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Waters — Technical Presentation (2013)

40 slides from Waters's technical presentation, 2013

  1. Slide 1: Charged Surface Hybrid C18 for High Resolution LC and LC/MS Peptide Separations

    Charged Surface Hybrid C18 for High Resolution LC and LC/MS Peptide Separations Higher Quality Peptide Separations Using Hybrid Particle-Based Reversed-Phase Columns and CSH Technology Matthew Lauber, Ph.D. Senior Applications Chemist ©2013 Waters Corporation 1

  2. Slide 2: Reversed Phase Peptide Separations

    Reversed Phase Peptide Separations  Peptide separations – critically important TFA – peptide mapping, bottom-up proteomics … LC performance – reversed phase – challenges remain MS performance TFA Peptide  Ionic analytes – peptides – Secondary interactions - Poor peak shape – Overloading at very lo

  3. Slide 3: Charged Surface Hybrid

    Charged Surface Hybrid ©2013 Waters Corporation 3

  4. Slide 4: Agenda

    Agenda  The Peak Shape Problem – Column Chemistries – CSH Technology – Peak Capacity  CSH130 C18 and separations without TFA – LC-MS of protein digests – Small protein separations  Peptide Mapping a Therapeutic mAb – CSH130 C18 for LC-UV-MS – Disulfides and Deamidation  Peptide Separation Techno

  5. Slide 5: The Peak Shape Problem

    The Peak Shape Problem ©2013 Waters Corporation 5

  6. Slide 6: Competitor's "Industry Standard" 5 µm Porous Silica C18

    Competitor’s “Industry Standard” 5 µm Porous Silica C18 3.0 0.1% TFA Ion Pairing 3 4 5 7 Strong 1 2 6 8 9 2.0 0.02% TFA 0.08% FA A214 3 4 7 8 2 5 6 9 1 1.0 0.1% FA Weak 1 2 3 4 5 6 7 8 9 – not detected 0.0 0 10 20 30 40 50 60 Time (min) Competitor’s “Industry Standard” C18 MassPREP™ Peptide Mixture

  7. Slide 7: Ethylene Bridged Hybrid - BEH Technology

    Ethylene Bridged Hybrid - BEH Technology™ U.S. Patent No. 6,686,035 B2 and others patent pending Bridged Ethanes In Silica Matrix Organo Silica Hybrid Particles – pH stability – Reduced ionic interactions – Basis of Peptide Separation Technology EtO CH2 CH2 OEt OEt O Si O Et Si Si EtO EtO OEt O O 4

  8. Slide 8: Small Particle Size

    Small Particle Size Mobile Phase 1500 Da Peptide Peptides 2 3.5 µm Porous Particle H (mm) Adsorption Equilibria Diffusion-related band broadening 1  Diffusion distances decrease 1.7 µm – Reduced Eddy diffusion – Improved mass transfer kinetics  Column efficiency 40 400  Narrower peaks µL/min µL/m

  9. Slide 9: Waters BEH130 C18 1.7 µm

    Waters BEH130 C18 1.7 µm 3.0 Competitor’s “Industry Standard” 0.1% TFA 5 µm C18 7 4 5 6 8 3 3 4 5 1 6 2 9 2.0 0.02% TFA 0.08% FA A214 7 8 2 3 4 5 6 3 4 1 5 6 9 1.0 0.1% FA 8 7 1 4 5 6 2 3 3 4 5 6 9 0.0 0 30 10 40 20 30 40 50 60 Time (min) Time (min) Peptide Sequence BEH130 C18 Waters ACQUITY UPLC BE

  10. Slide 10: A New Column Chemistry – CSH130 C18

    A New Column Chemistry – CSH130 C18 3.0 0.1% TFA 4 5 8 6 7 3 1 2 9 2.0 4 8 5 0.02% TFA 6 7 0.08% FA A214 3 2 1 9 1.0 Vo 1 4 5 7 8 0.1% FA 6 2 3 9 1 2 0.0 10 20 30 40 50 60 Time (min) Waters ACQUITY UPLC CSH130 C18 Peptide Sequence 2.1 x 150 mm, Porous 1.7 µm, 130Å 1 RASG-1 RGDSPASSKP 2 Angiotensin 1

