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AUTOMATE YOUR ANTIBODY PURIFICATION WORKFLOW AT PREPARATIVE AND ANALYTICAL SCALESKATIE SCHAEFER, PH.D. AND PETER WINSHIP, PH.D.
PART 1.TELEDYNE CETAC TECHNOLOGIES AND BIO-RADLABORATORIES:THE COMPANIES AND THE PRESENTERS
KATIE SCHAEFER, PH.D.BIO-RAD LABORATORIES
Based in Northern California, Katie Schaefer is the Global Product Manager forlaboratory-scale chromatography systems at Bio-Rad Laboratories. She is responsiblefor the NGC chromatography system and the accompanying ChromLab software. Katiebegan her research career as an undergraduate student and has over 12 years ofexperience in chromatographic techniques. Katie received her Ph.D. in Biochemistryand Molecular Biophysics from the California Institute of Technology in 2015, studyingthe biological implications of DNA-mediated charge transport. She began her careerwith Bio-Rad as a Proteomics Field Applications Scientist, training and supportingcustomers in chromatography, western blotting, digital imaging, and Bio-Plex assays.She now supports the NGC chromatography system and ChromLab software at Bio-Radheadquarters as a product manager and a technical application expert.
BIO-RAD LABORATORIESFounded in 1952
David and Alice Schwartz began Bio-Rad in a Quonset hut
Goal was to advance scientific discovery
Went public in 1966 with $1 million in sales
Global leader in clinical diagnostics and life science research
Located in Hercules, CA
PETER WINSHIP, PH.D.TELEDYNE CETAC TECHNOLOGIES
Based in the United Kingdom, Peter Winship is a Technical Product Manager atTeledyne CETAC Technologies and is responsible for the ASX-7000 Series automationproduct line. He joined the company in 2011. Pete has a degree in Chemistry, an MScin Analytical and Pharmaceutical Science and a Ph.D. in Analytical Chemistry. Pete hasworked with ICP-MS and ICP-OES instrumentation since 2001 and he has furtherexperience in the oil industry and biological and geological testing. Pete spent 5 yearsas a Senior Analyst at the Medical Research Council in the UK where he operated ICP-MS and ICP-OES instrumentation to study clinical and biological samples in support ofa number of research projects. He has been an author of, or contributor to, a numberof peer reviewed publications and has presented widely at international conferences.
TELEDYNE CETAC TECHNOLOGIESFounded in 1985, Ames, Iowa, USA
Began with introduction of the U5000 ultrasonic nebulizer.
First autosampler introduced in the early 90’s, this laid the foundation for the industry’s leader in autosamplers with a reputation for quality and reliability.
Sold to Steve Dwyer in 2000.
Acquired by Teledyne Technologies Inc. in 2013.
Today Teledyne CETAC Technologies manufactures automation and sampling handling/introduction systems, that support a gamut of analytical techniques, in Omaha, Nebraska, USA.
PART 2.PROTEIN/ANTIBODY PURIFICATION, AUTOMATION AND INSTRUMENTATION
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PROTEIN PURIFICATION: AN INTRODUCTION
Goal 1: Isolate one protein of interest from a biological expression platform
o Protein expression: E. coli, yeast, cell cultures, hybridoma cell lines
Goal 2: Purify large quantities of high purity proteins
Goal 3: Utilize these proteins in the research, development, and therapeutic treatment
Chromatographic protein purification workflows typically utilize multiple column chemistries
Preparative – small to large scale production
Analytical – high resolution separation and purity confirmation
Capture•Tagged Protein•Native Protein
Intermediate•Ion exchange•Desalting•Hydrophobic
Interaction
Polishing•High resolution
resins•Size Exclusion•Mixed Mode
ConfirmPurity
Purit
y
Procedure time
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MONOCLONAL IGG ANTIBODIES
Highly specific biomolecule from the immune response to an antigen
Highly favorable for scientific applications such as western blotting, ELISA, and flow cytometry
Monoclonal IgG antibodies are produced via hybridoma cell lines, phage display technologies and single-sorted human B cells
The high specificity of IgG antibodies makes them exceptional for therapeutic purposes - targeting a very specific antigen representative of a diseased state and eliciting an immune response
o Humira (adalimumab): Treatment for arthritis, Crohn’s disease, ulcerative colitis
o Rituxan (rituximab): Treatment for certain autoimmune diseases and cancers
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MONOCOLONAL ANTIBODY DEVELOPMENT (TWO WORKFLOWS)
Screening of Antibody Candidates – Express IgG antibody candidates on a small scale for efficacy testing
o ASX-500 Series autosampler + NGC with Sample Pump + Inlet Valve + Fraction Collector
Confirm Purity of Production – Analytical analysis for sample purity and integrity
o MVX-7100 µL Workstation + NGC with analytical flow cell
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BIO-RAD AND THE NGC CHROMATOGRAPHY SYSTEM
Customizable chromatography platform to meet your current and future purification needs
The system can change with your changing application needs
Medium-pressure system allows some for reversed-phase applications
Flexible fraction collection options
Intuitive ChromLab software
NGC Discover Pro
NGC Quest
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TELEDYNE CETAC TECHNOLOGIES AND AUTOMATION: WHY AUTOMATE PROTEIN PURIFICATION?
