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Life Cycle Management of Chromatographic Methods for Biopharmaceuticals

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The development and manufacture of biopharmaceuticals is a dynamic and rapidly growing industry. By the nature of their production, biopharmaceuticals are highly complex heterogeneous mixtures that require many analytical techniques for characterization and routine testing. As a result, many manufacturers incorporate life cycle management into their respective workflows to take advantage of newer technologies and methodologies to ensure efficacy and patient safety.

In this presentation, we will address the range of chromatographic categories – HPLC, UHPLC, and UPLC – and define the characteristics associated with each. The discussion will continue with several examples of methods transferred from legacy HPLC instrumentation to modern UHPLC and UPLC instruments. We will compare qualitative and quantitative data across each chromatographic class. Resolution, sensitivity, and overall run time will be used as metrics to assess the success of the method transfer to the respective LC platform, to ensure the transferred methods are in line with current acceptance criteria.

Learn:
- The importance of selecting the correct instrumentation to meet user needs.
- Which parameters influence method transfer from one LC platform to another.
- How workflows can benefit from features such as Multi-flow path technology and Gradient SmartStart when transitioning methods.

Interested in more detail? Watch the related on-demand webinar: http://view6.workcast.net/register?pak=3479247014905635

Published in: Science

Life Cycle Management of Chromatographic Methods for Biopharmaceuticals

  1. 1. ©2015 Waters Corporation 1 Brooke Koshel, Ph.D. Senior Scientist Waters Corporation Life Cycle Management of Chromatographic Methods for Biopharmaceuticals
  2. 2. ©2015 Waters Corporation 2 Outline  Global trend towards new technologies through life cycle management  Defining LC platforms for life cycle management of analytical procedures  Life cycle management of analytical procedures using UHPLC and UPLC systems – Method transfer of existing HPLC methods on the ACQUITY ARC UHPLC System – Method transfer of existing HPLC methods on the ACQUITY UPLC H-Class Bio System
  3. 3. ©2015 Waters Corporation 3 Current Global Environment Biopharmaceutical Industry  Highly competitive, regulated business environment – Lower costs without compromising product quality – Maintain regulatory and compliance requirements  Challenges to increase profitability: – Increasing regulatory pressures – Increased quality expectations and competitive pressures – Need for Efficiency : “Lean” laboratory operation  Need to deliver a sustainable competitive advantage – Invest in core competencies – Invest in technologies to achieve business objectives  Manufacturers are incorporating Life Cycle Management – Take advantage of newer technology and methodologies for increased ROI and quality standards
  4. 4. ©2015 Waters Corporation 4 ICH Q10 Incorporates Life Cycle Management into the Quality System ICH Q10 definition of Innovation : “The introduction of new technologies or methodologies”
  5. 5. ©2015 Waters Corporation 5 ICH Q6B Recommends New Technologies for Biologics
