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Waters — Technical Presentation (May 2013)

57 slides from Waters's technical presentation, May 2013

  1. Slide 1: Protein and Peptide Therapeutics: Addressing Bioanalytical Demands of Complicated Peptides

    Thank you for joining us, the session will begin shortly… Protein and Peptide Therapeutics: Addressing Bioanalytical Demands of Complicated Peptides Tips and Tricks for Troubleshooting and Optimization During Method Development Mary E. Lame Senior Applications Chemist Waters Corporation Milford, MA

  2. Slide 2: Friendly Reminders

    Friendly Reminders…  Please use text chat functionality to submit your questions today.  Erin Chambers, Principal Applications Chemist, Waters Corp.  “LIVE” Technical support during today’s event  Upon conclusion, follow up information will be available:  http://www.waters.com/May1  Recorded v

  3. Slide 3: Mary Lame, Sr. Applications Chemist

    Mary Lame, Sr. Applications Chemist  B.S. in Chemistry from the Western Connecticut State University (Danbury, CT)  M.S. in Chemistry from Central Connecticut State University (New Britain, CT).  She began her bioanalytical career in 1997 with a global Pharmaceutical company where she supported R

  4. Slide 4: Outline

    Outline  Introduction — Basic screening methods — Typical challenges faced  Understanding your peptide o pI, HPLC index, hydrophobic/hydrophilic  Understanding the challenges: symptoms, examples, and solutions for common problems  Conclusions ©2012 Waters Corporation 4

  5. Slide 5: Introduction: Why bioanalysis for peptides?

    Introduction  Why bioanalysis for peptides? 1. Drug discovery/development activities need to be performed o PK/PD, metabolic fate, bioequivalence, drug monitoring 2. Peptides as biomarkers 3. Signature peptides can be used to quantitate protein drugs and biomarkers in complex matrices, after digest

  6. Slide 6: Why LC-MS/MS?

    Why LC-MS/MS?  Why an LC-MS/MS based assay? — ELISA assays not practical for discovery, no antibodies available yet — Challenges with ELISA assays o time consuming, expensive to develop o require separate assay for each peptide o limited linear dynamic range o Possible cross reactivity  Benefits o

  7. Slide 7: Chromatographic Screening Protocol for Peptides

    Chromatographic Screening Protocol for Peptides  ACQUITY UPLC® BEH300 C18 2.1 X 50 mm, 1.7 µm Peptide Separation Technology (PST) Column — Columns are QC tested with peptide standards — 300Å PST column gave overall best performance (peak shape) for diverse peptides — 2.1 X 50 mm provides adequate t

  8. Slide 8: Single Screening Method: Diverse Peptides

    Single Screening Method: Diverse Peptides 1 2 3 5 MRM of 5 Channels ES+ 4 MS Data Peak Points Width Across Analyte MW (seconds) Peak 1. Vasopressin 1084 1.8 15 2. Angiotensin II 1046 2.2 15 % 3. Desmopressin 1069 2.2 18 4. Bivalirudin 2180 2.4 18 5. Enfuvirtide 4492 2.1 16 0 Time 0.20 0.40 0.60 0.80

  9. Slide 9: SPE Screening Protocol for Peptides

    SPE Screening Protocol for Peptides: Screening Complementary Sorbents Oasis® WCX Oasis® MAX µElution µElution Protocol Dilute plasma with 4% H3PO4 Condition MeOH/Equilibrate H2O Load Diluted Plasma Wash 1: 5% NH4OH Wash 2: 20% ACN Elution: 1% TFA in 75/25 ACN/H2O Dilute: H2O ©2012 Waters Corporation

  10. Slide 10: SPE Format: Oasis µElution Plates

    SPE Format : Oasis® µElution Plates Oasis® µElution plate technology  Up to 15X concentration without evaporation — Concentration often necessary to reach LOD’s with peptides  Minimizes analyte loss — Minimizes sticking to walls of collection plates — Eliminates problems re-solubilizing after dry-

  11. Slide 11: Final SPE Results after Methods Optimized

    Final SPE Results after BNP, Enfuvirtide and Somatostatin Methods Optimized 120 100 % SPE Recovery 80 Screening Protocol 60 Oasis® MAX 40 Screening Protocol Oasis® WCX 20 Modified Protocol 0 Minor, compound specific, modifications for 3 peptides result in excellent recovery for all peptides % Matrix