  11. Slide 11: A New Column Chemistry – CSH130 C18

    A New Column Chemistry – CSH130 C18 Competitor’s Competitor’s BEH130 C18 CSH130 C18 “Industry Standard” C18 Superficially Porous Porous (130Å) 1.7 µm Porous (130Å) 1.7 µm 2.1 x 150 mm 2.1 x 150 mm Porous (300Å) 5 µm “Peptide” C18 2.1 x 250 mm SPP (100Å) 1.7 µm 2.1 x 150 mm TFA UV absorbance (214 nm)

  12. Slide 12: Charged Surface Hybrid (CSH) Technology

    Charged Surface Hybrid (CSH) Technology patent pending Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography. P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter Waters White Paper 720003929EN 2011  Expands upon the robust BEH particle technology  CSH130 C18 = BEH130 ba

  13. Slide 13: Peak Capacity

    Peak Capacity  Peak Capacity = – The number of peaks that can be separated within a retention window Neue, U. D., J Chromatogr A 2005, 1079 (1-2), 153-61. – The best metric for determining the quality of gradient separations 100% 1 9 peaks 2.35σ Peak Height could resolve 50% wh ~300-400 4σ 13.4% w4

  14. Slide 14: Peak Capacity - FA vs TFA

    Peak Capacity - FA vs TFA Competitor’s “Industry Standard” Silica C18 5 µm 2.1 x 250 mm TFA FA % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 ©2013 Waters Corporation 14

  15. Slide 15: Peak Capacity - FA vs TFA

    Peak Capacity - FA vs TFA BEH130 C18 1.7 µm 2.1 x 150 mm Competitor’s “Industry Standard” Silica C18 5 µm 2.1 x 250 mm TFA FA % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 ©2013 Waters Corporation 15

  16. Slide 16: Peak Capacity - FA vs TFA

    Peak Capacity - FA vs TFA Competitor’s SPP “Peptide” C18 1.7 µm 2.1 x 150 mm BEH130 C18 1.7 µm 2.1 x 150 mm Competitor’s “Industry Standard” Silica C18 5 µm 2.1 x 250 mm TFA FA % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 ©2013 Waters Corporation 16

  17. Slide 17: Peak Capacity - FA vs TFA

    Peak Capacity - FA vs TFA CSH130 C18 1.7 µm 2.1 x 150 mm Competitor’s 20% SPP “Peptide” C18 1.7 µm 2.1 x 150 mm 90% BEH130 C18 1.7 µm 2.1 x 150 mm Competitor’s “Industry Standard” Silica C18 5 µm 2.1 x 250 mm TFA FA % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 ©2013 Waters Corporation 17

  18. Slide 18: MS Signal - FA vs TFA

    MS Signal - FA vs TFA Peak Capacity MS Signal CSH130 C18 1.7 µm Competitor’s SPP “Peptide” C18 1.7µm BEH130 C18 1.7 µm Competitor’s “Industry Standard” Silica C18 5 µm TFA TFA FA FA % TFA 0.00 0.05 0.10 % TFA 0.00 0.05 0.10 % FA 0.10 0.05 0.00 % FA 0.10 0.05 0.00 ©2013 Waters Corporation 18

  19. Slide 19: Loadability

    Loadability Attribute – how much analyte can be loaded before peak shape deteriorates B LowLowMass Load A Typical Mass Load 450 450 0.6 µg ofLoad Mass mixture 6 µg ofMass High mixture Load *Previously shown 8 CSH130CSH C18 C18 1.7 µm CSH C18 8 BEH C18 BEH C18 400 400 CSH130 C18 1.7 µm 350 350 BEH130

  20. Slide 20: CSH130 C18 and Separations without TFA

    CSH130 C18 and Separations without TFA ©2013 Waters Corporation 20

  21. Slide 21: LC-MS of Enolase Tryptic Digest

    LC-MS 2E+6 Enolase Tryptic Digest CSH130 C18 1.7 µm T6 0.1% FA Wh,avg= 4.0 s T38 T14 Pc,4σ = 532 Intensity T51 T23 1E+6 T35 T42 T45 T27 T40 T37 T10 0.1% TFA 0E+0 1 11 21 31 41 51 10x drop in Waters ACQUITY UPLC CSH130 C18 Time(min) 2.1 x 150 mm, 1.7 µm, 130Å sensitivity 2% ACN for 1 min, then to 50%