  6. 6. ©2015 Waters Corporation 6 US FDA Emphasis on New Technologies http://www.fda.gov/downloads/drugs/guidancecomplianceregula toryinformation/guidances/ucm386366.pdf Over the life cycle of a product, new information (e.g., a better understanding of product CQAs or awareness of a new impurity) may warrant the development and validation of a new or alternative analytical method. “New technologies may allow for greater understanding and/or confidence when ensuring product quality. Applicants should periodically evaluate the appropriateness of a product’s analytical methods and consider new or alternative methods.” VIII. LIFE CYCLE MANAGEMENT OF ANALYTICAL PROCEDURES
  7. 7. ©2015 Waters Corporation 7 Life Cycle Management of Analytical Procedures with New Technologies  New technologies allow for greater understanding when ensuring product quality. – Reduces exposure to unnecessary compliance risk – Reduces validation costs – Patients get quicker and safer access to drugs  Gives regulators the confidence that industry can be responsible for greater self-management of improvements and changes – Companies with good quality management systems – Well controlled processes and products – Appropriate technologies are being used for product safety  Increases ROI by decreasing equipment down time, overheads, solvent usage, etc. Proper management of the analytical equipment lifecycle is required to meet business and regulatory requirements
  8. 8. ©2015 Waters Corporation 8 Considerations for Life Cycle Management of LC-Technology Performance Confidence in technology Ease of Use Reduces training Avoidance of Human Error Method Transfer Ease of transfer Current vs. new methods Revalidation/refiling Flexibility Interface with other labs CRO/CMO/R&D, Dev./QC etc. Future-Proofing Incorporate technology advances while maintaining workflow Robustness Long term reliability of methods Informatics Ease of data processing Degree of compliance
  9. 9. ©2015 Waters Corporation 9 Outline  Global trend towards new technologies through life cycle management  Defining LC platforms for life cycle management of analytical procedures  Life cycle management of analytical procedures using UHPLC and UPLC systems – Method transfer of existing HPLC methods on the ACQUITY ARC UHPLC System – Method transfer of existing HPLC methods on the ACQUITY UPLC H-Class Bio System
  10. 10. ©2015 Waters Corporation 10 Platforms for Life Cycle Management LC Separation Categories ACQUITY ArcAlliance® HPLC ACQUITY UPLC H-Class Bio Chromatographic Resolution Increases Overall Run Time Decreases Method Sensitivity Increases
  11. 11. ©2015 Waters Corporation 11 Platforms for Life Cycle Management LC Separation Categories Chromatographic Resolution Increases Overall Run Time Decreases Method Sensitivity Increases AU(x10-3) -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Retention Time (min) 15 20 25 30 AU(x10-3) 0.0 1.0 2.0 3.0 4.0 5.0 1.0 1.5 2.0 2.5 Retention Time (min) HPLC UPLC * *
  12. 12. ©2015 Waters Corporation 12 What is at the Root of the Performance Differences Across the LC Categories?  Dispersion – n. Broadening of an analyte band due to both on-column effects (diffusion and mass transfer kinetics which are both dependent on particle size and linear velocity) and system effects (tubing internal diameter (I.D.) and length, connections, detector flow cell volumes, etc.)  True separation performance is governed by the system dispersion paired with a flow rate range that yields the highest possible efficiency for a given analytical column Read more at : https://www.waters.com/waters/Chromatographic-Bands,-Peaks-and-Band- Spreading/nav.htm?cid=134803614
