Biopharmaceutical Formulation Development CM3 Implementation and Initial Testing
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The document describes the Freeslate CM3 biologics formulation development workstation. The CM3 allows for automated sample preparation, processing, analysis, and experimental design for preformulation studies. It integrates liquid handling, analytical instruments, and software for designing and executing formulation experiments. Validation studies are performed to ensure reliable data from the liquid handling and analytical instruments. Buffer preparation and pH verification are provided as an example application.
Biopharmaceutical Formulation Development CM3 Implementation and Initial Testing
- 1. Biopharmaceutical Formulation Development CM3 Implementation and Initial Testing The KBI Experience Daniel Kinder 23.MARCH.2016
- 2. a customer and science-focused contract development & manufacturing organization
- 3. Boulder, Colorado - Microbial Process Development - Microbial cGMP Manufacturing - Analytical Development Durham, North Carolina - Cell Line Development - Cell Culture cGMP Manufacturing - Microbial cGMP Manufacturing - Analytical QC, Formulation, Stability RTP, North Carolina - Cell Culture Process Development - Microbial Process Development - Analytical Development Our Locations
- 4. Our Services Cell Line Development Upstream Process Development API Manufacturing Downstream Process Development Analytical & Formulation
- 5. Preformulation Services Biophysical Screening Scout the various biophysical techniques (DSC, DLS, FTIR, CD) to determine which are most informative about the protein’s thermal and conformational properties Effect of pH, buffer type, and excipients on thermal and conformational stability Solubility Studies Effect of pH, buffer type, ionic strength, excipients Surfactant Screening Studies Effect of surfactant type and concentration on aggregate and particle formation DOE Accelerated Stability Studies Effect of pH, buffer type, ionic strength, and excipients on chemical stability utilizing statistical design of experiments (DOE) Forced Degradation Studies Oxidation, acid & base hydrolysis, deamidation, heat, agitation, light, freeze/thaw
- 6. Automation Opportunities • Meet the increasing demand for more drug candidates with faster formulation and assessment • Conduct larger DOE’s earlier to reduce the risk of late stage formulation failures • High throughput experimental design and data management to accelerate decision making Automated sample preparation, processing and analysis Electronic experimental design and data management
- 10. Freeslate CM3 Biologics Formulation Development Workstation
- 11. CM3 Deck Layout
- 12. CM3 Workstation Devices 6-Channel Pipette • Air-displacement disposable tips • Variable pitch for access to a range of vial sizes • Multiple tip sizes enable 10-1000μL pipetting with ~1% cv precision Positive Displacement Pipette • Precise liquid handling ideal for even viscous samples • Tip sizes enable 10μL- 10mL pipetting 4-Channel Solid State pH Meter • 4-channel or single channel mode • 96 samples can be read in 30 minutes with ±0.1 pH accuracy • The 3mm probes can easily access 96 well plates
- 13. CM3 Workstation Devices Vial/plate Moving • Vial/Plate Gripper moves vials, microtiter plates and pipette tip racks • Robotic hand performs capping/uncapping and removes plate covers Storage Carousel • Carousel hotel provides storage for up to 32 racks/plates • Barcode enabled Viscosity Measurement • Measurement requires only 100μL • Shear rate measurement to evaluate Newtonian fluid behavior • Temperature control 4-40ºC
- 14. CM3 Workstation Devices Appearance Testing with Image Station • 2, 4, 5, 10 and 20mL vials • Quantitative color matching to Eur. Pharm. BY1-BY7 standards • Calibrated turbidity measurement 10- 1000 NTU • Particle Counting with digital image analysis detects sizes from 80μm
- 15. CM3 On-Board Analysis pH Color Analysis Viscosity Particle Count Turbidityuv/vis
- 16. CM3 Integrated Analysis Formulation Experiment Master Plate Uv/vis Plate DLS Plate Chromatography Plates (SEC, CEX, RP, etc.) IEF Plate CGE Plate
- 17. CM3 Software Suite: Library Studio® • Design complex experiments with realistic graphic display and text recipes • Assign sample group names and sample ID barcodes • Automatically design volume gradients, scale calculations, mathematical functions and more
- 18. CM3 Software Suite: Automation Studio® • Executes experimental workflows • Controls all integrated analytical instruments • Allows direct control over the CM3 devices
- 19. CM3 Software Suite: Automation Studio® • Quick review of data from individual experiments by query of date, data type, library ID, experiment conditions, etc.
- 20. CM3 Software Suite: PolyView® • View all data from each experimental condition in one organized spreadsheet • Graphical data and images displayed as spreadsheet • Query data from multiple experiments for trend analysis and reporting * Images from www.Freeslate.com * *
- 21. Questions asked by colleagues: • What does it do? • How fast? • How many? • How reliable is the data? How do you know? • How do I get my data? • How much effort to use?