  22. Slide 22: CSH130 C18 vs Other Chemistries

    CSH130 C18 vs Other Chemistries 2E+6 0.1% FA BEH130 BEH C18 C18 1.7 1.7 µmµm P = 399 c,4σ = 399 Pc,4σ Intensity 1E+6 0E+0 0 10 20 30 40 50 60 2E+6 Competitor’s Superficially Competitor A Porous “Peptide” C18 1.7 µm Superficially Porous C18 1.7 µm Intensity 1E+6 PPc,4σ = 405 c,4σ = 405 0E+0 0 10 20 3

  23. Slide 23: Retention and Selectivity

    LC-MS Retention and Selectivity 1E+6 BEH130 C18 T3 SVYDSR T12 ANIDVK T19 HLADSK More positive T10 GVLHAVK charge T19 T40 T40 IATAIEK T12 T3 T10 0E+0 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5 Competitor’s Superficially Porous “Peptide” C18 1E+6 Intensity T40 T3 T12 T19 T10 0E+0 5.5 6.5 7.5 8.5 9.5 10.5 11.5

  24. Slide 24: Large Peptides/Small Proteins

    Large Peptides/Small Proteins 0.1% FA Peptide/Protein kDa 1 Bradykinin 1.1 1.2 2 2 Renin Substrate 1.8 CSH130 C18 1.7 µm 1 3 Ubiquitin 8.6 5 Cytochrome C 4 12.4 0.8 (Equine) Insulin 1 A214 0.6 5 5.7 (Bovine) 0.4 3 6 6 Melittin 2.8 0.2 4 130 Å 0 1.2 10 15 20 25 30 35 40 45 50 BEH130 C18 1.7 µm Time (

  25. Slide 25: Peptide Mapping of a Therapeutic mAb

    Peptide Mapping of a Therapeutic mAb ©2013 Waters Corporation 25

  26. Slide 26: Peptide Mapping a Therapeutic mAb

    Peptide Mapping a Therapeutic mAb  Trastuzumab (Herceptin; Genentech) – Breast Cancer, Anti-HER2 – One of the highest grossing therapeutic mAbs (~5 billion $/yr) – Biosimilars – Basis for a new antibody drug conjugate (ADC; Trastuzumab emantansine) o Phase III clinical trials completed  High peak

  27. Slide 27: Non-Reduced Lys-C Peptide Mapping

    Non-Reduced Lys-C Peptide Mapping  Non-Reduced Lys-C Digests – Minimal complexity + disulfides preserved – 27 different linear peptides – 8 different disulfide linked peptides – 150 to 11,000 Da A recent Amgen protocol: Anal Biochem 2011, 411 (2), 284-91. S-S S-S Lys-C Cleavage L: 1-42 43-45 46-103

  28. Slide 28: Method Considerations

    Method Considerations ACQUITY UPLC BEH130 C18 1.7µm 2.1 x 150 mm ACQUITY UPLC CSH130 C18 1.7µm  Optimize peak capacity Elevated Temperature (60°C) 0.02% TFA  Small compromise to  Improved peak shape and recovery 0.08% FA MS Sensitivity  5-11 kDa species 400 CSH130 C18 2 5-11 kDa Species BEH130 C

  29. Slide 29: Non-Reduced Lys-C Peptide Maps Trastuzumab

    Non-Reduced Lys-C Peptide Maps Trastuzumab 1.1 BEH130 C18 0.02% TFA Peak Capacity = 207 0.08% FA 0.8 97.1% Coverage 5 A210 0.5 4 2 3 0.2 1 -0.1 0 10 20 30 40 50 60 70 80 90 100 110 1.1 CSH130 C18 wh (sec) Peak Capacity = 394 5 Peak BEH130 C 18 C SH130 C 18 0.8 96.7% Coverage 1 33.7 15.4 2 16.1 9.7 4

