Increasing the Throughput of Challenging Assays by

Automation

Malcolm CrookProcess 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

AssayTarget 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 ?

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 • 85Rb+• 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 chamberand nebulizer

Hollow cathodelamp source

FlameMonochromator

Photomultiplierdetector

Data processingand instrument

control

Detection

Manual Assay

AtomicAbsorption

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

Rob

ot

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 friendly5. Safety6. 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-timeOVERLORD Workstation

pre-emptiveOVERLORD 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 switch

Site computer services

Instrument control – Round Robin

Load, unloadbusy, error ?

AA #2 nodeReady ?

Error

AA #3 nodeReady ?

AA #4 nodeReady ?

Start RunRun AAS

Load plateAA #1 nodeReady ?

Data Handling

Cycle

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

LuminexxMAP 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

ShakerDeliddin

g

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 !