Increasing the Throughput of Challenging Assays by Automation Malcolm Crook Process Analysis & Automation Ltd Farnborough

What is a challenging assay ? Challenging instrument to automate –Physically –Software control, no automation interface Multiple PCs required

Why are we doing this Assay Development Low/High Throughput Screening

 Bioinformatics  Molecular biology  Expression studies  Stable cell generation  Bioinformatics  Molecular biology  Expression studies  Stable cell generation  Potency  Selectivity  Potency  Selectivity  Multiple platforms  Multiple readouts  Multiple platforms  Multiple readouts  'Big pharma' systems  Library choice  'Big pharma' systems  Library choice  Functional effects  Target-related  HERG  Cytotoxicity  Apoptosis  Cell proliferation  CYP450  Functional effects  Target-related  HERG  Cytotoxicity  Apoptosis  Cell proliferation  CYP450 Assay Target HTS Hit Evaluation Lead Optimisation Drug Discovery Process

Requirements for an Assay High-throughput Low false-positive rate Low false-negative rate Direct measure of function Good correlation with electrophysiology Reliability Reproducibility Amenable to miniaturization Low cost

Ion Channels by Atomic Absorption At BioFocus, Cambridge

assembly of proteins circular arrangement of proteins closely packed around water-filled pore large-pore channels permit –passage of ions –more or less indiscriminate What is an ion channel ?

archetypal channel pore –one or two atoms wide –conducts a specific species of ion –sodium or potassium –conveyed through membrane single file –fast –access to the pore is governed by a "gate“ –opened or closed by chemical/electrical signals Temperature mechanical force

Redistribution voltage-dependent dyes FRET-based technology Radioligand binding Automated two-electrode voltage clamp Automated whole-cell patch clamp Planar patch clamp Radiometric ion flux Non-radiometric ion flux Measuring Ion Channels Non-radiometric ion flux Radioactive Cost Low throughput

Chosen method Atomic absorption spectrometry 85 Rb+ Hollow cathode Rubidium lamp Air/acetylene flame

Advantages of AAS Health and Safety Ease of handling Cost of components Cost of disposal Environmental Impact Sensitivity No time limits to read samples Decay or Licence constraints

Manual Assay Processing electronics Spray chamber and nebulizer Hollow cathode lamp source Flame Monochromator Photomultiplier detector Data processing and instrument control

Detection

Manual Assay Atomic Absorption Burner Autosampler

Automated Assay Ion Channel Screening Cells processed using appropriate automation Supernatants analysed for Ion Content –Single burner system (low throughput) –Multi burner system

Automated Platform: Reader platform initial design SOLAAR S AAS #1 AutoSampler 4 Position #1 SOLAAR S AAS #2 AutoSampler 4 Position #2 SOLAAR S AAS #3 AutoSampler 4 Position #3 SOLAAR S AAS #4 AutoSampler 4 Position #4 Linear Track Robotic arm 80 microplate On-line Storage Operating system e.g. OVERLORD™ Data Processing Activity Base All equipment “off the shelf”

Schematic AA #3 AA #4 AA #2 AA #1 Robot bc stacker

What is the challenge for this assay ? 1.Scheduler has to be flexible to time delays 2.SOLAAR software-no automation interface 3.Four AAs required to achieve throughput 4.Autosampler was not robot friendly 5.Safety 6.Data handling/data quality

1. Scheduler needs to be flexible AA run times not the same Throughput critical Error trapping and recovery a necessity

Scheduler control options Time resolved –Static –Pre-emptive –All decisions are taken before the run starts. i.e. before the “GO” button is pressed Event driven –Real time –Dynamic –All decisions are taken during the time frame of the run, i.e. once the “GO” button is pressed Process Analysis & Automation have both !

Time resolved Advantages: –one or more accurate timings are guaranteed –easy to set up Disadvantages –inflexible –decisions can not be taken during the run –error recovery more difficult

Event Driven Advantages: –flexible –decisions can be taken during the run –error recovery easy Disadvantages –accurate timings are more difficult to set up –two accurate timings in a run are impossible

Scheduling - the Choice real-time OVERLORD Workstation pre-emptive OVERLORD Scheduler either

Real Time Instrument Control Options Sequential Round robin

Control system Main controller & Data processing OVERLORD AA#1 instrument control NetLORD node 1 AA#2 instrument control NetLORD node 2 AA#3 instrument control NetLORD node 3 AA#4 instrument control NetLORD node 4 Multiple port switchSite computer services

Instrument control – Round Robin Load, unload busy, error ? AA #2 node Ready ? Error AA #3 node Ready ? AA #4 node Ready ? Start Run Run AAS Load plate AA #1 node Ready ? Data HandlingCycle Run complete ? Unload plate Busy

2. SOLAAR software Requires automation interface There isn’t one !!!! Use the OVERLORD keystroke API Emulates a user

3. Four AAs required for throughput Use NetLORD Remote OVERLORD nodes Makes control structure easier –Node in 4 states Ready for load Ready for unload Busy Error state NetLORD nodes addressed by IP address Common folders for data transfer Redundancy

4. Autosampler was not robot friendly

5. Safety Hazards –AAS instrument Locked room Air conditioning –Acetylene gas Only run during the day Gas detectors Time out on AAS flames –Robot Hamilton SWAP stops when touched Hamilton SWAP can be recovered

6. Data handling/data quality Barcodes Data file renaming for integrity Data transfer to the server Real time data analysis for quality check

Luminex Instrument

Luminex 100 Bead based with 100 assays per well Small sample volume 1000 samples a day reproducible

Higher throughput Traditional ELISA 1 measurement/well xMAP technology 100 measurements/well

Analysis method Beads sucked up past fluorescence source Fluorescence detected

Luminex xMAP Technology Comparison Q-PCR ELISA Microarray Multiplex Throughput

What is the challenge for this assay ? Luminex software - no automation interface Luminex instrument – not robot friendly Caliper Twister I Lids

Luminex instrument – not robot friendly Plate tray only suitable for fingers –Modify the tray with a hacksaw Software –Version 1.7 no automation interface –Keyboard control possible

Caliper Twister I Limited control system –OVERLORD has own Twister I control Lids –Can be a problem –Relidding especially

Luminex Shaker Delidding Twister I

Conclusions Instruments can be automated –Keyboard control –Plate access Unusual assays –Real time scheduler –Round robin method Twister I robots good tools –Need more than basic software OVERLORD can control > 220 instruments And remember …..

Real high throughput screening !