Biopharmaceutical Formulation Development CM3 Implementation

and Initial Testing

The KBI Experience

Daniel Kinder23.MARCH.2016

a customer and science-focused contract development & manufacturing organization

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

Our Services

Cell Line Development

Upstream Process Development

API Manufacturing Downstream Process Development

Analytical & Formulation

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

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

Peter Huefner!1.5” Clearance!

Freeslate CM3 Biologics Formulation Development Workstation

CM3 Deck Layout

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

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

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

CM3 On-Board Analysis

pH

Color Analysis Viscosity

Particle CountTurbidityuv/vis

CM3 Integrated AnalysisFormulation Experiment Master Plate

Uv/vis Plate

DLS Plate

Chromatography Plates(SEC, CEX, RP, etc.)

IEF PlateCGE Plate

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

CM3 Software Suite: Automation Studio®

• Executes experimental workflows• Controls all integrated analytical instruments• Allows direct control over the CM3 devices

CM3 Software Suite:Automation Studio®

• Quick review of data from individual experiments by query of date, data type, library ID, experiment conditions, etc.

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?

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

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)

LiHa Event Sequence

Computer Digital Controller Voltage

MotorGearSyringe

Valve 6’ of Tubing

Pipet Tip Sample

System Fluid

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)

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

Colorimetric LiHa OptimizationSTEP 1: Set dispense parameters to a null value

Set these values to null(Slope = 1, Intercept = 0)

Colorimetric LiHa Optimization STEP 2: Dispense stock dye solution into uv/vis plate

Library Studio® Design SpectraMax® O.D. data

Manually pipet this row

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

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

Example LiHa OptimizationSTEP 4: Plot resulting volumes to derive LiHa curve

Optimization curve for 200μL ADT

Intercept -0.191Slope 1.0273

LiHa Function

Colorimetric LiHa OptimizationSTEP 5: Input optimized values for dispense category

Plug in Slope and Intercept

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

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

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 ! )

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.

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

Example: Buffer Prep and pH Library Studio® Design of Buffer Prep

Example: Buffer Prep and pH Mixtures from buffer stock solutions

BufferAcid

BufferBase

Target pH

CM3 preparing citrate buffer mixtures in 96-DWP

Example: Buffer Prep and pH

Plot of pH vs. Phosphate Base added( NaH2PO4/Na2HPO4 )

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

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

Example: Buffer Prep and pH

Histidine.HCl/Histidine Citric Acid/Trisodium Citrate

Other Examples:

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

Acknowledgements:

• KBI Biopharma, Inc. for the vision to invest in automation

• Freeslate/Unchained Labs for great engineering and great support