 300 Agilent AdvanceBio LC Columns Agilent 1290 Infinity II For Research Use Only. Not for use in diagnostic procedures.

14. Intact Protein Characterization Sample Prep Separation Detection Analysis Intact protein spectral clarity (10E-8 vacuum) Large molecule SWARM autotune 30k m/z mass range Vent-free capillary removal Quick start methods with PLRP-S column For Research Use Only. Not for use in diagnostic procedures.

15. Intact Protein Characterization Sample Prep Separation Detection Analysis For Research Use Only. Not for use in diagnostic procedures.

16. Deconvolved Spectrum Raw Spectrum NEW: Select for Relative Quant Glycoforms and other PTMs For Research Use Only. Not for use in diagnostic procedures.

17. Agenda The Right Tools for the Job Tackling Intact Proteins Break it Down Peptides and PTMs Sweet Success Released Glycan Profiling Conclusion For Research Use Only. Not for use in diagnostic procedures.

18. Peptide Sequence Mapping and PTM Identification Sample Prep Separation Detection Analysis Preconfigured protocols that required no programming language Get reproducible digestion Run parallel digestions for method development or multiple enzyme preparations For Research Use Only. Not for use in diagnostic procedures.

19. Peptide Sequence Mapping and PTM Identification Sample Prep Separation Detection Analysis AdvanceBio Peptide Mapping Tried and true for routine peptide mapping applications. NEW AdvanceBio Peptide Plus The latest AdvanceBio column specifically designed for LC-MS peptide applications. The charged surface provides sharp symmetrical peaks with just formic acid and provide alternate separation for critical PTMs at higher mass loads Agilent AdvanceBio LC Columns Agilent 1290 Infinity II For Research Use Only. Not for use in diagnostic procedures.

20. Peptide Sequence Mapping and PTM Identification Sample Prep Separation Detection Analysis Quick-start peptide mapping method w/columns Identification confidence (<1 ppm mass error, 50k resolution) One-click optimization with SWARM autotune IterativeMS/MS mode Agilent Jet Stream ion source For Research Use Only. Not for use in diagnostic procedures.

21. Peptide Sequence Mapping and PTM Identification Sample Prep Separation Detection Analysis MassHunter BioConfirm 1. Find Peptides 2. Identify Peptides 3. Assign Possible PTMs 4. Confirm Sequence AUTOMATICALLY For Research Use Only. Not for use in diagnostic procedures.

22. Chromatograms Red/Yellow/Green Confirmation of Protein Annotated MS/MS Spectrum MS Spectrum Sequence Coverage Map Peptides Found For Research Use Only. Not for use in diagnostic procedures. Agilent Restricted For Research Use Only. Not for use in diagnostic procedures

23. Iterative MS/MS When you need to dig deeper LC/MS Run 1 m/z LC/MS Run 2 ASMS Poster WP-694 Enhanced Analysis of Host Cell Proteins from CHO Cell Cultured mAb Using a Newly Developed Q-TOF Instrument, Linfeng Wu m/z Additional Runs Precursors selected for MS/MS Rolling excluded precursors For Research Use Only. Not for use in diagnostic procedures.

24. New Sequence Coverage Map Display multiple data files (e.g. different enzyme digests or Iterative MS/MS) For Research Use Only. Not for use in diagnostic procedures.

25. Agenda The Right Tools for the Job Tackling Intact Proteins Break it Down Peptides and PTMs Sweet Success Released Glycan Profiling Conclusion For Research Use Only. Not for use in diagnostic procedures.

26. AssayMAP Glycan Profiling Preparation Sample Prep Separation Detection Analysis Intact mAb HC & LC Deglycosylated mAb Deglycosylated HC & LC ProZyme InstantPC glycan tag or 2-AB glycan tag Fc F(ab) 2 Fd & LC For Research Use Only. Not for use in diagnostic procedures.

27. AdvanceBio Glycan Mapping Column For Research Use Only. Not for use in diagnostic procedures.

28. Released Glycan Workflow BioConfirm runs Find By Formula algorithm to find glycan features Found glycans are reported in the Biomolecule Table Use AM database for formula match Optional: choose glycan tag For Research Use Only. Not for use in diagnostic procedures.

29. Agenda The Right Tools for the Job Tackling Intact Proteins Break it Down Peptides and PTMs Sweet Success Released Glycan Profiling Conclusion For Research Use Only. Not for use in diagnostic procedures.

30. Tools to address protein characterization at multiple levels Efficiency Results you can trust For Research Use Only. Not for use in diagnostic procedures.