Display and Antibody Screening Jason Graves September 20, 2011 AAS - PowerPoint PPT Presentation

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

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

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

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

BioCel #1

BioCel #1

BioCel #1

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

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

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

First Pass Yields – 24 Well Transfection Automated vs Manual Transfection Yield Comparison 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 G5_BP1 G5_BP2 G5_BP3 G5_BP4 G5_M0hr G5_M3hr G5_M6hr

Sequence and Yield Correlation DNA Concentration (ug/mL) • Sequences that binned 1 2 3 4 5 6 7 8 9 10 11 12 A 256 243 245 249 252 255 254 267 277 276 282 5 produced similar expression B 265 243 146 236 181 255 233 264 264 258 11 199 levels as IgG C 278 287 242 260 279 273 264 246 287 271 304 265 • Wells with no antibody have D 272 274 246 339 218 246 278 252 98 268 187 274 E 231 292 277 283 249 267 260 261 285 291 282 298 sequences with stop codons F 306 317 298 276 294 266 295 285 282 304 308 188 G 301 249 321 317 299 325 309 315 335 318 329 345 H 5 350 328 328 327 307 319 310 330 304 325 349 Antibody Concentration (ug/mL) 1 2 3 4 5 6 7 8 9 10 11 12 A 12 0 13 15 67 104 0 0 0 0 27 0 B 0 1.4 0 0 0 0 0 0 13 12 0 0 C 0 51 0 0 12 11.8 0 0 0 0 0 0 D 0 0 0 32 25 0 5.4 0 12 12 0 7.3 E 0 0 0 0 0 53.2 0 5.9 62 24 0 12 F 0 0 7.5 0 0 63.9 12 8.4 0 0 6 0 G 0 0 4.1 0 2.6 6.97 0 0 9.5 0 11 5.6 H 0 23 14 0 14 13.3 0 11 0 0 0 9.4

Yield Comparison Format vs Yield 250 200 150 100 50 0 96 Well 48 Well 24 Well 30 mL

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

BioCel #2

BioCel #2

BioCel #2

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

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 μ g. • 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 .

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

Lab Automation 2011

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

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

Head to Head with Magnetic Beads Magnetic Beads AssayMap Clone Input Output %Rec Input Output %Rec Clone # 1 675 179 27 243 194 80 Clone # 2 760 166 22 273 158 58 Clone # 3 775 143 18 279 184 66 Clone # 4 675 94 14 243 141 58 Clone # 5 925 89 10 333 241 72 Clone # 6 770 139 18 277 173 62 Clone # 7 865 157 18 311 218 70 Avg 18 Avg 67 • Note: Post buffer-exchange

Low Input Purification Input (μg) Avg Recov (μg) %Recov N 20 15.8 78.8 24 10 7.3 73.3 24 5 3.3 65.0 32

Cartridge Use Limit 18 16 14 Avg Output (ug) 12 10 8 6 4 2 0 0 2 4 6 8 10 12 14 16 Plate Number

AssayMap Purification • Price • Reasonable (~ $170/ 96 wells – 10-15 uses per box) • Automation • Fully automated platform • Input volumes • up to 200 μ L per load step • Endotoxin • None from cartridges • Recovery • Excellent!! ~ 80% consistently