AUTOMATE YOUR ANTIBODY PURIFICATION WORKFLOW AT PREPARATIVE AND - - PowerPoint PPT Presentation

AUTOMATE YOUR ANTIBODY PURIFICATION WORKFLOW AT PREPARATIVE AND ANALYTICAL SCALES

KATIE SCHAEFER, PH.D. AND PETER WINSHIP, PH.D.

PART 1. TELEDYNE CETAC TECHNOLOGIES AND BIO-RAD LABORATORIES: THE COMPANIES AND THE PRESENTERS

KATIE SCHAEFER, PH.D. BIO-RAD LABORATORIES

Based in Northern California, Katie Schaefer is the Global Product Manager for laboratory-scale chromatography systems at Bio-Rad Laboratories. She is responsible for the NGC chromatography system and the accompanying ChromLab software. Katie began her research career as an undergraduate student and has over 12 years of experience in chromatographic techniques. Katie received her Ph.D. in Biochemistry and Molecular Biophysics from the California Institute of Technology in 2015, studying the biological implications of DNA-mediated charge transport. She began her career with Bio-Rad as a Proteomics Field Applications Scientist, training and supporting customers in chromatography, western blotting, digital imaging, and Bio-Plex assays. She now supports the NGC chromatography system and ChromLab software at Bio-Rad headquarters as a product manager and a technical application expert.

BIO-RAD LABORATORIES

Founded 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 at Teledyne CETAC Technologies and is responsible for the ASX-7000 Series automation product line. He joined the company in 2011. Pete has a degree in Chemistry, an MSc in Analytical and Pharmaceutical Science and a Ph.D. in Analytical Chemistry. Pete has worked with ICP-MS and ICP-OES instrumentation since 2001 and he has further experience in the oil industry and biological and geological testing. Pete spent 5 years as 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 of a number of research projects. He has been an author of, or contributor to, a number

• f peer reviewed publications and has presented widely at international conferences.

TELEDYNE CETAC TECHNOLOGIES

Founded 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

K

PROTEIN PURIFICATION: AN INTRODUCTION



Goal 1: Isolate one protein of interest from a biological expression platform

• 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 re

• Tagged Protein
• Native Protein

Intermediat ate

• Ion exchange
• Desalting
• Hydrophobic

Interaction

Polishi hing

• High resolution

resins

• Size Exclusion
• Mixed Mode

Confirm Purity ty

Purity Procedure time

K

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

• Humira (adalimumab): Treatment for arthritis, Crohn’s disease, ulcerative colitis
• Rituxan (rituximab): Treatment for certain autoimmune diseases and cancers

K

MONOCOLONAL ANTIBODY DEVELOPMENT (TWO WORKFLOWS)



Screeni ning o

• f A

Anti ntibody y Cand ndidates – Express IgG antibody candidates

• n a small scale for efficacy testing
• ASX-500 Series autosampler + NGC with Sample Pump + Inlet Valve +

Fraction Collector



Confir irm P Purit ity o

• f Productio

ion – Analytical analysis for sample purity and integrity

• MVX-7100 µL Workstation + NGC with analytical flow cell

K

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 C Disco cover P Pro NGC GC Q Ques est K

TELEDYNE CETAC TECHNOLOGIES AND AUTOMATION: WHY AUTOMATE PROTEIN PURIFICATION?



Flexible automation to meet the requirements of the protein purification workflows

• mL sample introduction scale (ASX-500 Series autosampler)
• µL sample introduction scale (MVX-7100 µL Workstation)



Configurable, robust and reliable



Undertake unattended chromatography instrument operation

• 24 hours a day, 7 days per week operation (improve efficiency)
• Allow purification of 84 samples in two days



Accurate and precise sample uptake and introduction

• Minimize sample introduction error

MVX VX-7100 µ µL Works ksta tati tion ASX ASX-500 S Seri ries s Autosa sample ler P

PART 3. PREPARATIVE PROTEIN PURIFICATION WORKFLOW

K

MONOCOLONAL ANTIBODY DEVELOPMENT (TWO WORKFLOWS)

K



Screeni ning o

• f A

Anti ntibody y Cand ndidates – Express IgG antibody candidates

• n a small scale for efficacy testing
• ASX-500 Series autosampler + NGC with Sample Pump+ Inlet Valve +

Fraction Collector



Confir irm P Purit ity o

• f Productio

ion – Analytical analysis for sample purity and integrity

• MVX-7100 µL Workstation + NGC with analytical flow cell

PREPARATIVE PROTEIN PURIFICATION WORKFLOW

K

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

K

PREPARATIVE PURIFICATION: SAMPLES TRANSFERRED TO ASX-500 SERIES AUTOMATION



ASX-280 a auto tosample ler (2 sample rack capacity)



ASX-560 a auto tosample ler (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 ASX-560 A Autos

