Transcript Slide 1

Automated Potency Assays:
Platforms for Binding ELISAs and Cell-Based Assays
Markus Wendeler, Novartis Pharma – Technical R&D Biologics
BEBPA 2013, Basel
Agenda
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Automated platforms for analytics
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Automated binding ELISA for potency determination
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Flexibility and method performance
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Automated cell-based potency assays – concept
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Summary
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Automated Platforms for Analytics
Automated Phys.-Chem Analytics
• SEC, Reverse Phase, CEX, CE-SDS (Caliper)
• pH,Turbidity, DLS
• Carbohydrate Pattern
Automated Bioanalytics
• Impurity ELISA (Host Cell Proteins, protein A)
• Standard Binding ELISA (potency, identity)
• Cell-based Bioassay (potency, identity)
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Automated Bioanalytics Platforms
(~1500 samples/year)
• Performs HCP impurity analytics for all CHO-derived development
projects on a routine basis
• Performs Protein A impurity analytics for all mAbs development
projects on a routine basis
Automated
Potency Binding
ELISA
• Generic potency binding ELISA for characterization of early mAb
development projects.
• System and method currently qualified to perform potency binding
ELISAs for release and stability QC analytics
Automated
Cell-Based
Potency Assays
• System currently being implemented to support parallel processing of
automated cell-based-potency assays for different projects
• Full automation of cell culture maintenance and preparation of cells
for assays foreseen in a second step
Automated
Impurity ELISA
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Robotic system: Hamilton® and Tecan® Systems
Source of image: http://www.tecan.com/
Source of images: http://www.hamiltonrobotics.com/
Fully automated system for binding ELISAs comprises:
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Robotoc systems for liquid, sample, and plate handling
Balance for gravimetric dilution
Plate shaker and reagent cooling device
Plate washer (96 and 384 well plates)
Plate reader (absorption, fluoresecence, luminescence)
Automated data capture, analysis and assay documentation
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Generic automated Binding ELISA
Plate layout and assay setup
Increase number
of dosage points
in linear part
asymptotes
BL: blank control
N: negative control
A/B/C/D(1-8): dose-response curve of sample A-D in duplicate
R(1-8): dose-response curve of reference in duplicate
Sample dilution (fully automated):
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1. dilution gravimetrically
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following dilutions volumetrically
Coating reagents:
projec-specific
Analyte:
projec-specific
Detection antibody:
generic
Potency Analysis:
Detection:
generic
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Parallel line analysis (linear fit or 4P fit)
Coating/Wash/Assay buffer:
generic
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Range of 50% to 200%
Dilution and Assay Plates:
generic
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Plate and sample SST criteria
Coating/Blocking/Incubation times:
generic
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Automated assay documentation
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Automated Binding ELISA: Capacity + Flexibility
Source of image:
http://www.hamiltonrobotics.com/
3 plates per robot; 4 samples per plate
12 samples + 3x Ref
project X, Y or Z
Plate 1
4 samples + Ref
project A
Plate 1
Plate 2
Plate 2
4 samples + Ref
project B
blocking
Plate 3
4 samples + Ref
project C
samples
blocking
Plate 3
detection
samples
blocking
Washing + pipetting steps
samples
Preparation of samples
~6 h
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detection
Incubation steps
detection
Stop solution + data capture
Automated Binding ELISA: Capacity + Flexibility
Maximum capacity
per day
Maximum Flexibility
per day
Run1 : Plate 1 -6
24 samples of project A
Run 1: Plate 1-6
4 sample each of 6 different projects (A-F)
Second run,
2 robots in
parallel
Run2 : Plate 1 -6
24 samples of project A
48 samples of
the same project
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Run 2: Plate 1-6
4 sample each of 6 different projects (G-L)
4 samples each of
12 different projects
(currently 8 different binding ELISAs running on the robot)
Source of robot images: http://www.hamiltonrobotics.com/
First run,
2 robots in
parallel
Automated Binding ELISA
Automated vs. manual ELISA performance
Comparison of binding ELISA performed manually and on a robotic system
Binding ELISA 1, (n=4)
50%
100%
200%
manual
automated
manual
automated
manual
automated
Accuracy (%)
95
110
93
99
99
96
Precisison; GRSD (%)
6
1
9
3
13
1
Binding ELISA 2, (n=4)
50%
manual
automated
100%
manual
automated
200%
manual
automated
Accuracy (%)
100
100
100
100
100
103
Precision; GRSD (%)
4
4
4
2
4
4
manual method:
automated method:
validated, performed with qualified instruments
scientifically sound, robotic system not qualified
Automated binding ELISA performs with similar or higher quality when
compared to the validated manual binding ELISA.
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Automated Binding ELISA:
Qualification of the robotic system and validation of the generic ELISA
Binding ELISA 1
200% and 50%
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8 different binding ELISAs are
currently running on the robotic
platform
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Robotic binding ELISA platform
currently being qualified to support
release and stability analyses.
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Validation of 3 binding ELISA
methods on robot (generic
automated binding ELISA)
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For a newly developed binding
ELISA feasible to run with the
generic setup only
 coating reagent concentration
 analyte starting concentration
 dose-response curve
need to be adapted.
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Automated Binding ELISA: Homogeneity on plate
Dose response curves from one plate
(samples and reference all 100%)
3.5
3.0
Response
2.5
2.0
1.5
Sample A
Sample B
Reference
Sample C
Sample D
1.0
0.5
0.0
0.1
1
10
100
1000
Analyte concentration (ng/ml)
The sample location on the assay plate
has no influence on the potency results
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Automated Cell-based Potency Assay
Concept: Automated assay performance + cell culture maintenance
Robotic System 1: Potency Assay Performance
Allows the parallel performance of a certain subset of
• reporter gene assays,
• kinase receptor activation assay
• cytokine release assay
• cytotoxicity/proliferation assays
Source of image: http://www.tecan.com/
Short term solution for seeding of analytical cells:
• Manual seeding of cells or
• Use of read-to-use cryopreserved cell aliquots
Mid to long term solution
Robotic System 2: Cell Culture Maintenance
• Culture and amplification of analytical cell lines
• Prepares assay plates for assays running on
System 1
• Prepares analytical cell banks
Source of image: http://www.tecan.com/
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Automated Cell-based Potency Assay
Analytical cells for automated assays – cryopreserved single-use aliquots
Use cryopreserved ready-to-use analytical cell
aliquots:
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No constant cell culture maintenance
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Flexible assay start independent of cell availability
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Assay to assay results more reproducable (no passage)
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Need of suitable freezing medium ensuring cell viability
and performance in potency assay
Differences in freezing medium and thawing procedure
shows no impact on EC50
EC50 [ng/mL]
Procedure 1
Higher Pos/Neg
due to lower
assay background
Procedure 3
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Medium A
Cell Culture
Medium B
Cell Culture
Medium A
Cell Culture
Medium B
Cell Culture
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24
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24
23
19
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Summary
 Flexible ELISA platforms for impurity and potency determination
 Increase of analytical capacity by centralization and generalization
 Flexible project support with generic automated ELISA
 High quality data of the automated method
 Current concept of automated cell-based potency assays
 Ready-to-use frozen aliquots of analytical cells
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Acknowledgements
Guillaume Rey
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Excel and robot programming,
Performance of experiments
Christian Kaluschke, Cécile Willauer, Bernadette Hauss
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Performance of experiments
Olivier Graf and Kamal Egodage
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Automation expertise and financial support
Tom Millward, Christoph Bächler, and Irmgard Hofmann
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Helpful bioanalytical discussions
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