  12. Slide 12: When the Basic Methods Don't Work: Typical Challenges Faced

    When the Basic Methods Don’t Work: Typical Challenges Faced  Peptide Loss  Poor Sensitivity  Solubility  Specificity/Selectivity  Chromatography  Protein Binding  Non-specific Binding  Low Extraction Recovery  Inadequate Retention  Chemical Instability ©2012 Waters Corporation 12

  13. Slide 13: Outline

    Outline  Introduction — Basic screening methods — Typical challenges faced  Understanding your peptide o pI, HPLC index, hydrophobic/hydrophilic  Understanding the challenges: symptoms, examples, and solutions for common problems  Conclusions ©2012 Waters Corporation 13

  14. Slide 14: Understanding Your Peptide

    Understanding Your Peptide 1. Obtain the peptide sequence 2. Calculate pI and HPLC index 3. Assess nature by evaluating the residues 4. Look for any potential stability problems ©2012 Waters Corporation 14

  15. Slide 15: Chemical Properties of Therapeutic Peptides

    Chemical Properties of Therapeutic Peptides Peptide MW pI # of Residues HPLC Index* Octreotide 1019 9.3 8 40.8 Angiotensin II 1046 7.35 8 38.3 Desmopressin 1069 8.6 9 16.8 Vasopressin 1084 9.1 9 7.6 Goserelin 1270 7.3 10 31.7 Angiotensin I 1296 7.51 10 56.2 Somatostatin 1638 10.4 14 52.6 Neurotensin

  16. Slide 16: Understanding Your Peptide: Step 1

    Understanding Your Peptide: Step 1  Teriparatide (1-34 fragment of human parathyroid hormone) example: Obtain sequence 1. ©2012 Waters Corporation 16

  17. Slide 17: Understanding Your Peptide: Step 2

    Understanding Your Peptide: Step 2  Teriparatide (1-34 fragment of human parathyroid hormone) example: Calculate pI and HPLC index 2. Teriparatide is basic and sticky ©2012 Waters Corporation 17

  18. Slide 18: Understanding Your Peptide: Step 3

    Understanding Your Peptide: Step 3  Teriparatide (1-34 fragment of human parathyroid hormone) example: Assess nature by evaluating the residues 3. Teriparatide sequence: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF Property Amino Acid Hydrophobic A, F, I, L, M, P, V, W, Y Moderate C, G Hydrophilic D, E, H, K

  19. Slide 19: Understanding Your Peptide: Step 4

    Understanding Your Peptide: Step 4  Teriparatide (1-34 fragment of human parathyroid hormone) example: Look for any potential stability problems 4. Teriparatide sequence: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF Property Amino Acid Hydrophobic A, F, I, L, M, P, V, W, Y Moderate C, G Hydrophilic D, E, H,

  20. Slide 20: How Can I Use This Information?

    How Can I Use This Information?  pI — Helps to determine solubility — Helps identify best SPE and chromatographic conditions/solvents  HPLC Index — Indicates if polar or more hydrophobic o Can guide troubleshooting • Hydrophobic: may lead to non-specific binding, protein binding, carryover, low SP

  21. Slide 21: Outline

    Outline  Introduction — Basic screening methods — Typical challenges faced  Understanding your peptide o pI, HPLC index, hydrophobic/hydrophilic  Understanding the challenges: symptoms, examples, and solutions for common problems — Solubility — Poor Sensitivity — Specificity — Chromatography and

  22. Slide 22: Specific Challenges in Bioanalysis of Peptides: Solubility

    Specific Challenges in Bioanalysis of Peptides: Solubility  When is Solubility Important? – In initial solubilization of powder – During sample preparation o Pre-treatment, wash steps, elution – In mobile phase, at flow rate you are running – In injection solvent  Symptoms of inadequate/incomplete

  23. Slide 23: General Guidelines for Solubilizing Powder

    General Guidelines for Solubilizing Powder 1. If a peptide is < 5 residues, it will likely dissolve in aqueous solutions unless the sequence is entirely comprised of hydrophobic residues. 2. Peptides containing >25% charged residues and < 25% hydrophobic residues generally dissolve in aqueous soluti

  24. Slide 24: General Guidelines for Solubilizing Powder

    General Guidelines for Solubilizing Powder 4. Peptides containing >50% hydrophobic residues may be only slightly soluble or insoluble in aqueous solutions. Hydrophobic peptides are best solubilized in DMSO, DMF, strong acid solutions (TFA, formic, acetic), or isopropanol. For cysteine- containing pe

