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 of 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.

K 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 o Goal 2: Purify large quantities of high purity proteins  Goal 3: Utilize these proteins in the research, development, Confirm  Polishi hing Purity ty Purity and therapeutic treatment •High resolution resins Intermediat ate •Size Exclusion •Mixed Mode •Ion exchange Chromatographic protein purification workflows typically utilize •Desalting  •Hydrophobic Capture re Interaction multiple column chemistries •Tagged Protein •Native Protein Preparative – small to large scale production  Analytical – high resolution separation and purity confirmation  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 o Rituxan (rituximab): Treatment for certain autoimmune diseases and cancers o

K MONOCOLONAL ANTIBODY DEVELOPMENT (TWO WORKFLOWS) Screeni ning o of A Anti ntibody y Cand ndidates – Express IgG antibody candidates  on a small scale for efficacy testing ASX-500 Series autosampler + NGC with Sample Pump + Inlet Valve + o Fraction Collector Confir irm P Purit ity o of Productio ion – Analytical analysis for sample purity  and integrity MVX-7100 µL Workstation + NGC with analytical flow cell o

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

P 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) o µL sample introduction scale (MVX-7100 µL Workstation) o Configurable, robust and reliable  Undertake unattended chromatography instrument operation  MVX VX-7100 µ µL Works ksta tati tion 24 hours a day, 7 days per week operation (improve efficiency) o Allow purification of 84 samples in two days o Accurate and precise sample uptake and introduction  Minimize sample introduction error o ASX ASX-500 S Seri ries s Autosa sample ler

K PART 3. PREPARATIVE PROTEIN PURIFICATION WORKFLOW

K MONOCOLONAL ANTIBODY DEVELOPMENT (TWO WORKFLOWS) Screeni ning o of A Anti ntibody y Cand ndidates – Express IgG antibody candidates  on a small scale for efficacy testing ASX-500 Series autosampler + NGC with Sample Pump+ Inlet Valve + o Fraction Collector Confir irm P Purit ity o of Productio ion – Analytical analysis for sample purity  and integrity MVX-7100 µL Workstation + NGC with analytical flow cell o

K PREPARATIVE PROTEIN PURIFICATION WORKFLOW Sample uptake Sample Transfer to ASX- and introduction preparation/pre- 500 Series to the NGC treatment automation instrument Tandem Repeated Fraction chromatography tandem collection (Protein A to chromatography Desalting) Purity Data generation confirmation

K SCREENING OF ANTIBODY CANDIDATES -- SAMPLE PRODUCTION

P 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  ASX ASX-280 A Autos osampler 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 osampler

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  Air S Sensor or 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 

K TANDEM CHROMATOGRAPHY: PROTEIN A TO DESALTING IgG P Protein Protein A A El Eluti ution B Buf uffer Prot otein A n A Desalting ng Fr Fractions Captures IgG Size Separation pH 7.3 Elutes at pH 3.0 pH 7.3

K NGC AND ASX FOR AUTOMATED TANDEM CHROMATOGRAPHY Sample Protein A elution Desalting Column Desalting column Protein A column Clean system for Application Protein A column + desalting Elution and equilibration equilibration next sample wash ASX + Inlet Valve + column sample Fraction from ASX Sample Pump application Collection Repeat t for a all s sampl mples es o on ASX

K OVERLAY OF AUTOMATED IGG PURIFICATION SERIES WITH ASX H 7. 7.3 pH Conductivity mS/cm A 280 nm (mAU) A 280 nm (mAU) ity 22 22 mS Conductiv ivit mS/cm /cm “Me Method is is eas asy t to u use an and truly han ands-of off” Volume