  13. 13. ©2015 Waters Corporation 13 Defining the LC Categories: Power Range vs. Dispersion  LC systems trying to cover a wide “power range” (flow rate / pressure envelope) end up compromising extra-column dispersion, and therefore performance, in efforts to accommodate both sub 2 µm and traditional column technologies.  Flow rate range and available pressure alone have little bearing on the actual separation power of the system and do not provide an appropriate measurement of system performance. The difference between UHPLC and UPLC True separation performance is governed by system dispersion mAU 0.00 200.00 400.00 AU 0.00 0.20 0.40 Minutes 1.10 1.20 1.30 1.40 1.50 1.60 Rs = 0.52 Rs = 1.53 Rs = 1.44 Rs = 2.84 Vendor A UHPLC with Higher ‘Power Range’ ACQUITY UPLC H-Class with Lower ‘Power Range’
  14. 14. ©2015 Waters Corporation 14 Defining the LC Categories by Dispersion Dispersion > 30 µL Columns accepted: • 3.0 – 4.6 mm ID • 3 - 10 µm particles Optimal: • 4.6 mm ID, 5 µm Typical operating pressure: • < 6,000 PSI Dispersion 12 - 30 µL Columns accepted: • 2.1 - 4.6 mm ID • 1.7 - 5 µm particles Optimal: • 3.0 mm ID, 2.x µm Typical operating pressure: • 6,000 – 15,000 PSI Dispersion < 12 µL Columns accepted: • 1.0 - 4.6 mm ID • 1.6 - 5 µm particles Optimal column: • 2.1 mm ID, 1.7 µm Typical operating pressure: • 9,000 – 15,000 PSI HPLC UHPLC UPLC Alliance HPLC 34 μL Shimadzu Prominence 35 μL ACQUITY Arc 25 µL Agilent 1260 SL 25 µL ACQUITY UPLC 10 μL H-Class CH-A 7 μL I-Class CH-A 5 μL All dispersion values measured at 5 σ
  15. 15. ©2015 Waters Corporation 15 Bridging The Performance Gap Between HPLC and UPLC Technology HPLC UPLC UHPLC Extends the ACQUITY family into laboratories requiring method compatibility with HPLC and UHPLC (2.x µm) separations Reasons For Slow Adoption •Not yet evaluated UPLC technology •Do not require a UPLC level of performance •Budget •Training
  16. 16. ©2015 Waters Corporation 16 Outline  Global trend towards new technologies through life cycle management  Defining LC platforms for life cycle management of analytical procedures  Life cycle management of analytical procedures using UHPLC and UPLC systems – Method transfer of existing HPLC methods on the ACQUITY ARC UHPLC System – Method transfer of existing HPLC methods on the ACQUITY UPLC H-Class Bio System
  17. 17. ©2015 Waters Corporation 17 Lifecycle Management of an Analytical Procedure using UHPLC HPLC UHPLC HPLC Methods or Updated UHPLC Methods Isocratic Methods OR Gradient Methods
  18. 18. ©2015 Waters Corporation 18 ACQUITY Arc System Comprehensive detector portfolio -UV/Vis -Photodiode Array -Fluorescence -Refractive Index -Evaporative Light Scattering -Mass Detection Negligible carryover Flow-through-needle design with user definable wash settings Thermal management options -Heating or heating/cooling -Supports columns up to 300 mm -Optional column switching Auto•Blend Plus™ Technology Automated online solvent blending at specific pH and ionic strength that supports reversed phase, SEC, and IEX Quaternary solvent management Precise and accurate blending of up to 4 solvents with automated solvent compressibility. Optional 6 solvent select valve expands flexibility Gradient SmartStart Counteract system dwell volume differences without altering the gradient table. Minimize cycle times by managing gradient start and pre- injection steps in parallel Arc Multi-flow path™ Technology Plug-and-play method compatibility with HPLC or UHPLC. Replicate methods by selecting the most appropriate flow path.