Flexible automation to meet the requirements of the protein purification workflows
o mL sample introduction scale (ASX-500 Series autosampler)
o µL sample introduction scale (MVX-7100 µL Workstation)
Configurable, robust and reliable
Undertake unattended chromatography instrument operation
o 24 hours a day, 7 days per week operation (improve efficiency)
o Allow purification of 84 samples in two days
Accurate and precise sample uptake and introduction
o Minimize sample introduction error
MVX-7100 µL Workstation
ASX-500 Series Autosampler
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PART 3.PREPARATIVE PROTEIN PURIFICATION WORKFLOW
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MONOCOLONAL ANTIBODY DEVELOPMENT (TWO WORKFLOWS)
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Screening of Antibody Candidates – Express IgG antibody candidates on a small scale for efficacy testing
o ASX-500 Series autosampler + NGC with Sample Pump+ Inlet Valve + Fraction Collector
Confirm Purity of Production – Analytical analysis for sample purity and integrity
o MVX-7100 µL Workstation + NGC with analytical flow cell
PREPARATIVE PROTEIN PURIFICATION WORKFLOW
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Sample preparation/pre-
treatment
Transfer to ASX-500 Series automation
Sample uptake and introduction
to the NGC instrument
Tandem chromatography
(Protein A to Desalting)
Fraction collection
Repeated tandem
chromatography
Data generation Purity confirmation
SCREENING OF ANTIBODY CANDIDATES -- SAMPLE PRODUCTION
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PREPARATIVE PURIFICATION: SAMPLES TRANSFERRED TO ASX-500 SERIES AUTOMATION
ASX-280 autosampler (2 sample rack capacity)
ASX-560 autosampler (4 sample rack capacity)
Robust and reliable XYZ automation for 10’s and 100’s mL sample volumes
Numerous sample tube/bottle options: 7 mL to 250 mL sizes
Rinse station (peristaltic pump filled/drained)
Resistant to chaotropic agents, organic solvents, and detergents
Sample probe connected to the NGC sample inlet valve
ASX-560 Autosampler
ASX-280 Autosampler
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ASX-500 SERIES AUTOMATION AND NGC INSTRUMENT: INTERFACE
AScript Operating Software
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SAMPLE UPTAKE AND INTRODUCTION PROCESS
Method begins for column equilibration and to zero baseline
In method, sample loading phase sends trigger signal received by ASX-500 Series autosampler
Sample probe moves to the appropriate sample/standard position
NGC Sample pump turns ON
Sample pump draws up sample and loads directly to column
Air sensor triggers and terminates the loading process
Resume remainder of purification method
ASX-500 Series probe washed in preparation for next sample
Air Sensor
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TANDEM CHROMATOGRAPHY: PROTEIN A TO DESALTING
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IgG Protein
Protein A Elution Buffer
Protein ACaptures IgG
Elutes at pH 3.0
DesaltingSize Separation
pH 7.3
FractionspH 7.3
NGC AND ASX FOR AUTOMATED TANDEM CHROMATOGRAPHY
Desalting column equilibration
Protein A column equilibration
Clean system for next sample
from ASX
Protein A column wash
Sample Application
ASX + Inlet Valve + Sample Pump
Protein A elution+ desalting
column sample application
Desalting ColumnElution and
Fraction Collection
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Repeat for all samples on ASX
OVERLAY OF AUTOMATED IGG PURIFICATION SERIES WITH ASX
“Method is easy to use and truly hands-off”A
280
nm (m
AU)
Conductivity mS/cm
Volume
pH 7.3
Conductivity 22 mS/cm
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280
nm (m
AU)
NGC FRACTION COLLECTORCollects into multiple sized vessels within a method from 96 well plates to 250ml bottles
Peltier Cooling option
Supports flow rates up to 200 ml/min
High quality racks
Factory calibrated
Triggers for collection including slope, pH, & conductivity
NGC will support up to two fraction collectors per machine
Designed to fit into both swing door and sliding door deli-fridges
Tube Type Tube/Rack FC Capacity
13 mm 96 384
16 mm 75 300
15 ml (18 mm) 70 280
50 ml (30 mm) 27 108
96-well 2 8
2 ml microtube 96 384
250 ml bottle 4 16
Prep-Rack 20 40
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SAMPLE PURITY WITHOUT CROSS-CONTAMINATION
Purity is >95% No cross contamination detected by LC-MS
Ab1
Ab2
Ab3
Typical yield is 2-6 mg recovered which is sufficient to enable secondary screening procedure (functional assay)
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AUTOMATED SCREENING SOLUTION