• sampler

ASX ASX-280 A Autos

• sampler

P

ASX-500 SERIES AUTOMATION AND NGC INSTRUMENT: INTERFACE

ASc AScrip ipt Operatin ing So Software P

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 S Sensor

• r

P

TANDEM CHROMATOGRAPHY: PROTEIN A TO DESALTING

K IgG P Protein Protein A A El Eluti ution B Buf uffer

Prot

• tein A

n A

Captures IgG Elutes at pH 3.0

Desalting ng

Size Separation pH 7.3

Fr Fractions

pH 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 Column Elution and Fraction Collection

K Repeat t for a all s sampl mples es o

• n ASX

OVERLAY OF AUTOMATED IGG PURIFICATION SERIES WITH ASX

“Me Method is is eas asy t to u use an and truly han ands-of

• ff”

A 280 nm (mAU) Conductivity mS/cm Volume

pH H 7.

7.3

Conductiv ivit ity 22

22 mS

mS/cm /cm

K

A 280 nm (mAU)

NGC FRA C FRACTI CTION CO COLLECTO CTOR

Collects 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

Tub Tube Ty Type Tube/Rack ck FC C Capa paci city 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

K

SAMPLE PURITY WITHOUT CROSS-CONTAMINATION

Purity is >95% No cross c contam amin inat atio ion d n detected b by L LC-MS MS Ab1 Ab2 Ab3  Typical yield is 2-6 mg recovered which is sufficient to enable secondary screening procedure (functional assay) K

AUTOMATED SCREENING SOLUTION



We have implemented a robust purification method for Antibodies candidate



The method is fully aut utomated 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) P

PART 4. ANALYTICAL SEPARATION/PURIFICATION WORKFLOW

P



Screeni ning o

• f A

Anti ntibody y Cand ndidates – Express IgG antibody candidates

• n a small scale for efficacy testing
• ASX-500 Series autosampler + NGC with Sample Pump + Inlet Valve +

Fraction Collector



Confir irm P Purit ity o

• f Productio

ion – Analytical analysis for sample purity and integrity

• MVX-7100 µL Workstation + NGC with analytical flow cell

MONOCOLONAL ANTIBODY DEVELOPMENT (TWO WORKFLOWS)

K

ANALYTICAL SEPARATION/PURIFICATION WORKFLOW

K

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 automation

• < 5 µL to 950 µL sample volumes



Different sample vial and well plate options

• 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 evaporation

MVX VX-7100 µ µL Works ksta tati tion P

MVX-7100 AUTOMATION AND NGC INSTRUMENT: INTERFACE

CETAC W Workstat atio ion n Operat atin ing S Softwar are P

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

P

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

• f the samples and to configure the rinsing processes



Use the pre-configured operating methods for full sample loop and partial sample loop sample injections

P

CETAC WORKSTATION SOFTWARE



Full sample loop injection

• Completely fill a sample loop with sample (e.g. 100 µL, 500 µL)
• Slight excess of sample on either side of the sample loop sent to waste
• Example: 27 µL of sample used to fill 25 µL sample loop with 2 µL sample excess



Partial sample loop injection

• Partially fill a sample loop with sample (e.g. 10 µL of sample in a 500 µL loop)
• Buffer solution on either side of the sample to ensure the loop is full of liquid
• Particularly applied to very low sample volumes

Full S Sampl ple L Loop U p Upt ptake Partial S al Sample le L Loop

• p Up

Uptak ake

P

DATA FROM MVX-7100 : 48 RUNS

K

Full S Sampl ple L Loop U p Upt ptake Varied concentrations of a standard 25 µl full loop injection

MVX-7100 10 UL INJECTIONS: SEC STANDARD ON ENRICH 650

K

Parti tial S Sample L Loop Upta take ke

A, thyroglobulin; B, gamma globulin; C, ovalbumin; D, myoglobin; E, vitamin B12.

A B C D E

Gel Filtration Standard 10 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.

• 24 hour, 7 days per week unattended operation
• Maintain accuracy and precision of sample introduction
• Automation to meet the full sample volume range requirements



Use the Bio-Rad NGC chromatography system for separation and purification of protein/antibody species.

• Intuitive system that adapts and grows with your purification needs
• Automate your purification for increased sample consistency and reproducibility
• NGC platform can be used for preparative as well as analytical separations

THANK YOU FOR YOUR ATTENTION



Katie S Schaefer, P Ph.D. D.

• Global Product Manager: Laboratory Scale Chromatography Systems
• katie_schaefer@bio-rad.com
• www.bio-rad.com/ngc



Peter W Winship, p, P Ph.D. D.

• Technical Product Manager: ASX-7000 Series Automation
• pete.winship@teledyne.com
• www.teledynecetac.com

QUESTIONS??????