  19. 19. ©2015 Waters Corporation 19 Transfer HPLC Method to ACQUITY Arc Arc Multi-flow path Technology For UHPLC Separations Lower System Volume To injector and column For HPLC Separations Higher System Volume Choose the path that best fits your application Path 1: 1100uL Path 2: 700uL
  20. 20. ©2015 Waters Corporation 20 ACQUITY Arc System: Quaternary Solvent Manager-R Pressure transducers Gradient proportioning valve Solvent degasser Passive check valves Arc Multi-flow path Technology Seal wash pump Optional solvent select valve
  21. 21. ©2015 Waters Corporation 21 Transfer HPLC Method to H-Class Transferring Isocratic Methods From HPLC to UHPLC Application Example Size Exclusion Chromatography (SEC) Scenarios Method Equivalency Method Improvement
  22. 22. ©2015 Waters Corporation 22 Transfer HPLC Method to ACQUITY Arc Isocratic: SEC Instrument HPLC (Quaternary) UHPLC ACQUITY Arc (Quaternary) Column Chemistry TOSOH Biosciences G3000SWXL, 5 um (250 Å pore size) No Change Dimensions 7.8 mm ID x 300 mm No Change Mobile Phase 0.02 M sodium phosphate, 0.3 M sodium chloride, pH 6.8 No Change Flow Rate 500 ml min-1 No Change Temperature 30 oC No Change Injection Volume 30 ml No Change Run Time 35 min No Change CDS Empower® Empower Sample: Rituximab
  23. 23. ©2015 Waters Corporation 23 AU 0.00 0.10 0.20 0.30 AU 0.00 0.10 0.20 0.30 Retention Time (min) 15 30 Sample: Rituximab Acquity Waters Waters Waters Acquity Acquity Waters System Mode Rs Relative Peak Area (%) HMW Species Monomer x̅ σ x̅ σ Arc HPLC 1.5 1.29 0.01 98.65 0.01 Agilent 1100 HPLC 1.5 1.28 0.01 98.63 0.01 HMWPeak Dimer Monomer AU -0.001 0.000 0.001 0.002 0.003 0.004 Minutes 10.00 15.00 20.00 25.00 30.00 AU -0.001 0.000 0.001 0.002 0.003 0.004 Minutes 10.00 15.00 20.00 25.00 30.00 HMWPeak Dimer Monomer Transfer HPLC Method to ACQUITY Arc Isocratic: SEC
  24. 24. ©2015 Waters Corporation 24 Injection Area HMW Species 1 80304 2 81069 3 81890 4 81153 5 82012 Mean 81285.6 Std. Dev. 693.00 %RSD 0.85 Inj Rs Monomer- Dimer 1 1.51 2 1.54 3 1.52 4 1.58 5 1.55 AU -0.001 0.000 0.001 0.002 0.003 0.004 Minutes 10.00 15.00 20.00 25.00 30.00 5 Injections AU 0.00 0.05 0.10 0.15 0.20 Retention Time (min) 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 HMWPeak Dimer Monomer Transfer HPLC Method to ACQUITY Arc Isocratic: SEC
  25. 25. ©2015 Waters Corporation 25 Transfer HPLC Method to H-Class Transferring Isocratic Methods From HPLC to UHPLC Application Example Size Exclusion Chromatography (SEC) Scenarios Method Equivalency Method Improvement
  26. 26. ©2015 Waters Corporation 26 Update HPLC Method to UHPLC Improvements in Separations Dimer Monomer Method Scaled for new column dimensions AU -0.001 0.000 0.001 0.002 0.003 0.004 Minutes 6.00 8.00 10.00 12.00 14.00 AU -0.001 0.000 0.001 0.002 0.003 0.004 Minutes 12.00 15.00 18.00 21.00 HPLC UHPLC Resolution 95.0 95.5 96.0 96.5 97.0 97.5 98.0 98.5 99.0 99.5 100.0 Arc (HPLC) Agilent 1100 Arc (UHPLC) PeakArea(%) 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 Arc (HPLC) Agilent 1100 Arc (UHPLC) PeakArea(%) 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 Arc (HPLC) Agilent 1100 Arc (UHPLC) Sample: Rituximab Column TOSOH Biosciences G3000SWXL 7.8 mm ID x 300 mm Column Waters XBridge® Protein BEH 7.8 mm ID x 300 mm 3.5 mm5 mm
  27. 27. ©2015 Waters Corporation 27 Transfer HPLC Method to H-Class Transferring Gradient Methods From HPLC to UHPLC Application Examples Example 1: Ion Exchange Chromatography(IEC) Example 2: Peptide Mapping Scenarios Method Equivalency Method Improvement