We have implemented a robust purification method for Antibodies candidate
The method is fully automated and can handle 84 Ab over a weekend
The described method is easy to implement
Can easily be adapted to more elaborate purification scheme (multi-dimensional)
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PART 4.ANALYTICAL SEPARATION/PURIFICATION WORKFLOW
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Screening of Antibody Candidates – Express IgG antibody candidates on a small scale for efficacy testing
o ASX-500 Series autosampler + NGC with Sample Pump + Inlet Valve + Fraction Collector
Confirm Purity of Production – Analytical analysis for sample purity and integrity
o MVX-7100 µL Workstation + NGC with analytical flow cell
MONOCOLONAL ANTIBODY DEVELOPMENT (TWO WORKFLOWS)
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ANALYTICAL SEPARATION/PURIFICATION WORKFLOW
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Sample preparation/pre-
treatment
Transfer to MVX-7100 µL
Workstation
Sample uptake and introduction
to the NGC instrument
High-resolution purification on
NGC
Small-scale fraction
collection
Repeated High-resolution
purification on NGC
Data generation Purity confirmation
SAMPLES TRANSFERRED TO MVX-7100 AUTOMATION
Accurate, precise and robust XYZ automationo < 5 µL to 950 µL sample volumes
Different sample vial and well plate optionso VT54, 96 well plates and 384 well plates
Fine control of sample aliquot uptake
Temperature control (4°C to 40°C)
Rinse station
Resistant to chaotropic agents, organic solvents, and detergents
Plates can be foil covered to prevent sample evaporationMVX-7100 µL Workstation
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MVX-7100 AUTOMATION AND NGC INSTRUMENT: INTERFACE
CETAC Workstation Operating Software
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SAMPLE UPTAKE AND INTRODUCTION PROCESS
Trigger signal received by MVX-7100
Sample probe moves to the sample/standard position
Syringe pump pulls sample onto MVX-7100 sample loop
MVX-7100 is connected to the inlet valve of NGC
Sample loaded into mobile phase through NGC loop port and sent to column
Remaining method resumed
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CETAC WORKSTATION SOFTWARE
Operating software for the MVX-7100 µL Workstation
Select sample locations, sample injection volume and uptake flow rates
Use the CETAC Workstation software to control the temperature of the samples and to configure the rinsing processes
Use the pre-configured operating methods for full sample loop and partial sample loop sample injections
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CETAC WORKSTATION SOFTWARE
Full sample loop injection
o Completely fill a sample loop with sample (e.g. 100 µL, 500 µL)
o Slight excess of sample on either side of the sample loop sent to waste
o Example: 27 µL of sample used to fill 25 µL sample loop with 2 µL sample excess
Partial sample loop injection
o Partially fill a sample loop with sample (e.g. 10 µL of sample in a 500 µL loop)
o Buffer solution on either side of the sample to ensure the loop is full of liquid
o Particularly applied to very low sample volumes
Full Sample Loop Uptake
Partial Sample Loop Uptake
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DATA FROM MVX-7100 : 48 RUNS
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Full Sample Loop Uptake
Varied concentrations of a standard25 µl full loop injection
MVX-7100 10 UL INJECTIONS: SEC STANDARD ON ENRICH 650
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Partial Sample Loop Uptake
A, thyroglobulin; B, gamma globulin; C, ovalbumin; D, myoglobin; E, vitamin B12.
A
BC
D
E
Gel Filtration Standard10 µl partial loop uptake
PART 5.CONCLUSIONS
CONCLUDING COMMENTS SLIDE
Teledyne CETAC Technologies automation can easily streamline the protein/antibody purification workflows of the Bio-Rad NGC instrument.
o 24 hour, 7 days per week unattended operation
o Maintain accuracy and precision of sample introduction
o Automation to meet the full sample volume range requirements
Use the Bio-Rad NGC chromatography system for separation and purification of protein/antibody species.
o Intuitive system that adapts and grows with your purification needs
o Automate your purification for increased sample consistency and reproducibility
o NGC platform can be used for preparative as well as analytical separations
THANK YOU FOR YOUR ATTENTION
Katie Schaefer, Ph.D.
o Global Product Manager: Laboratory Scale Chromatography Systems
o www.bio-rad.com/ngc
Peter Winship, Ph.D.
o Technical Product Manager: ASX-7000 Series Automation
o www.teledynecetac.com
QUESTIONS??????