Using a BioCel System and AssayMap Technology in Phage Display and Antibody Screening. Jason Graves September 20, 2011 AAS User Group Meeting

Transcription

1 Using a BioCel System and AssayMap Technology in Phage Display and Antibody Screening Jason Graves September 20, 2011 AAS User Group Meeting

2 My Background 15 Years of Automation and Related Experience Parke-Davis/Pfizer Used automation to support High Throughput Screening Implemented new technologies from companies such as Aurora and Rosys Developed and implemented database tools for data interpretation, upload and retrieval to and from corporate databases Rosetta (Merck) Developed and implemented new automation processes to support lab operations Quality control of automation and instrumentation Troubleshooting processes and equipment

3 My Background 15 Years of Automation and Related Experience Novo Nordisk Inflammation Research Center Evaluate processes for automation Identify and acquire necessary equipment Develop methods and supporting tools Maintain equipment Used many different platforms and equipment: Agilent (V11), Aurora, Beckman Coulter, BioTek, Hamilton, Matrix, Perkin Elmer, Rosys, Tecan, Thermo Scientific, TomTec, Zymark

4 Novo Nordisk First Big Task Integrated System that can do Tissue Culture Work 24-Well Plate Transfections Incubator Liquid Handler(s) (96 wells and Individual Channels) Sterile Environment Plate and Tip Capacity

5 BioCel #1

6 BioCel #1

7 BioCel #1

8 Phage display selections are an iterative process A selectable phenotype is linked to a genotype by inserting a displayed protein into the phage genome. In this case a large library of random antibody sequences. (Click any key to continue) An antigen is immobilized to a surface. The library is queried by exposure to the antigen of interest. Antibodies which stick, carry phage that code for them. Others are washed away, enriching the remaining phage for specific binding. The genotypes of the antibodies are rescued by infecting E. coli with the phage. New Phage are produced and the cycle is repeated After 2-3 rounds, the antibodies are produced in soluble form, assayed, and hits are chosen VL VH RBS VH CH GIII

9 First Protocol 24 Well Transfections IgG conversions of antibodies from phage display DNA combined with fectin reagent and media on Bravo Plate incubates in plate hotel Cells (1ml) come out of incubator and go to Hamilton DNA is distributed to four 24 well plates on Hamilton Cells get put back into incubator Process repeats Once finished all cells go into a shaking incubator for five days

10 Second Protocol 24 Well Harvest Cells come out of shaking incubator and go back into BioCel incubator after five days Four 24 well plates get reformatted back into a 96 well deep well block on Hamilton 96 well deep well block gets spun down in centrifuge Supernatant gets pulled off into a new storage plate on Bravo All plates get put away Process repeats

11 First Pass Yields 24 Well Transfection Automated vs Manual Transfection Yield Comparison G5_BP1 G5_BP2 G5_BP3 G5_BP4 G5_M0hr G5_M3hr G5_M6hr

12 Sequence and Yield Correlation DNA Concentration (ug/ml) A B C D E F Sequences that binned produced similar expression levels as IgG Wells with no antibody have sequences with stop codons G H Antibody Concentration (ug/ml) A B C D E F G H

13 Yield Comparison Format vs Yield Well 48 Well 24 Well 30 ml

14 Transfection and Harvest Numbers We have the capacity to process up to 5,000 clones a week (~8 hour day) ~3.5 hours/10 DNA plates transfection ~2.5 hours/10 DNA plates harvest We ve processed just over 30,000 clones in the first year of production

15 BioCel #2

16 BioCel #2

17 BioCel #2

18 High Throughput Protein Purification Unpurified IgG supernatants are fine for some ELISA and flow cytometry assays. Purified IgGs are required to run in most functional screens There is a desire to have the capability to purify large numbers of antibodies on a smaller volume scale than what is normally done by the protein purification group

19 What do we need? We need the ability to purify up to 5 ml of IgG or Fab supernatants with a minimum output of 10 μg. The method should be plate based in 96 well format to be able to be truly high throughput. The method should be endotoxin free. The method should be reasonably inexpensive.

20 What have we tried? Purification Tips Purification Plates Magnetic Beads Cost ~$150/96w ~$750/96w ~$1200/96w % Recovery 20-30% 10-15% 20-30% Automatable Yes Not really Semi Notes >20μg input -- >20μg input

21 Lab Automation 2011

22 First Run Minimal Optimization 300 μl/well of IgG clone split over two plates Equilibrate Cartridges Load Plate 1 Load Plate 2 Wash Elute Neutralize Strip Cartridges Equilibrate Cartridges

23 First Run Minimal Optimization Quantified on the Octet Red Input per well 62 μg Average Output 52 +/- 3 μg (n=62) Average % Recovery 84%

24 Head to Head with Magnetic Beads Magnetic Beads AssayMap Clone Input Output %Rec Input Output %Rec Clone # Clone # Clone # Clone # Clone # Clone # Clone # Note: Post buffer-exchange Avg 18 Avg 67

25 Low Input Purification Input (μg) Avg Recov (μg) %Recov N

26 Cartridge Use Limit Avg Output (ug) Plate Number

27 AssayMap Purification Price Reasonable (~$170/96 wells uses per box) Automation Fully automated platform Input volumes up to 200 μl per load step Endotoxin None from cartridges Recovery Excellent!! ~80% consistently