  30. Slide 30: LC-UV-MS with an MS-Compatible Mobile Phase

    LC-UV-MS with an MS-Compatible Mobile Phase CSH130 C18 0.02% TFA MS 0.08% FA Low TFA mobile phase Optimized Peak Capacity + MS signal ≥ UV signal UV Peak Area MS-98701 UV-96527 ACQUITY H-Class Bio CSH130 C18 1.7 µm UV Detector 3+ ESI-MS 2+ Xevo G2 QTof m/z 200 400 600 800 1000 1200 1400 1600 1800 30

  31. Slide 31: Disulfide Characterization

    Disulfide Characterization CSH130 C18 - Optimized Gradient Non-Reduced L:208-214 x H:222-225 H:137-150 x H:151-213 H:252-277 x H:324-325 H:226-251 x H:226-251 H364-373 x H:418-442 L:127-145 x L:191-207 1.1 H:1-30 x H:77-124 L:1-42 x L:46-103 0.9 0.7 A210 0.5 0.3 0.1 Blank -0.1 -0.3 A210 -0.5 S-S S-S

  32. Slide 32: Assaying Deamidation

    Assaying Deamidation CSH130 C18 - Optimized Gradient 44 GLEWVARIYPTNGYTRYADSVK65  Binding region from the heavy chain  Prone to deamidation 0.5 3E+5 0.4 H:44-65 92 % Asn Unmodified 7 % isoAsp Deamidated 0E+0 853 854 855 856 0.3 1 % Asp 2E+4 Mass shift A210 Intensity * +0.98 Da 0.2 0E+0 853 854 855

  33. Slide 33: Conclusions

    Conclusions  CSH130 C18 Peptide Separations – Improved loadability and greater peak capacity vs. other C18 columns – Excellent peak shape with both TFA and FA mobile phases (highly compatible with MS) – 130Å pore size optimal for species up to 8-10 kDa – Unique selectivity (basic residues) o Less r

  34. Slide 34: Peptide Separation Technology Columns

    Peptide Separation Technology Columns ©2013 Waters Corporation 34

  35. Slide 35: New Addition to the Suite of Waters Peptide Separation Technology

    New Addition to the Suite of Waters Peptide Separation Technology  Peptide Separation Technology – Peptide C18 Columns – QC Tested with Digests  BEH Technology – BEH130 C18 and BEH300 C18 – Industry Leading Performance for Most Applications – Two Pore Sizes – Particle Sizes: 1.7 µm, 3.5 µm, 5 µm –

  36. Slide 36: UPLC and HPLC

    UPLC and HPLC CSH130 C18 High peak capacity separations not limited to UPLC 2.1 x150 mm 1.0 1.0 0.1 % FA 0.1 % TFA 0.8 0.8 0.6 ~8000 0.6 1.7 µm psi A214 A214 CSH130 C18 Peptide Separation 0.4 0.4 Technology Columns 0.2 0.2 Available 0.0 Now: Upcoming: Method 0.0 Analytical Columns Nano (75, 20150, T

  37. Slide 37: Quality Control

    Quality Control CSH130 C18 0.1 % Formic Acid ©2013 Waters Corporation 37

  38. Slide 38: Quality Control

    Quality Control 1.20 T12 Batch 110 T12-T13 1.00  CSH130 C18 0.80 AU T9-T10 0.60 T13-T14 T10 T8 T14 T19C T15  Batch-to-Batch T4 0.40 T19 T1 Reproducibility 0.20 0.00 1.20 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00  Cytochrome C 1.00 Batch 108 Minutes Digest, 0.1% 0.80 AU 0.60 Formic Acid 0.40 0.20 

  39. Slide 39: Analytical Standards and Reagents (ASR)

    Analytical Standards and Reagents (ASR) MassPREP Peptide Mixture Digestion Standards Cytochrome C Digestion Standard Part Number: 186006371 Rapigest SF Amgen Digestion Protocol Anal Chem. 2009 81(4):1686-92 ©2013 Waters Corporation 39

  40. Slide 40: Useful Literature

    Useful Literature  Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography – P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter – Waters White Paper 720003929EN 2011  Increasing Peak Capacity in Reversed Phase Peptide Separations with Charged Surface Hybrid (CSH) C18 Col

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