1. Automated Potency Assays: Platforms for Binding ELISAs and Cell-Based Assays
Markus Wendeler, Novartis Pharma – Technical R&D Biologics
BEBPA 2013, Basel

2. Agenda Automated platforms for analytics
Automated binding ELISA for potency determination
Flexibility and method performance
Automated cell-based potency assays – concept
Summary
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

3. 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)
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

4. Automated Bioanalytics Platforms
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
Impurity ELISA
(~1500 samples/year)
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
Potency Binding ELISA
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
Cell-Based Potency Assays
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

5. Robotic system: Hamilton® and Tecan® Systems
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Source of images:
Fully automated system for binding ELISAs comprises:
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
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

6. Generic automated Binding ELISA Plate layout and assay setup
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
Increase number of dosage points in linear part
asymptotes
Coating reagents: projec-specific
Analyte: projec-specific
Detection antibody: generic
Detection: generic
Coating/Wash/Assay buffer: generic
Dilution and Assay Plates: generic
Coating/Blocking/Incubation times: generic
Potency Analysis:
Parallel line analysis (linear fit or 4P fit)
Range of 50% to 200%
Plate and sample SST criteria
Automated assay documentation
Sample dilution (fully automated):
1. dilution gravimetrically
following dilutions volumetrically
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

7. Automated Binding ELISA: Capacity + Flexibility
Washing + pipetting steps
Incubation steps
Preparation of samples
Stop solution + data capture
blocking
samples
detection
Plate 1
Plate 2
Plate 3
4 samples + Ref
project A
project B
project C
3 plates per robot; 4 samples per plate
12 samples + 3x Ref
project X, Y or Z
~6 h
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| BEBPA 2013 | Markus Wendeler | Novartis Pharma

8. Automated Binding ELISA: Capacity + Flexibility
First run,
2 robots in parallel
Second run,
Run1 : Plate 1 -6
24 samples of project A
Maximum capacity per day
Run2 : Plate 1 -6
48 samples of
the same project
Run 1: Plate 1-6
4 sample each of 6 different projects (A-F)
Run 2: Plate 1-6
4 sample each of 6 different projects (G-L)
Maximum Flexibility per day
4 samples each of
12 different projects
(currently 8 different binding ELISAs running on the robot)
Source of robot images:
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

9. 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
Accuracy (%) 95
110 93 99 96
Precisison; GRSD (%) 6 1 9 3 13
Binding ELISA 2, (n=4)
50%
100%
200%
manual
automated
Accuracy (%)
100
103
Precision; GRSD (%) 4 2
manual method: validated, performed with qualified instruments
automated method: scientifically sound, robotic system not qualified
Automated binding ELISA performs with similar or higher quality when compared to the validated manual binding ELISA.
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

10. Automated Binding ELISA: Qualification of the robotic system and validation of the generic ELISA
8 different binding ELISAs are currently running on the robotic platform
Robotic binding ELISA platform currently being qualified to support release and stability analyses.
Validation of 3 binding ELISA methods on robot (generic automated binding ELISA)
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.
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

11. Automated Binding ELISA: Homogeneity on plate
Dose response curves from one plate
(samples and reference all 100%)
The sample location on the assay plate has no influence on the potency results
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

12. 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
Short term solution for seeding of analytical cells:
Manual seeding of cells or
Use of read-to-use cryopreserved cell aliquots
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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
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| BEBPA 2013 | Markus Wendeler | Novartis Pharma

13. Automated Cell-based Potency Assay Analytical cells for automated assays – cryopreserved single-use aliquots
Use cryopreserved ready-to-use analytical cell aliquots:
No constant cell culture maintenance
Flexible assay start independent of cell availability
Assay to assay results more reproducable (no passage)
Need of suitable freezing medium ensuring cell viability and performance in potency assay
Higher Pos/Neg due to lower assay background
Differences in freezing medium and thawing procedure shows no impact on EC50
EC50 [ng/mL]
Procedure 1
Medium A 19
Cell Culture 24
Medium B 18 20
Procedure 3 23
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

14. 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
| BEBPA 2013 | Markus Wendeler | Novartis Pharma

15. Acknowledgements Guillaume Rey
Excel and robot programming,
Performance of experiments
Christian Kaluschke, Cécile Willauer, Bernadette Hauss
Olivier Graf and Kamal Egodage
Automation expertise and financial support
Tom Millward, Christoph Bächler, and Irmgard Hofmann
Helpful bioanalytical discussions
| BEBPA 2013 | Markus Wendeler | Novartis Pharma