- 22. CM3 Analytical Verification • Liquid Handling (LiHa) Verification • Volumes confirmed by calibrated devices • Specific to current application • pH Verification • Daily calibration/verification • Comparison with calibrated glass electrode • Tip-to-tip variance • Time-dependent drift • Viscometer verification • Calibration with standards • Protein or glycerol routine injections
- 23. LiHa precision/accuracy affected by: • Tip size (be sure to use the right one) • Syringe size (max motor/drive length) • Dispense categories (LiHa parameters) • Touch-off • Aspirate/dispense heights, speeds • Delays, air gaps, etc. • Nature of sample (e.g. viscosity, surface tension) • Nature of vessels (source and target) • Order of addition (what’s already in the target vial)
- 24. LiHa Event Sequence Computer Digital Controller Voltage MotorGearSyringe Valve 6’ of Tubing Pipet Tip Sample System Fluid
- 25. LiHa Verification - Methods • Gravimetric • Slow but sure • Use good technique, correct density for temperature • Automated weighing a plus • Unlimited volume range • Colorimetric • Uv/vis or fluorescent dyes (Orange-G, Me-Orange, Eosin-Y) • Must calibrate with a verified pipet • Very rapid read (96-wells in 10 seconds) • Limited to volume of uv/vis plate (~200μL)
- 26. Example LiHa Optimization (6-Tip, Gravimetric) 1000µL ADT Target (μL) Actual (μL) %CV %∆ 225 225.6 ± 0.8 0.4 0.3 450 451.1 ± 2.0 0.44 0.2 675 677.4 ± 1.1 0.16 0.4 900 902.6 ± 3.5 0.39 0.3
- 27. Colorimetric LiHa Optimization STEP 1: Set dispense parameters to a null value Set these values to null (Slope = 1, Intercept = 0)
- 28. Colorimetric LiHa Optimization STEP 2: Dispense stock dye solution into uv/vis plate Library Studio® Design SpectraMax® O.D. data Manually pipet this row
- 29. Example LiHa Optimization STEP 3: Determine abs-to-volume conversion function Beer’s Law plot for 200μL ADT Intercept -3.137 Slope 184.06 RSQ 1.0000 Standard Function Target volume (μL) Average (AU) SD 45.0 0.26 0.01 90.0 0.51 0.00 135.0 0.75 0.00 180.0 1.00 0.00 Standard curve
- 30. Example LiHa Optimization “Null” Values 200µL ADT Target (μL) Actual (μL) %CV %∆ 45 44.1 ± 0.4 0.9 2 90 87.6 ± 0.4 0.4 3 135 131.6 ± 0.7 0.5 3 180 175.4 ± 0.6 0.3 3
- 31. Example LiHa Optimization STEP 4: Plot resulting volumes to derive LiHa curve Optimization curve for 200μL ADT Intercept -0.191 Slope 1.0273 LiHa Function
- 32. Colorimetric LiHa Optimization STEP 5: Input optimized values for dispense category Plug in Slope and Intercept
- 33. Example LiHa Optimization Corrected Values 200µL ADT Target (μL) Actual (μL) %CV %∆ 45 44.6 ± 0.2 0.4 0.9 90 89.6 ± 0.3 0.3 0.4 135 135.0 ± 0.5 0.4 0.0 180 180.4 ± 0.9 0.5 0.2
- 34. Example LiHa Optimization Target Actual %CV %∆ Target Actual %CV %∆ Target Actual %CV %Δ 11 10.5 2 5% 45 44.6 0.50 1% 225 225.6 0.4 0% 22 21.6 1 2% 90 89.6 0.40 0% 450 451.1 0.5 0% 33 32.9 0.8 0% 135 135.0 0.40 0% 675 677.4 0.2 0% 45 44.8 0.8 0% 180 180.4 0.50 0% 900 902.6 0.4 0% Target Actual %CV %Δ Target Actual %CV %Δ Target Actual %CV %Δ 11 11.4 2.1 4% 22 22.1 1.9 0% 50 50.1 0.6 0% 22 21.9 2.5 0% 45 45.0 1.0 0% 100 101.4 0.3 1% 34 34.1 0.6 0% 70 70.6 0.9 1% 150 151.4 0.5 1% 45 44.6 0.5 1% 90 90.9 0.4 1% 200 199.9 0.2 0% Target Actual %CV %Δ Target Actual %CV %Δ Target Actual %CV %Δ 225 224.5 0.3 0% 1100 1117.5 2.9 2% 2250 2215.2 0.9 2% 450 449.9 0.2 0% 2200 2230.3 1.0 1% 4500 4474.7 0.2 1% 675 673.7 0.1 0% 3300 3274.4 0.3 1% 6750 6727.9 0.3 0% 900 898.4 0.0 0% 4400 4342.2 0.3 1% 9000 9003.0 0.0 0% 1mL PDT 5mL PDT 10mL PDT 50µL ADT 200µL ADT 1000µL ADT 50µL PDT 100µL PDT 250µL PDT
- 35. What about viscous samples? Pipet neat glycerol to prepare mixtures and measure viscosity: %wt Glycerol Viscosity (CM3) 0.0% 0.82 18.8% 1.51 40.3% 3.52 50.5% 5.69 60.1% 9.66 64.4% 13.14 67.0% 15.83 69.8% 19.55 71.9% 23.34 74.8% 29.71 76.7% 35.79 79.8% 47.14 ( Viscosity of glycerol = 1412 cP ! )
- 36. Literature Resources • “A Simple Method for Validation and Verification of Pipettes Mounted on Automated Liquid Handlers”, Michael Stangegaard et al, Journal of Laboratory Automation, October, 2011, pp 381- 386. • “Practical Considerations of Liquid Handling Devices in Drug Discovery”, Sergio C. Chai et al, (online book) http://dx.doi.org/10.5772/52546. • “Improving Accuracy by Use of Technique Calibration”, Lisa Knapp, et al, (Beckman Coulter technical bulletin) https://www.beckmancoulter.com/wsrportal/bibliography?docnam e=T-1915A.pdf.