  25. Slide 25: Teriparatide Solubilization Solvent Assessment

    Teriparatide Solubilization Solvent Assessment ) MRM of 4 Channels ES+ 100 0.88 824.32 > 983.78 (Teriparatide 5+) 47318 6.41e5 Area % DMSO 0 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 MRM of 4 Channels ES+ 100 0.86 824.32 > 983.78 (Teriparatide 5+) 47286 6.44e5 Area 1% TFA, 50% ACN % 0 0

  26. Slide 26: Protein Precipitation and Solubility of Large Peptides

    Protein Precipitation and Solubility of Large Peptides % Teriparatide Recovery 120 100 80 60 40 20 0 1:1 ACN 2:1 ACN 1:1, 1% FA 1:1, 1% 1:1, 1% AA 1:1, 5% 2:1, 5% in ACN TFA in ACN in ACN NH4OH NH4OH ACN ACN Too much organic causes peptide loss, pH impacts recovery ©2012 Waters Corporation 26

  27. Slide 27: Improving Solubility in Mobile Phase B, Reducing Carryover

    Improving Solubility in Mobile Phase B, Reducing Carryover 07-Jun-2012 MRM of 2 Channels ES+ 824.351 > 983.774 (Teriparatide) 100 3.49e5 Area 1.05 9940 % Mobile phase B= 0.1% FA in ACN 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 MRM of 2 Channels ES+ 1.00 824.351 > 983.77

  28. Slide 28: Specific Challenges in Bioanalysis of Peptides: Specificity

    Specific Challenges in Bioanalysis of Peptides Specificity  Possible Causes — Many peptides, high abundance proteins in sample — Interference by similar endogenous compounds — Biomarker Analysis o Difficult to get blank matrix for use in method development/validation • Use of stripped or surrogate

  29. Slide 29: Specificity in Human Plasma: Impact of Pretreatment Prior to SPE

    Specificity in Human Plasma: Impact of Pretreatment Prior to SPE Plasma diluted with 10mM TRIS Base 1: MRM of 4 Channels ES+ 4.48 TIC 100 % 2.86 4.59 3.95 5.50 6.30 8.21 7.12 0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 1: MRM of 4 Channels ES+ 100 Plasma diluted with 1% TFA 5.76 TIC 4.46 % 4.56 8

  30. Slide 30: Lack of Specificity in an MRM assay for Trastuzumab

    Lack of Specificity in an MRM assay for Trastuzumab 1 nM Trastuzumab in ~1500 area counts Solvent A human serum digest 1 nM Trastuzumab in serum ~500-700 area counts digest = 2-3X lower! Addressing the problems……… ©2012 Waters Corporation 30

  31. Slide 31: Mixed-mode Cation Exchange SPE Clean-up for Protein Digests

    Mixed-mode Cation Exchange SPE Clean-up for Protein Digests Before SPE Increase in Signal Reduction in Background Removal of Digest Reagents After SPE Reduction of Plasma Phospholipids Improved Specificity Digest Concentration ©2012 Waters Corporation 31

  32. Slide 32: MS Specificity: Avoiding Immonium Ion Fragments

    MS Specificity: Avoiding Immonium Ion Fragments 02-Mar-2012 1: MRM of 4 Channels ES+ 0.99 867 > 136 (Lantus) 100 9.40e5 1.68 867 -> 136 (tyrosine immonium ion) % 1.58 1.15 1.20 1.30 1.40 1.49 Lack of Specificity 0.56 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3

  33. Slide 33: MS Specificity: Use of Higher m/z Precursors and Fragments

    MS: Specificity: Use of Higher m/z Precursors and Fragments Human Plasma Extract MRM of 2 Channels ES- 2.48 1031.5 > 1026.8 3.20e4 4th charge state 1031.5 -> 1026.8 % 3.36 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 MRM of 2 Channels ES- 2.48 825 > 821.3 3.26e4 1.67 1.22 1.63 5th charge state % 1.96 2.49 8

  34. Slide 34: Specific Challenges: Protein Binding

    Specific Challenges in Bioanalysis of Peptides Protein Binding  Symptoms – Decreases solid phase extraction recovery o Peptide passes through on load step with associated protein – Results in underestimation of peptide concentration  Possible Causes – More pronounced in stickier/more hydrophobic p

