Ensure your Success with Agilent s Biopharma Workflows

Transcription

1 Ensure your Success with Agilent s Biopharma Workflows Steve Madden Software Product Manager Agilent Technologies, Inc. June 5, 2018

2 Agenda Agilent BioPharma Workflow Platform for LC/MS Intact Proteins and ADCs Protein Digests Peptides and PTMs Released Glycan Profiling Host Cell Proteins Conclusion

3 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?

4 Multiple Experiments One Set of Tools Sample Preparation Separation Data Processing Detection

5 Agilent BioPharma Workflow Platform for LC/MS Sample Prep Separation Detection Data Analysis Agilent AssayMAP Bravo InfinityLab LC Series and AdvanceBio Columns 6545XT AdvanceBio LC/Q-TOF MassHunter BioConfirm

6 Agenda Agilent BioPharma Workflow Platform for LC/MS Intact Proteins and ADCs Protein Digests Peptides and PTMs Released Glycan Profiling Host Cell Proteins Conclusion

7 New Features in BioConfirm B Intact Protein Workflow Our BioConfirm Intact Protein workflow has been being significantly enhanced by: Labeling of glycoforms on the mass deconvoluted mass spectrum Relative quantitation of PTMs, with easy editing by the user Sequence Table that can be can be filtered so that the relative quant information for the same modification can be seen and easily compared between two data files Easier to set up disulfide bonds by just putting in their number (unspecified)

8 Intact Protein Workflow New Unspecified Disulfide Linkages Unspecified disulfide linkages This new feature allows the software to calculate the correct intact mab mass without requiring the user to do the tedious work of manually inputting the disulfide linkages. ~ Requested by AE and field teams

9 Intact Protein Workflow Video

10 Intact ADC Drug Antibody Ratio (DAR) Calculation calculated DAR DAR peak list shows all components and % area for each species

11 Reduced ADC DAR Calculation heavy chain Total DAR from both HC and LC light chain

12 Agenda Agilent BioPharma Workflow Platform for LC/MS Intact Proteins and ADCs Protein Digests Peptides and PTMs Released Glycan Profiling Host Cell Proteins Conclusion

13 New Features in BioConfirm B Protein Digest Workflow Our Protein Digest workflow is being significantly enhanced by: New peptide mapping algorithm that uses MS/MS for higher confidence identifications New False Discovery Rate (FDR) and Score (Bio) to assess the quality of identifications Faster performance by optimizing the search for PTMs Cumulative protein coverage from multiple experiments in Sequence Table (e.g. because of multiple enzymes or Iterative MS/MS) Graphically annotated Sequence Coverage Map

14 Red/Yellow/Green Confirmation of Protein Chromatograms MS Spectrum Annotated MS/MS Spectrum Sequence Coverage Map Peptides Found Agilent Restricted use in diagnostic procedures

15 Protein Digest Workflow Video

16 Agenda Agilent BioPharma Workflow Platform for LC/MS Intact Proteins and ADCs Protein Digests Peptides and PTMs Released Glycan Profiling Host Cell Proteins Conclusion

17 New BioConfirm B Released Glycans Workflow New fully curated Glycans Personal Compound Database (PCD) Ability to choose a tag (i.e. chemical label) or add a custom tag Improved Find-by-Formula algorithm with multiple adduct support

18 Released Glycan Workflow Use AM database for formula match Optional: choose glycan tag BioConfirm runs Find By Formula algorithm to find glycan features Found glycans are reported in the Biomolecule Table 18 June 17, 2018 Agilent Restricted use in diagnostic procedures

19 Released Glycans Workflow Video 19 June 17, 2018 Agilent Restricted use in diagnostic procedures

20 Agenda Agilent BioPharma Workflow Platform for LC/MS Intact Proteins and ADCs Protein Digests Peptides and PTMs Released Glycan Profiling Host Cell Proteins Conclusion

21 Challenges for LC/MS Analysis of HCPs Low abundant HCP peptides co-elute with very intense product mab peptides Need broad dynamic range and better separation

