Development of Pfizer's Third Generation Turbidimetric Solubility Assay - An Engineering Perspective
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Development of Pfizer's Third Generation Turbidimetric Solubility Assay - An Engineering Perspective

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Development of Pfizer\'s Third Generation Turbidimetric Solubility Assay - An Engineering Perspective Presented at ISLAR 2002

Development of Pfizer\'s Third Generation Turbidimetric Solubility Assay - An Engineering Perspective Presented at ISLAR 2002

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  • 1. Development of Pfizer's Third Generation Turbidimetric Solubility Assay - An Engineering Perspective Brian Bissett Molecular Properties Group Pfizer Global R&D Groton CT
  • 2. Presentation Overview
    • History
    • New Assay Overview
    • Engineering: Design Considerations and Obstacles
    • Testing Performance and Repeatability
    • Project Management Considerations
  • 3.
    • Purpose: The turbidimetric solubility assay mimics typical biology lab solubilization procedures for an in vivo experiment and thus is relevant to early discovery stage absorption problems.
    • Turbidimetric solubilities are most useful in the very early stages of discovery. Their value declines as in vivo activity is detected and as more manning intensive, more accurate drug metabolism and pharm R&D resources are invoked.
    Why do we need solubility data anyway?
  • 4. History of Turbidimetric Assays at Pfizer
    • This first “primitive” assay could only run 6 compounds at a time!
  • 5. History of Turbidimetric Assays at Pfizer
    • G2483B – The first “high throughput” solubility assay at Pfizer.
  • 6. Desired Improvements over existing 2483B Assay
    • Septum Piercing Needle Delivery of Compounds in DMSO (Safety Issue) .
    • Automated Data Reporting (Oracle Database Upload). Previous Assay Required Manually Run Macro.
    • Prior Assay could run only one Plate of 45 compounds at a time, new assay can run up to 4 Plates of 90 compounds or 360 Consecutive Solubilities.
    • Automated Generation of Files for High Dose Platereader Solubility Assay. Previous Assay required a separate program run after assay completion to generate these files.
    • Total Automation - Place compounds on rack, set up software and walk away.
  • 7. History of Turbidimetric Assays at Pfizer G5143A
  • 8. What advantages does this new Assay have?
    • It is Comparable to a Commercial Unit.
    • Increased Capacity (4X).
    • Increased Sensitivity.
    • Increased Safety.
    • Increased Reliability.
    • Reduced Operator Time.
  • 9. System Model of Turbidimetric Solubility Assay
  • 10. Experimental Differences with Previous Assay
    • Custom Designed Cuvette (Design done internally) Fabricated by Starna Cells, Inc.
    • Custom Designed Septum Piercing Needle (Design done internally) Fabricated by Popper & Sons, Inc.
    • Compound Added Directly to Cuvette. No Secondary Mixing Chamber.
    • Mixing done within the Cuvette via Microstirbar.
    • Longer Equilibration Time 90 sec. vs. 60 sec.
    • Rinse Solvent Changed to DMSO vs. MeOH.
    • Multiple Rinse Methods Dependent Upon Baseline:
      • Simple DMSO
      • DMSO and Glass Cleaner with EDTA
      • Wash Cycle, Multiple DMSO & Glass Cleaner Rinses
  • 11. Experimental Differences with Previous Assay
    • Off Axis & Forward Detector Blocked on original G2483B Assay.
  • 12. Software Interface for Assay
  • 13. Hach Controller Software GUI
  • 14. Obstacle: Designing the Septum Piercing Needle
    • Design Requirements:
    • Must be Septum Piercing
    • Coefficients of Variation < 5.0% between successive additions.
    • Ideally all the compound to be utilized in the assay should be contained within the needle as opposed to being drawn into the supporting tubing.
  • 15. Calculating the volume capacity of a Needle
    • Assume Needle is a right circular cylinder of height h and radius r .
    Example: Needle diameter = 150 microns and height = 100mm.
  • 16. Obstacle: Designing the Cuvette Holder
    • Hach Designed for Round Cell
    • Light Ports Must Minimize Noise Created By Backscatter from Square Cell Edges.
    • Design Requirements:
    • Must have Septum Piercing Top
    • Dual Ports for Solvent/Buffer Addition
    • Micro Magnetic Stirrer for Cuvette
    • Vent near top of cuvette to equilibrate pressure from evacuation and addition of liquids.
  • 17. Obstacle: Controlling the Hach 2100N Turbidimeter
    • The Hach Turbidimeter was designed to be operated by hand. A means had to be devised to remotely control the machine by computer.