  35. Slide 35: Specific Challenges: Non-specific Binding (NSB)

    Specific Challenges in Bioanalysis of Peptides Non-specific Binding (NSB)  Symptoms – Peptide loss – Loss of low end of standard curve – Non-linearity – Irreproducible chromatography – Loss of chromatographic peak – Poor LC or SPE recovery  Possible Solution – Requires carrier protein or surfactan

  36. Slide 36: Insulin Analogs: Testing for and Eliminating Non-Specific Binding

    Insulin Analogs: Testing for and Eliminating Non-Specific Binding Insulin Glargine solution: <30 min. on benchtop 1: MRM of 4 Channels ES+ 1.01 1011.2 > 1179 (Lantus) 100 469 Area 30% MeOH, 10% acetic acid, 0.05% rat plasma % 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.

  37. Slide 37: Carrier Protein Options

    Carrier Protein Options MRM of 4 Channels ES+ 1.06 824.25 > 983.795 (Teriparatide 5+) 100 5.57e5 Teriparatide + 40ug/ml Bovine Serum Albumin % 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 MRM of 4 Channels ES+ 100 824.25 > 983.795 (Teriparatide 5+) 5.57e5 1.

  38. Slide 38: Specific Challenges: Peptide Loss and Poor Sensitivity

    Specific Challenges in Bioanalysis of Peptides  Peptide loss — Potential losses during evaporation — Hydrophobic peptides stick to vials/collection plates o Concentration dependent — Addition of organic or acid helps maintain solubility for storage — Non-specific binding — Maintain solubility acros

  39. Slide 39: Specific Challenges: Chromatography

    Specific Challenges in Bioanalysis of Peptides: Chromatography  Problem: Poor peak shape — Possible Solutions o Increase temperature o Decrease flow rate o Increase stationary phase pore size o Change ligand or column chemistry o Add carrier protein to reduce NSB  Problem: Poor Sensitivity — Possi

  40. Slide 40: Improving Chromatography for Amyloid Beta Peptide 1-40: Temperature

    Improving Chromatography for Amyloid Beta Peptide 1-40, MW 4330: Temperature 52% increase in area counts at higher temperature, carryover also reduced MRM of 1 Channel ES+ 1.17 100 365 1.32e4 Area 365 area counts 60 C % 0 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 MRM of 1 Chan

  41. Slide 41: Improving Chromatography for Teriparatide: Column Chemistry

    Improving Chromatography for Teriparatide: Column Chemistry 0 MRM of 4 Channels ES+ 1.23 824.32 > 983.78 (Teriparatide 5+) 100 ACQUITY BEH C18 300A % 1.59 1.67 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 MRM of 4 Channels ES+ 0.78 824.32 > 983.78 (Teriparat

  42. Slide 42: Improving Chromatography: Ligand and Pore Size

    Improving Chromatography: Ligand and Pore Size Enfuvirtide: MW 4492 ACQUITY BEH 1.7 µm C18 130Å ACQUITY BEH 1.7 µm C18 300Å ACQUITY BEH 1.7 µm C4 300Å ©2012 Waters Corporation 42

  43. Slide 43: Chromatography: Use of Carrier Protein to Reduce NSB

    Chromatography: Use of Carrier Protein to Reduce NSB Teriparatide 18-May-2012 14:41:24 extracted soln 1ng rep 1 MRM of 2 Channels ES+ 0.95 TIC 100 2.56e5 Pure solution in 30% ACN, 1% TFA % 0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20

  44. Slide 44: Poor Peak Shape or No Peak: Column Conditioning

    Poor Peak Shape or No Peak: Column Conditioning Insulin Glargine MRM of 10 Channels ES+ 866.8 > 984 (Lantus) 100 2.85e6 After 9 injections of % precipitated plasma 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 MRM of 10 Channels ES+ 100 866.8 > 984 (Lantus) 2.85e6 2nd injec

  45. Slide 45: Chromatographic Carryover

    Chromatographic Carryover Problem: Carryover  Possible Causes of carryover — Improper injection solvent — Stuck on column — Stuck to LC tubing, needle etc.  Possible Solutions — Stuck on column: increase temperature, decrease flow rate, change mobile phase modifier, chromatographic pore size, CSH

  46. Slide 46: Improving Chromatography for Amyloid Beta Peptide 1-40: Flow Rate

    Improving Chromatography for Amyloid Beta Peptide 1-40, MW 4330: Flow Rate 48% increase in area counts at lower flow rate, carryover also reduced MRM of 1 Channel ES+ 100 385 1.32e4 Area 400 µL/min % 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 MRM of 1 Channel ES+ 10