  28. 28. ©2015 Waters Corporation 28 Transfer HPLC Method to ACQUITY Arc Gradient: Ion Exchange Instrument HPLC (Quaternary) UHPLC ACQUITY Arc (Quaternary) Column Chemistry Dionex ProPac WCX-10, 10 um No Change Dimensions 4 mm ID x 250 mm No Change Mobile Phase 0.2 M MES in water, pH 6.0 0.02 MES 0.4 M NaCl in water, pH 6.0 No Change Flow Rate 700 ml min-1 No Change Temperature 30 ºC No Change Run Time 115 min No Change Injection Volume 40 ml No Change CDS Empower Empower Sample: Rituximab Arc Multi-flow path Technology: HPLC Flow Path 1
  29. 29. ©2015 Waters Corporation 29 Arc Multi-flow path Technology  Flow Path 1: For HPLC Separation (Larger Dwell Volume) – Selectable dwell volume that emulates both system volume and mixing behavior  Does not impact the gradient table – Falls within USP <621> guidelines on transferring gradient methods between different chromatographic systems System Emulation with Arc Multi-flow path Technology Select Path 1
  30. 30. ©2015 Waters Corporation 30 AU 0.000 0.002 0.004 0.006 0.008 0.010 AU 0.000 0.002 0.004 0.006 0.008 0.010 Retention Time (min) 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 Acquity Waters Waters Waters Acquity Acquity Waters Transfer HPLC Method to ACQUITY Arc Gradient: Ion Exchange System Mode Rs Relative Peak Area (%) K1 K0 x̅ σ x̅ σ Arc HPLC 2.1 4.78 0.04 70.30 0.11 Agilent 1100 HPLC 1.8 5.14 0.05 69.67 0.28 Sample: Rituximab K1 K0 K1 K0
  31. 31. ©2015 Waters Corporation 31 Transfer HPLC Method to ACQUITY Arc Ion Exchange: High repeatability of results K0 AU 0.00 0.05 0.10 Retention Time (min) 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 6 Injections AU -0.0025 0.0000 0.0025 0.0050 0.0075 0.0100 Minutes 30.00 40.00 50.00 60.00 Inj Rs K0-K1 1 2.11 2 2.10 3 2.08 4 2.11 5 2.15 6 2.14 Injection Area K1 1 269505 2 272294 3 267541 4 266328 5 268459 6 265468 Mean 268265.8 Std. Dev. 2445.71 %RSD 0.91 K1 Sample: Rituximab K1 K0
  32. 32. ©2015 Waters Corporation 32 Transfer HPLC Method to H-Class Transferring Gradient Methods From HPLC to UHPLC Application Examples Ion Exchange Chromatography(IEC) Peptide Mapping Scenarios Method Equivalency Method Improvement
  33. 33. ©2015 Waters Corporation 33 Transfer HPLC Method to ACQUITY Arc Gradient: Peptide Mapping Instrument HPLC (Quaternary) UHPLC ACQUITY Arc (Quaternary) Column Chemistry XBridge BEH C18 130Å, 3.5 mm No Change Dimensions 4.6 mm x 100 mm No Change Mobile Phase H2O with 0.1% (v/v) TFA Acetonitrile with 0.1% (v/v) TFA No Change Flow Rate 500 ml min-1 No Change Temperature 40 ºC No Change Run Time 60 min No Change Injection Volume 75 ml No Change CDS Empower Empower Sample: Waters MassPREP™ Peptide Mixture (Infliximab) Arc Multi-flow path Technology: HPLC Flow Path 1 with Gradient Offset
  34. 34. ©2015 Waters Corporation 34 Transfer HPLC Method to ACQUITY Arc Gradient: Peptide Mapping Retention Time (min) 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 No Gradient Offset ACQUITY Arc Agilent 1100 Series Sample: Waters MassPREP™ Peptide Mixture Dwell volume differences between instruments results in retention time differences
  35. 35. ©2015 Waters Corporation 35 Arc Multi-flow path Technology  Flow Path 1: for HPLC Separations (Larger dwell volume)  Compensates for transferring methods from LC systems with variable volume  Adjust when the gradient starts relative to the injection sequence  No impact to gradient table System Emulation with Arc Multi-flow path Technology and Gradient SmartStart Select Path 1 Gradient SmartStart