- 37. Example: Buffer Prep and pH • Biologic pharmaceuticals are typically handled in formulations of very precise composition • pH control is maintained by buffer systems (phosphate, citrate, acetate, etc.) • An in-house table of buffer recipes to achieve target pH’s would be useful • Great CM3 training/verification activity
- 38. Example: Buffer Prep and pH Library Studio® Design of Buffer Prep
- 39. Example: Buffer Prep and pH Mixtures from buffer stock solutions Buffer Acid Buffer Base Target pH CM3 preparing citrate buffer mixtures in 96-DWP
- 40. Example: Buffer Prep and pH Plot of pH vs. Phosphate Base added ( NaH2PO4/Na2HPO4 )
- 41. Sample A (μl) B (μl) Target pH CM3 pH Δ 1 950 0 (Neat Acid) 4.47 NA 2 870 80 5.8 5.80 0.00 3 840 110 6.0 5.94 ‐0.06 4 810 140 6.2 6.03 ‐0.17 5 700 250 6.4 6.31 ‐0.09 6 600 350 6.6 6.58 ‐0.02 7 480 470 6.8 6.79 ‐0.01 8 370 580 7.0 6.98 ‐0.02 9 270 680 7.2 7.18 ‐0.02 10 180 770 7.4 7.41 0.01 11 120 830 7.6 7.62 0.02 12 80 870 7.8 7.86 0.06 13 50 900 8.0 8.05 0.05 14 0 950 (Neat Base) 9.14 NA 4.00 3.90 ‐0.10 7.01 7.02 0.01 10.04 10.00 ‐0.04 pH 4 Reference Buffer @ 21°C pH 7 Reference Buffer @ 21°C pH 10 Reference Buffer @ 21°C Example: Buffer Prep and pH Selected data for phosphate buffer prep compared with literature target pH’s
- 42. Sample A (μl) B (μl) Target pH CM3 pH Glass pH Δ 1 950 0 (Neat Acid) 4.47 4.50 0.03 2 870 80 5.8 5.80 5.79 ‐0.01 3 840 110 6.0 5.94 5.95 0.01 4 810 140 6.2 6.03 6.06 0.03 5 700 250 6.4 6.31 6.38 0.07 6 600 350 6.6 6.58 6.59 0.01 7 480 470 6.8 6.79 6.81 0.02 8 370 580 7.0 6.98 7.00 0.02 9 270 680 7.2 7.18 7.21 0.03 10 180 770 7.4 7.41 7.44 0.03 11 120 830 7.6 7.62 7.65 0.03 12 80 870 7.8 7.86 7.85 ‐0.01 13 50 900 8.0 8.05 8.07 0.02 14 0 950 (Neat Base) 9.14 9.13 ‐0.01 4.00 3.90 4.03 0.13 7.01 7.02 7.03 0.01 10.04 10.00 10.05 0.05 pH 4 Reference Buffer @ 21°C pH 7 Reference Buffer @ 21°C pH 10 Reference Buffer @ 21°C Example: Buffer Prep and pH Selected data for phosphate buffer prep compared with glass electrode
- 43. Example: Buffer Prep and pH Histidine.HCl/Histidine Citric Acid/Trisodium Citrate Other Examples:
- 44. Conclusions • Need to optimize or verify liquid handling, pH or viscosity? Let the robot do the work! • Buffer prep is a breeze with precise liquid handling and rapid pH measurement • Increased buffer prep throughput creates need for automated buffer exchange
- 45. Acknowledgements: • KBI Biopharma, Inc. for the vision to invest in automation • Freeslate/Unchained Labs for great engineering and great support