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Biopharmaceutical Formulation Development CM3 Implementation
and Initial Testing
The KBI Experience
Daniel Kinder23.MARCH.2016
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a customer and science-focused contract development & manufacturing organization
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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
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Our Services
Cell Line Development
Upstream Process Development
API Manufacturing Downstream Process Development
Analytical & Formulation
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Preformulation ServicesBiophysical ScreeningScout 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 StudiesEffect of pH, buffer type, ionic strength, excipients
Surfactant Screening StudiesEffect of surfactant type and concentration on aggregate and particle formation
DOE Accelerated Stability StudiesEffect of pH, buffer type, ionic strength, and excipients on chemical stability utilizing statistical design of experiments (DOE)
Forced Degradation StudiesOxidation, acid & base hydrolysis, deamidation, heat, agitation, light, freeze/thaw
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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
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Peter Huefner!1.5” Clearance!
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Freeslate CM3 Biologics Formulation Development Workstation
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CM3 Deck Layout
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CM3 Workstation Devices6-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
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CM3 Workstation DevicesVial/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
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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
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CM3 On-Board Analysis
pH
Color Analysis Viscosity
Particle CountTurbidityuv/vis
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CM3 Integrated AnalysisFormulation Experiment Master Plate
Uv/vis Plate
DLS Plate
Chromatography Plates(SEC, CEX, RP, etc.)
IEF PlateCGE Plate
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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
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CM3 Software Suite: Automation Studio®
• Executes experimental workflows• Controls all integrated analytical instruments• Allows direct control over the CM3 devices
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CM3 Software Suite:Automation Studio®
• Quick review of data from individual experiments by query of date, data type, library ID, experiment conditions, etc.
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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
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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?
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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
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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)
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LiHa Event Sequence
Computer Digital Controller Voltage
MotorGearSyringe
Valve 6’ of Tubing
Pipet Tip Sample
System Fluid
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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)
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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
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Colorimetric LiHa OptimizationSTEP 1: Set dispense parameters to a null value
Set these values to null(Slope = 1, Intercept = 0)
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Colorimetric LiHa Optimization STEP 2: Dispense stock dye solution into uv/vis plate
Library Studio® Design SpectraMax® O.D. data
Manually pipet this row
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Example LiHa OptimizationSTEP 3: Determine abs-to-volume conversion function
Beer’s Law plot for 200μL ADT
Intercept -3.137Slope 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 0.00180.0 1.00 0.00
Standard curve
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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
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Example LiHa OptimizationSTEP 4: Plot resulting volumes to derive LiHa curve
Optimization curve for 200μL ADT
Intercept -0.191Slope 1.0273
LiHa Function
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Colorimetric LiHa OptimizationSTEP 5: Input optimized values for dispense category
Plug in Slope and Intercept
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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
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Example LiHa Optimization
Target Actual %CV %∆ Target Actual %CV %∆ Target Actual %CV %Δ11 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 %Δ11 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 %Δ225 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
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What about viscous samples?
Pipet neat glycerol to prepare mixtures and measure viscosity:
%wt Glycerol Viscosity (CM3)0.0% 0.8218.8% 1.5140.3% 3.5250.5% 5.6960.1% 9.6664.4% 13.1467.0% 15.8369.8% 19.5571.9% 23.3474.8% 29.7176.7% 35.7979.8% 47.14
( Viscosity of glycerol = 1412 cP ! )
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Literature Resources• “A Simple Method for Validation and Verification of Pipettes
Mounted on Automated Liquid Handlers”, Michael Stangegaardet 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?docname=T-1915A.pdf.
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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
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Example: Buffer Prep and pH Library Studio® Design of Buffer Prep
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Example: Buffer Prep and pH Mixtures from buffer stock solutions
BufferAcid
BufferBase
Target pH
CM3 preparing citrate buffer mixtures in 96-DWP
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Example: Buffer Prep and pH
Plot of pH vs. Phosphate Base added( NaH2PO4/Na2HPO4 )
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Sample A (μl) B (μl) Target pH CM3 pH Δ1 950 0 (Neat Acid) 4.47 NA2 870 80 5.8 5.80 0.003 840 110 6.0 5.94 ‐0.064 810 140 6.2 6.03 ‐0.175 700 250 6.4 6.31 ‐0.096 600 350 6.6 6.58 ‐0.027 480 470 6.8 6.79 ‐0.018 370 580 7.0 6.98 ‐0.029 270 680 7.2 7.18 ‐0.0210 180 770 7.4 7.41 0.0111 120 830 7.6 7.62 0.0212 80 870 7.8 7.86 0.0613 50 900 8.0 8.05 0.0514 0 950 (Neat Base) 9.14 NA
4.00 3.90 ‐0.107.01 7.02 0.0110.04 10.00 ‐0.04
pH 4 Reference Buffer @ 21°CpH 7 Reference Buffer @ 21°CpH 10 Reference Buffer @ 21°C
Example: Buffer Prep and pHSelected data for phosphate buffer prep compared with
literature target pH’s
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Sample A (μl) B (μl) Target pH CM3 pH Glass pH Δ1 950 0 (Neat Acid) 4.47 4.50 0.032 870 80 5.8 5.80 5.79 ‐0.013 840 110 6.0 5.94 5.95 0.014 810 140 6.2 6.03 6.06 0.035 700 250 6.4 6.31 6.38 0.076 600 350 6.6 6.58 6.59 0.017 480 470 6.8 6.79 6.81 0.028 370 580 7.0 6.98 7.00 0.029 270 680 7.2 7.18 7.21 0.0310 180 770 7.4 7.41 7.44 0.0311 120 830 7.6 7.62 7.65 0.0312 80 870 7.8 7.86 7.85 ‐0.0113 50 900 8.0 8.05 8.07 0.0214 0 950 (Neat Base) 9.14 9.13 ‐0.01
4.00 3.90 4.03 0.137.01 7.02 7.03 0.0110.04 10.00 10.05 0.05
pH 4 Reference Buffer @ 21°CpH 7 Reference Buffer @ 21°CpH 10 Reference Buffer @ 21°C
Example: Buffer Prep and pHSelected data for phosphate buffer prep compared with
glass electrode
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Example: Buffer Prep and pH
Histidine.HCl/Histidine Citric Acid/Trisodium Citrate
Other Examples:
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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
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Acknowledgements:
• KBI Biopharma, Inc. for the vision to invest in automation
• Freeslate/Unchained Labs for great engineering and great support