  36. 36. ©2015 Waters Corporation 36 Accounting for Dwell Volume  Gradient dwell volume VD is the total volume of the system from where the mobile phase mixing occurs to the analytical column  Differences in dwell volume can lead to retention, selectivity and resolution differences t1/2 (1) t1/2 (2) 50% 100% tG UHPLC HPLC 𝑡 𝐷 = 𝑡1/2 − 1 2 𝑡 𝐺 𝑉𝐷 = 𝑡 𝐷 𝐹 Programmed gradient Gradient delay UHPLC Gradient delay HPLC
  37. 37. ©2015 Waters Corporation 37 Accounting for Dwell Volume Gradient SmartStart ACQUITY UPLC Quaternary Solvent Manager Gradient Start : “After injection” OR, enter volume Differences
  38. 38. ©2015 Waters Corporation 38 Transfer HPLC Method to ACQUITY Arc Gradient offset aligns chromatograms Retention Time (min) 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 No Gradient Offset ACQUITY Arc Agilent 1100 Series Retention Time (min) 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Agilent 1100 Series ACQUITY Arc Programmed Gradient Offset Sample: Waters MassPREP Peptide Mixture
  39. 39. ©2015 Waters Corporation 39 AU 0.00 0.20 0.40 AU 0.00 0.20 0.40 Retention Time (min) 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 AcquityWaters Waters Waters Acquity Acquity Waters Transfer HPLC Method to ACQUITY Arc Gradient: Peptide Mapping Sample: Infliximab
  40. 40. ©2015 Waters Corporation 40 Transfer HPLC Method to H-Class Transferring Gradient Methods From HPLC to UHPLC Application Examples Ion Exchange Chromatography(IEC) Peptide Mapping Scenarios Method Equivalency Method Improvement
  41. 41. ©2015 Waters Corporation 41 Update HPLC Method to UHPLC Gradient: Peptide Mapping Instrument HPLC (Quaternary) UHPLC ACQUITY Arc (Quaternary) Column Chemistry XBridge BEH C18 130 Å, 3.5 mm XBridge BEH C18 130 Å, 2.5 mm Dimensions 4.6 mm x 100 mm No Change Mobile Phase H2O with 0.1% (v/v) TFA Acetonitrile with 0.1% (v/v) TFA No Change Flow Rate 500 ml min-1 No Change Temperature 40 ºC No Change Run Time 60 min No Change Injection Volume 75 ml No Change CDS Empower Empower Sample: Infliximab Arc Multi-flow path Technology: UHPLC Flow Path 2
  42. 42. ©2015 Waters Corporation 42 Arc Multi-flow path Technology Flow Path 2 For UHPLC Separations (Lower Dwell volume) Update HPLC Method to UHPLC UHPLC Flow Path 2 Select Path 2
  43. 43. ©2015 Waters Corporation 43 ACQUITY QDa® Mass Detector for Mass Confirmation 2998 PDA New Low dispersion analytical flow cell 2489 UV/Vis New Low dispersion analytical flow cell 2414 RI 2475 FLR New Low dispersion analytical flow cell 2424 ELS ACQUITY QDa Additional High Performance Detection Options
  44. 44. ©2015 Waters Corporation 44 Sample: Tryptic digest of Infliximab Update HPLC Method to UHPLC Peptide Mapping: Improvements in Separations AU 0.00 0.10 0.20 0.30 AU 0.00 0.10 0.20 0.30 0.40 0.50 Intensity(x10)6 1.0 2.0 3.0 4.0 5.0 6.0 Retention Time (min) 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 Agilent 1100 Series HPLC System 3.5 µm ACQUITY Arc System, UV/Vis 2.5 µm ACQUITY Arc System, QDa 2.5 µm
  45. 45. ©2015 Waters Corporation 45  UHPLC separations enables better chromatographic resolution of peptide variants over HPLC  ACQUITY QDa provides the specificity and sensitivity for relative quantification of peptides  Mass confirmation for increased confidence 5.7% 94.3%SIR TUV TIC AU 0.05 0.10 0.15 0.20 0.25 Intensity 1x106 2x106 3x106 4x106 5x106 6x106 7x106 8x106 Intensity 0.0 5.0x105 1.0x106 1.5x106 2.0x106 Retention Time (min) 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 Sample: Tryptic digest of Infliximab Native Peptide Oxidized Peptide Native Oxidized Update HPLC Method to UHPLC Quantification using ACQUITY QDa