  47. Slide 47: Teriparatide Chromatography: Improving Diffusion/Solubility

    Teriparatide Chromatography: Improving Diffusion/Solubility Column temp 60C Flow rate 0.4ml/min MRM of 4 Channels ES+ 1.08 824.25 > 983.795 (Teriparatide 5+) 100 22737 2.98e5 Area 15-50% B in 2 min 72% Increase in area counts % 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

  48. Slide 48: Low SPE Recovery

    Low SPE Recovery Problem: Low SPE Recovery  Possible Causes of Low Recovery – Extraction Device Capacity – Poor Retention – Chemical Stability o Apparent low recovery – Non-specific binding – Protein binding – Not strong enough elution solvent or enough volume ©2012 Waters Corporation 48

  49. Slide 49: Low SPE Recovery

    Low SPE Recovery  Possible Solutions for Low Recovery – Extraction Device Capacity o Load less sample, increase sorbent bed mass o Change pre-treatment to eliminate interferences – Poor Retention o Use appropriate mixed-mode sorbent and ensure charge through proper pretreatment o Eliminate protein

  50. Slide 50: Troubleshooting Recovery

    Troubleshooting Recovery 1ngmL_5050_N4OH_020810_001 MRM of 3 Channels ES- SPE Recovery = 94% 5.69 TIC 4.23e4 5.42 5.44 SPE Recovery = 92% 5.44 3 peptides of the same class, extracted from human CSF 5.45 5.71 SPE Recovery = 64% Elution solvent contains 65% ACN 5.67 and 5% NH4OH 5.88 % 5.40 5.89 5.39

  51. Slide 51: Sample Extraction: Final Method

    Sample Extraction: Final Method Oasis® MCX μElution Plate Amyloid β % SPE Peptide Recovery Condition, Equilibrate 1-38 94% Load: 150 μL diluted, pretreated sample 1-40 92% 1-42 90% Wash 1 and 2 Elute: 2 X 25 µL 75/10/15 ACN/conc. NH4OH/water (by volume) Fully solubilizes aβ 1-42 Dilute: 25 µL water

  52. Slide 52: Troubleshooting Recovery

    Troubleshooting Recovery  Recovery is 94% in human CSF but 40% in human plasma  What is the problem? — Most likely to be protein binding  What can we do?  Modify pretreatment — Dilute with stronger acid — Dilute with base — Denature with guanidine HCl (typically 5M, 9:1 guanidine:plasma) ©2012 W

  53. Slide 53: Amyloid Peptides: Troubleshooting Non-specific Binding in SPE Recovery

    Amyloid Peptides: Troubleshooting Non- specific Binding in SPE Recovery  Solvent standards used for initial SPE method development % SPE Recovery 100 — SPE recovery = 60% 90 80 70  Amyloid Beta Peptides 60 50 — >4000 MW 40 — > 50% hydrophobic residues 30 20 10 0  Mass Balance Results Solvent Std

  54. Slide 54: Outline

    Outline  Introduction — Basic screening methods — Typical challenges faced  Understanding your peptide o pI, HPLC index, hydrophobic/hydrophilic  Understanding the challenges: symptoms, examples, and solutions for common problems  Conclusions ©2012 Waters Corporation 54

  55. Slide 55: Conclusions

    Conclusions  Using predefined guidelines to learn about your peptide can help predict potential problems and can guide you towards solutions.  Many problems stem from 5 major causes: protein binding, non-specific binding, solubility, specificity and stability.  If you are aware of what the challe

  56. Slide 56: Acknowledgements

    Acknowledgements Waters Corporation ICON PLC  Erin Chambers  Jon Bardsley  Debadeep Battacharya  Eileen Collins  Catalin Doneanu  Sally Hannam  Kenneth Fountain  Liz Thomas  Gordon Fujimoto  Joanne Mather  Paul Rainville  Martha Stapels  Hua Yang ©2012 Waters Corporation 56

  57. Slide 57: Thank You! Questions?

    Thank You!  Questions?  Landing Page…  http://www.waters.com/May1 – Promotional Offers on Products for Peptide Separation and Sample Preparation – Full Webinar Recording of Today’s Session – PDF Slide Deck – Compilation of Literature, White Papers, Brochures, Application Notes  General Questions

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