    • Signals are taken directly from the Hach’s internal Circuit Boards and routed to a 50 pin Connector.
    • Signals Pass out the connector to a ribbon cable and are connected to the Interface and Relay Boards.
    • 32 Channel Relay board is used to simulate Manual Keypresses on the Hach, automating tasks intended to be done by hand.
  • 18. Obstacle: Controlling the Hach 2100N Turbidimeter
    • Design Requirements:
    • Complete Computer Control of Hach 2100N Turbidimeter by Software, a machine designed to be Operated only by Hand.
    • Interface Board with 40 Operational Amplifiers Filters Signals from Hach Turbidimeter and Translates them into TTL digital logic signals which are read by a Data Acquisition Board.
    • Interface Board allows for Error and State Checking via Software, something not possible with the original unit.
  • 19. Obstacle: Keeping the Cuvette Clean
    • Previous Assay had Separate Mixing Chamber to which Compound was Added. Compound mixed in buffer would then be pumped into Hach Chamber for Reading.
    • Tars and “Gummy” Compounds would stick to the Mixing Chamber but not be pumped into the Cell in the Hach for Reading. Result, Hach cell rarely got dirty but accuracy on some measurements suffered.
    • New Assay Delivers Compounds directly to the Cell in the Hach Turbidimeter. But the Cell gets Dirty more often. Solution: More Aggressive Cleaning Methods ……….
  • 20. Obstacle: Keeping the Cuvette Clean
    • Solution:
    • Replace MeOH Rinse with DMSO, DMSO Rinse works 85% of the Time.
    • Additional Solvent used for “difficult” compounds (tars & gums). Tried several formulations including DMA, Acetone, and other acids and bases. Cheap Glass Cleaner was found to be the most effective.
    • Wash Cycle which mimics a washing machine is used for compounds which leave residues difficult to remove. Wash cycle utilizes both Solvents and lasts approximately 20 minutes.
    © 1999 Procter & Gamble
  • 21. Just how effective is this cleaning method ? When the Baseline “creeps” upward the wash cycles beat it back down preserving sensitivity.
  • 22. Obstacle: Effective Mixing Within the Cuvette 1 2 3
  • 23. Obstacle: Termination of Cable to Syringe Pumps
    • Problem: Line which carries signals to Syringe Pumps is long enough that reflections occur.
    • Signals take time to propagate.
    • Propagating Signals have energy.
    • All Wires have an inherent resistance, inductance and capacitance. Such parameters form the “characteristic impedance” (Z 0 ) of the Transmission line.
    • If a line is short enough, the inherent resistance, inductance, and capacitance is small enough to be ignored.
    • Ideally, the Load attached to the end of the line must match the “characteristic impedance” (Z 0 ) of the Transmission line, in which case the line is said to be “matched”. Matching is accomplished through “termination”, which is adding a load which may be composed of inductors, capacitors, and resistors.
  • 24. Obstacle: Reflections on communication lines.
    • Without proper termination, energy reaching the end of a transmission line has nowhere to go, so it reflects back to the source. Just as a rubber ball will bounce off a solid surface.
  • 25. Reflections can Imitate Legitimate Control Signals
  • 26. Solution: Termination of Cable to Syringe Pumps
    • Solution: “match” the line to the load so that reflections do not occur. RC termination was determined to be the best solution.
    RC Network will act as LPF
  • 27. Obstacle: Valve Problems
    • Defective Valves Received from General Valve Corporation, pull in Voltage exceeded that of ability of Driver to Actuate the Valve.
    • Prior Assay Drove Valves with +24VDC. Valves ran hot reducing their life and possibly having unknown thermal effects on the liquids that passed through them.
    • Valves on New Assay Driven with Specified Valve Driver which Plugs into Custom Designed Board. Each Board contains 4 Valve Drivers which are independently fused. Valve Drivers can easily be replaced in the event of Failure by simply plugging in a new module.
  • 28. Obstacle: Firmware Incompatibility
    • Kloehn Syringes used in Assays because they provide twice the precision as Cavro Syringe Pumps (48,000 steps vs. 24,000 steps).
    • But Tecan RSP9000 series Robots are designed to be used with Cavro Syringes. A Firmware (“a hardware timing problem”) occurs when more than one Kloehn Syringe is connected to the RS-485 bus off the RSP-9000 Robot. This problem has been verified by Cavro’s Engineer Roger McIntosh.
    • Solution: A new driver was written to control Multiple Syringe Pumps via an independent RS-485 Card plugged into an ISA slot on the host computer. Handshaking is performed between the Syringe Control Application, the Robotic Control Application, and the Hach Instrument Control Application.