  46. 46. ©2015 Waters Corporation 46 HPLC or UHPLC Methods on UHPLC Platform Benefits: – Replicate established HPLC assays without compromise o System-to-system transfer o “Method Transfer” – Improve productivity with modern UHPLC column technology o “Method Improvement” – Increase confidence with ACQUITY QDa mass detection HPLC Methods or Improved UHPLC Methods Replicate HPLC assays or improve methods regardless of the LC platform used for method development
  47. 47. ©2015 Waters Corporation 47 Outline  Global trend towards new technologies through life cycle management  Defining LC platforms for life cycle management of analytical procedures  Life cycle management of analytical procedures using UHPLC and UPLC systems – Method transfer of existing HPLC methods on the ACQUITY ARC UHPLC System – Method transfer of existing HPLC methods on the ACQUITY UPLC H-Class Bio System
  48. 48. ©2015 Waters Corporation 48 Lifecycle Management of an Analytical Procedure using UPLC HPLC UPLC ACQUITY UPLC H-CLASS Bio HPLC Methods or Updated UPLC Methods Isocratic Methods OR Gradient Methods
  49. 49. ©2015 Waters Corporation 49 ACQUITY UPLC H-CLASS BIO: HPLC Simplicity UPLC Performance Reproduce established HPLC &UHPLC methods Enables seamless transfer to UPLC The system of choice for method development Flexibility for HPLC , UHPLC & UPLC Methods Biocompatible system for analysis in high salt mobile phases The system of choice for method Transfer Low dispersion True UPLC performance with band spread of less than 10μL for highest chromatographic resolution Auto-Blend Plus Automated online solvent blending at specific pH and ionic strength that supports reversed phase, SEC, and IEX
  50. 50. ©2015 Waters Corporation 50 Transfer HPLC Method to H-Class Transferring Isocratic Methods From HPLC to UPLC Application Example Size Exclusion Chromatography (SEC) Scenarios Method Equivalency Method Improvement ACQUITY UPLC H-CLASS Bio
  51. 51. ©2015 Waters Corporation 51 Transfer HPLC Method to H-Class Isocratic: SEC Instrument HPLC (Quaternary) ACQUITY UPLC H-Class Bio (Quaternary) Column Chemistry Waters BioSuite™ SEC 250Å, 10 mm No Change Dimensions 7.5 mm x 300 mm No Change Mobile Phase 20 mM Phosphate, 200 mM NaCl, pH 6.8 No Change Flow Rate 400 ml min-1 No Change Temperature Ambient No Change Injection Volume 20 ml No Change Run Time 35 min No Change CDS Empower Empower Sample: Infliximab
  52. 52. ©2015 Waters Corporation 52 AU 0.00 0.05 0.10 0.15 0.20 AU 0.00 0.10 0.20 0.30 0.40 Retention Time (min) 10 20 30 AU(x10-3) -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 AU(x10-3) -1.0 0.0 1.0 2.0 3.0 4.0 Retention Time (min) 15 20 25 30 Retention Time (min) 15 20 25 30 Transfer HPLC Method to H-Class Resolution Maintained Sample: Infliximab System Mode Rs Relative Peak Area (%) Dimer Monomer x̅ σ x̅ σ HPLC HPLC 1.72 0.48 0.01 99.5 0.01 H-Class Bio HPLC 1.77 0.47 0.00 99.5 0.00
  53. 53. ©2015 Waters Corporation 53 Transfer HPLC Method to H-Class Transferring Isocratic Methods From HPLC to UPLC Application Examples Size Exclusion Chromatography (SEC) Scenarios Method Equivalency Method Improvement ACQUITY UPLC H-CLASS Bio
  54. 54. ©2015 Waters Corporation 54 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Rs(m,d) 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 PeakArea(%) 99.0 99.1 99.2 99.3 99.4 99.5 99.6 99.7 99.8 99.9 100.0 PeakArea(%) Update HPLC Method to UPLC Improvements in Separations AU(x10-3) -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Retention Time (min) 15 20 25 30 AU(x10-3) 0.0 1.0 2.0 3.0 4.0 5.0 1.0 1.5 2.0 2.5 Retention Time (min) HPLC UPLC RsDimer Monomer * * * Increased resolution of low level clipsMethod Scaled for new column dimensions Column ACQUITY UPLC Protein BEH SEC 200Å 4.6 mm x 150 mm Column Waters BioSuite SEC 250Å 7.5 mm x 300 mm 1.7 mm10 mm
