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

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

Jan 29, 2024 243 likes •530 views

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

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Using a BioCel System and AssayMap Technology in Phage Display and Antibody Screening

Jason Graves September 20, 2011 AAS User Group Meeting

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SLIDE 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
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SLIDE 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

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SLIDE 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
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SLIDE 5

BioCel #1

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SLIDE 6

BioCel #1

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SLIDE 7

BioCel #1

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SLIDE 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)

VL VH RBS VH CH GIII

  • 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

  • An antigen is immobilized to a

surface.

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SLIDE 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

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SLIDE 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
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SLIDE 11

First Pass Yields – 24 Well Transfection

Automated vs Manual Transfection Yield Comparison

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 G5_BP1 G5_BP2 G5_BP3 G5_BP4 G5_M0hr G5_M3hr G5_M6hr

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SLIDE 12

Sequence and Yield Correlation

  • Sequences that binned

produced similar expression levels as IgG

  • Wells with no antibody have

sequences with stop codons

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 B 265 243 146 236 181 255 233 264 264 258 11 199 C 278 287 242 260 279 273 264 246 287 271 304 265 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 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 1 2 3 4 5 6 7 8 9 10 11 12 A 12 13 15 67 104 27 B 1.4 13 12 C 51 12 11.8 D 32 25 5.4 12 12 7.3 E 53.2 5.9 62 24 12 F 7.5 63.9 12 8.4 6 G 4.1 2.6 6.97 9.5 11 5.6 H 23 14 14 13.3 11 9.4

DNA Concentration (ug/mL) Antibody Concentration (ug/mL)

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SLIDE 13

Yield Comparison

Format vs Yield

50 100 150 200 250 96 Well 48 Well 24 Well 30 mL

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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

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BioCel #2

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BioCel #2

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SLIDE 17

BioCel #2

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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

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SLIDE 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.
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SLIDE 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
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SLIDE 21

Lab Automation 2011

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SLIDE 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
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SLIDE 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%
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SLIDE 24

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
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SLIDE 25

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

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SLIDE 26

Cartridge Use Limit

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

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SLIDE 27

AssayMap Purification

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