  • 29. Testing Performance of the New Assay: First a standard had to be created which would add a known quantity of Turbidity (NTUs) with each addition to the test solution (H 2 0) contained within the cuvette. Formula (from Hach Manual):
  • 30. Testing Performance of the New Assay: Example: A single 1.0 µL addition of 1000NTU solution to a cuvette with a total volume of 2.5 mL.
  • 31. 3rd Generation Turbidimetric Solubility Assay
    • Based on 1.0 µL 1000 NTU additions into a 2.5mL cuvette:
      • Each addition causes a &quot;jump&quot; of .003 NTU on the display.
      • Each 1.0 µL addition causes a 0.2 NTU rise turbidity.
      • (0.2 NTU) / 3  0.066 NTU
    • A 0.001 change on the modified Hach 2100N Turbidimeter represents an &quot;actual&quot; change of approximately 0.066 NTU’s. The variation from the “true” value is the result of using a square cuvette instead of the circular test tube which the Hach Turbidimeter was designed to work with.
  • 32. Revising the Hach 2100N Turbidimeter Specs Revised Hach 2100N Turbidimeter Specifications: * The standard deviation of each successive addition was 0.001 NTUs. Original Hach 2100N Turbidimeter Specifications: 0.2 ±0.003* Repeatability .066 0.001 Resolution True NTU Value Displayed NTU Value Measurement N/A ±0.01 or 1.0% > of Repeatability N/A 0.001 Resolution True NTU Value Displayed NTU Value Measurement
  • 33. Repeatability: 1000 NTU Testing CV’s 1.4% ~ 1.84%
  • 34. Testing Assay Performance on “Real” Compounds
    • “ Ideal Conditions” - Compounds are diluted with DMSO, shaken, and run on G2483B and then immediately run on G5143A - slack time between runs kept to a minimum .
    • “ Non Ideal Conditions” - Compounds are diluted with DMSO, shaken, and run on 2483B. Compounds then sit in racks (diluted) for an indeterminate period of time (days to several weeks) before being assayed again on G5143A. (Crystal Formation Possible).
    Parallel Runs with Existing 2483B Assay
  • 35. Parallel Run (2483B Assay) “Ideal” Conditions
  • 36. Parallel Run (2483B Assay) “Non-Ideal” Conditions Although 40% of the compounds had a different solubility, more often than not (75%) the difference was only 5 - 10 µg/µL.
  • 37. Parallel Run (2483B Assay) “Non-Ideal” Conditions
  • 38. Repeatability: Midrange Solubility Variation  g/  L
  • 39. “ Binning” the results – What do they mean? Retrain Your Chemists <15 >50 >15 <50 Potential Problems Drug Like Material
  • 40. Project Management Considerations for New Assays
    • Control of Deliverables
      • What will be needed ?
      • Who should supply it ?
      • Can they Supply it on time ?
        • “ past performance is the single best predictor of future behavior”
    • Accommodation (to Infrastructure)
      • What needs to be changed to bring this screen online ?
      • Who will make these changes ?
      • Can the changes be made within the specified time frame ?
      • Will the changes be made within the specified time frame ?
  • 41. Project Management Considerations Infrastructure Specific
    • Compound Management
      • Does an Existing Method Exist for Compound Delivery ?
      • Is the Sample Repository Currently Preparing Samples in the desired format ?
      • Turnaround Time Required ?
      • Degradation & Stability Issues ?
        • Crystal Formation
        • Solubility in Solvent (DMSO, MeOH, etc.)
        • Light Sensitivity
      • Information Transfer of Delivered Compounds ?
        • Files (xls, csv, txt, etc.)
        • Web based ?
      • Will “Backdoor” submissions be allowed ?
        • Flexibility vs. Conformity issues.
        • The best and brightest want their results NOW.
        • Such capability = More Work.
  • 42. Project Management Considerations Software “The Big 3”
    • Controller Software for Hardware
    • Data Analysis Software
      • Reports for Legal Notebooks
      • Files for Database Uploading/Results Archival
    • Database Upload Software
      • Drag and Drop
      • Web Based File Loading
    • Who is going to write the software ?
    • Compatibility Issues ?
    • Outsourcing Concerns
      • Who Owns the source code ?
      • What you know we know - and eventually so will your competitors.
      • No Vested Interest – It Just has to work upon Delivery.
  • 43. Project Management Considerations Hardware
    • The Hardware must be designed.
    • The Hardware must be prototyped (bread boarded).
    • The Prototype must be tested.
    • The Prototype must be Laid out using some form of Hardware Design Tool such as schematic capture.