22 New Host Cell Protein Analysis Workflow Summary Protein Reduction, Alkylation and Digestion 6545XT AdvanceBio LC/Q-TOF Using Iterative MS/MS Match MS/MS Spectra to Database

23 Protein 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

24 Protein Identification Sample Prep Separation Detection Analysis AdvanceBio Peptide Mapping Tried and true for routine peptide separation applications. NEW AdvanceBio Peptide Plus Agilent 1290 Infinity II Agilent AdvanceBio LC Columns 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

25 Excellent Chromatography Reproducibility Overlay chromatograms of triplicate runs Peptide Precursor ion (m/z) Mass error (ppm) Intensity Intensity %RSD Spiking Protein Level (ppm) ALELFR E % 8 Myoglobin TIAQDYGVLK E % 10.4 Peroxiredoxin 1 Protein name SAVTALWGK E % 74.8 Hemoglobin subunit beta EPQVYTLPPSR E % 1.0E+06 mab

26 Protein Identification Sample Prep Separation Detection Analysis Broad dynamic range (5 orders) Identification confidence (<1 ppm mass error, 50k resolution) One-click optimization with SWARM autotune Iterative MS/MS mode Agilent Jet Stream ion source 6545XT AdvanceBio LC/Q- TOF

27 Automated Iterative MS/MS Acquisition LC/MS Injection 1 m/z LC/MS Injection 2 m/z Additional Injections Precursors selected for MS/MS Rolling excluded precursors

28 Protein Identification, Quantification and Comparison Sample Prep Separation Detection Analysis Raw file Report in minutes Confident protein identification from leading MS/MS search engine Streamlined quantification and report generation Facilities cross-sample quantitative comparison and consistent reporting

29 Export Table of Normalized Protein XIC per Sample from PM software 8 ppm spiking level Positive identification of all the spiking proteins above 8 ppm with 1D-LC workflow coupling with Iterative MS/MS acquisition

30 Iterative MS/MS Decision Engine Improves Protein Identification Comparison of Unique Peptide Sequences between Iterative MS/MS and auto MS/MS using 1D-LC/MS Protein name Protein Spiking Level (ppm) 3 Injections per method Iterative MS/MS Auto MS/MS mab_hc NA mab_lc NA ALBU_HUMAN_spike CAH2_HUMAN_spike CAH1_HUMAN_spike LEP HUMAN_spike HBB_HUMAN_spike HBA_HUMAN_spike UBIQ_HUMAN_spike CO5_HUMAN_spike CATA_HUMAN_spike SUMO1_HUMAN_spike NQO1_HUMAN_spike PRDX1_HUMAN_spike PPIA_HUMAN_spike MYG_HUMAN_spike Iterative MS/MS acquisition method identified more unique peptide sequences

31 Further Improve Identification by Fractionation using AssayMAP Bravo Protein Alias name Protein Spiking Level (ppm) # Unique Peptide Sequences ALBU_HUMAN_spike CAH2_HUMAN_spike CAH1_HUMAN_spike LEP_HUMAN_spike HBB_HUMAN_spike HBA_HUMAN_spike UBIQ_HUMAN_spike CO5_HUMAN_spike CATA_HUMAN_spike SUMO1_HUMAN_spike NQO1_HUMAN_spike PRDX1_HUMAN_spike PPIA_HUMAN_spike MYG_HUMAN_spike CYB5_HUMAN_spike EGR_HUMAN_spike SYHC_HUMAN_spike KCRM_HUMAN_spike ppm spiking level Positive identification of all the spiking proteins above 2 ppm with on-cartridge HPRP fractionation using AssayMAP Bravo coupling with three injections of Iterative MS/MS acquisition

32 Agenda Agilent BioPharma Workflow Platform for LC/MS Intact Proteins and ADCs Protein Digests Peptides and PTMs Released Glycan Profiling Host Cell Proteins Conclusion

33 Four Workflows to Ensure Your Success! Trusted Results for: Intact Proteins Protein Digests Released Glycans Host Cell Proteins