  55. 55. ©2015 Waters Corporation 55 Transfer HPLC Method to H-Class Transferring Gradient Methods From HPLC to UPLC Application Example Peptide Mapping Scenarios Method Equivalency ACQUITY UPLC H-CLASS Bio
  56. 56. ©2015 Waters Corporation 56 Transfer HPLC Method to H-Class Gradient: Peptide Mapping Instrument HPLC (Quaternary) ACQUITY UPLC H-Class Bio (Quaternary) Column Chemistry XBridge BEH C18 130Å, 3.5 mm No Change Dimensions 4.6 mm x 100 mm No Change Mobile Phase H2O with 0.1% (v/v) TFA Acetonitrile with 0.1% (v/v) TFA No Change Flow Rate 500 ml min-1 No Change Temperature 40 ºC No Change Run Time 60 min No Change Injection Volume 20 ml No Change CDS Empower Empower
  57. 57. ©2015 Waters Corporation 57 VD1 – VD2 (360 ml) Accounting for Dwell Volume Gradient SmartStart ACQUITY Quaternary Solvent Manager Gradient Start : “After injection” Enter volumetric Differences
  58. 58. ©2015 Waters Corporation 58 Transfer HPLC Method to H-Class Gradient offset aligns chromatograms * Retention Time (min) 10 15 20 25 30 35 40 45 Retention Time (min) 10 15 20 25 30 35 40 45 No Gradient Offset HPLC H-Class Bio Sample: Waters MassPREP Peptide Mixture * Programmed Gradient Offset HPLC H-Class Bio * is a system peak observed on the instrument
  59. 59. ©2015 Waters Corporation 59 0.5 1.0 1.5 2.0 2.5 3.0 1 5 9 14 18 22 26 30 34 39 44 48 52 63 RRT Peak Number Transfer HPLC Method to H-Class Gradient: Peptide Mapping A total of 56 peptide peaks were selected for monitoring 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1819 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 AU 0.00 0.05 0.10 0.15 AU 0.02 0.04 0.06 0.08 0.10 0.12 Retention Time (min) 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 1 2 3 4 5 6 7 8 9 10 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 3738 39 40 41 42 43 44 4546 47 48 49 50 51 52 53 54 55 56 11 12 Sample: Infliximab H-Class Bio HPLC
  60. 60. ©2015 Waters Corporation 60 HPLC or UPLC methods on UPLC Platform Benefits:  Flexibility – Run both legacy HPLC methods and UPLC methods  Future-proofing your laboratory – Taking advantage of sub 2 µm particle technology for separation efficiency  Improve critical performance parameters – Resolution and sensitivity – Reduce analysis time ACQUITY UPLC H-CLASS Bio HPLC Methods or Improved UPLC Methods
  61. 61. ©2015 Waters Corporation 61 Summary  Global trend towards new technologies through life cycle management – Driven by industry and regulators – Increased ROI and quality standards  Three tiers of LC categories : HPLC, UHPLC, UPLC – Increased resolution, sensitivity and reduced run time – Dispersion (not pressure/flow envelope )characteristics of instruments govern performance  UPLC provides largest scientific and business benefits  UPLC and UHPLC can be seamlessly incorporated into life cycle management strategy  Waters has solutions at every LC category for reliable robust and reproducible solutions Versatility without Compromise Performance that enhances your laboratory Dependability HPLC Methods HPLC/UHPLC Methods HPLC/UHPLC/UPLC Methods
  62. 62. ©2015 Waters Corporation 62 ACQUITY ARC System www.waters.com/arc
  63. 63. ©2015 Waters Corporation 63

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