    • The Physical Board must be routed.
    • The Gerber Files must be generated and sent to a board house for fabrication.
      • Populated ?
      • If Populated, Surface Mount Components ?
    • Does the manufactured Board still work? No – Goto Step 4!
    • Common Reasons for Failure.
      • Poor Decoupling and/or Noise Issues.
      • Timing Issues (Often Due to Trace Routing)
      • Grounding Issues (Ground Loops, Lack of Ground Planes, etc.)
  • 44. Project Management Considerations Sphere of Influence
    • Prior to System Integration, the Software, Hardware, Infrastructure, and Compound Management issues must be resolved.
    • In other words, your deliverables must be delivered.
    • Delivered and Working.
    • The “ Sphere of Influence” a person can have will in all probability not span the entire group of disciplines.
    • Thus any resource required will need to be managed and sufficiently motivated to deliver on time.
    • Resources fall into two categories:
      • Those you have direct control over. (Groups and People under your direct supervision.)
        • Rewards, Motivation, and occasionally Punishment.
      • Those you have limited control over. (Contractors and those supervised by others.)
        • Reasoning, Praise, and Coercion.
  • 45. Project Management Considerations System Integration
    • Murphy’s Law of Project Management: “All the pieces may work perfectly separately , but they will never work together perfectly on the first try.”
    • System Integration is the art of getting all the components you so meticulously had designed to work in harmony together.
    • The single most difficult aspect of system integration is estimating an accurate time frame in which a working system can be developed.
    • This is because in a system of any complexity dependencies will exist, and should a redesign become necessary of any subsystem, its dependant components cannot be tested until the redesign is complete.
  • 46. Great Project Management Blunders
    • OSFA - One Size Fits All
    • “ It fits my all, but something else is hanging out the back.”
    • - Buddy Hackett
    • Example: A Pharmaceutical Company Creates a Research Database to store Global Data, yet it is initially modeled using only one type of screen.
    • Result: Screens with contain any differing parameters cannot have their data uploaded or retrieved until numerous revisions are made.
  • 47. Great Project Management Blunders
    • NIH – Not Invented Here
    • Example: A Pharmaceutical Company Creates an Automated Compound handling system from scratch using an industrial contractor rather than purchase a proven commercial compound handling system utilized by its largest competitors.
    • Result: The contractor was accustomed to working on inventory systems where a lag time of 3 days was sufficient for checking a part in or out of the system. As a result, in the original system, a compound could not be requested if it had been requested by any other lab 36 hours prior to the current request. This was unacceptable and resulted in costly fixes both in terms of redesign and wasted resources.
  • 48. Project Management Considerations: Communication
    • Many Project Management Failures are a result of poor Communication.
    • Communication Failures result from a variety of reasons including:
      • Information was missing
      • Timing was poor
      • Key people are absent
      • Stress and Tension
      • Sobering Statistic “31% of all communications that occur in Operating Rooms could be categorized as a failure in some way and more than a third of these failures had negative effects on what was happening.”
      • Source: “Communication failures in the operating room: an observational classification of recurrent types and effects.”
  • 49. 3rd Generation Turbidimetric Solubility Assay
    • Tecan RSP9000 Robotic Sample Processor
    • Kloehn 48,000 Step Syringe Pumps (3 per Assay)
    • Hach 2100N Turbidimeter (Modified)
    • Keithley Metrabyte Metrabus Industrial Data Acquisition Interface
      • Digital I/O Card (64 TTL Level Inputs/Outputs)
      • 8 Channel High Current Relay Card
      • 32 Channel Standard Relay Card
    • Custom Designed Interface Cards
    Hardware Specifications:
  • 50. Acknowledgements
    • Molecular Properties Group:
    • Dr. Christopher Lipinski – Adj. Senior Research Fellow
    • Dr. Franco Lombardo – Associate Research Fellow
    • Andrea Bunger (Davis) – First Solubility Assay
    • Paul Feeney – 2 nd Generation Solubility Assay
    • Gus Campos – Molecular Properties Group
    • Chris Caldwell – Molecular Properties Group
    • Victor Donahue Esq. – Pfizer Legal Dept. NYC
    • Stephen Gilbert Esq. – Bryan Cave LLP NYC
    • Thomas Freehill P.E. – Eastern CT Microelectronics
    • Roger McIntosh – McIntosh Analytical Systems
  • 51. Additional Resources - USPTO
    • United States Patent Application
    • “ Automated Kinetic Solubility Assay Apparatus and Method”
    • Application Number 20040076546
    • Filed Oct 18 2002
  • 52. Additional Resources