Markus Wendeler, Novartis Pharma – Technical R&D Biologics BEBPA 2013, Basel Automated Potency...
-
Upload
edith-spike -
Category
Documents
-
view
224 -
download
2
Transcript of Markus Wendeler, Novartis Pharma – Technical R&D Biologics BEBPA 2013, Basel Automated Potency...
Markus Wendeler, Novartis Pharma – Technical R&D Biologics
BEBPA 2013, Basel
Automated Potency Assays:Platforms for Binding ELISAs and Cell-Based Assays
| BEBPA 2013 | Markus Wendeler | Novartis Pharma2
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 Pharma3
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 Pharma4
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
Automated
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
Automated
Cell-Based Potency Assays
| BEBPA 2013 | Markus Wendeler | Novartis Pharma5
Robotic system: Hamilton® and Tecan® Systems
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
Source of images: http://www.hamiltonrobotics.com/
Source of image: http://www.tecan.com/
| BEBPA 2013 | Markus Wendeler | Novartis Pharma6
Generic automated Binding ELISAPlate layout and assay setup
BL: blank controlN: negative controlA/B/C/D(1-8): dose-response curve of sample A-D in duplicateR(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 Pharma7
Automated Binding ELISA: Capacity + Flexibility
Washing + pipetting steps Incubation stepsPreparation of samples Stop solution + data capture
blocking samples detection
blocking samples detection
blocking samples detection
Plate 1
Plate 2
Plate 3
Plate 14 samples + Ref
project A
Plate 24 samples + Ref
project B
Plate 34 samples + Ref
project C
3 plates per robot; 4 samples per plate12 samples + 3x Ref
project X, Y or Z
~6 h
Source of image:http://www.hamiltonrobotics.com/
| BEBPA 2013 | Markus Wendeler | Novartis Pharma8
Automated Binding ELISA: Capacity + Flexibility
First run,2 robots in
parallel
Second run,2 robots in
parallel
Run1 : Plate 1 -624 samples of project A
Maximum capacity per day
Run2 : Plate 1 -624 samples of project A
48 samples ofthe same project
Run 1: Plate 1-64 sample each of 6 different projects (A-F)
Run 2: Plate 1-64 sample each of 6 different projects (G-L)
Maximum Flexibility per day
4 samples each of12 different projects
(currently 8 different binding ELISAs running on the robot)
Sou
rce
of r
obot
imag
es:
http
://w
ww
.ham
ilton
robo
tics.
com
/
| BEBPA 2013 | Markus Wendeler | Novartis Pharma9
Automated Binding ELISAAutomated vs. manual ELISA performance
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% 100% 200%
manual automated manual automated manual automated
Accuracy (%) 100 100 100 100 100 103
Precision; GRSD (%) 4 4 4 2 4 4
Comparison of binding ELISA performed manually and on a robotic system
manual method: validated, performed with qualified instrumentsautomated 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 Pharma10
Automated Binding ELISA:Qualification of the robotic system and validation of the generic ELISA
Binding ELISA 1200% and 50% • 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 Pharma11
Automated Binding ELISA: Homogeneity on plate
0.1 1 10 100 10000.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Sample A Sample B Reference Sample C Sample D
Re
sp
on
se
Analyte concentration (ng/ml)
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 Pharma12
Automated Cell-based Potency AssayConcept: 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
Mid to long term solutionRobotic 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
Short term solution for seeding of analytical cells:• Manual seeding of cells or• Use of read-to-use cryopreserved cell aliquots
Source of image: http://www.tecan.com/
Source of image: http://www.tecan.com/
| BEBPA 2013 | Markus Wendeler | Novartis Pharma13
Automated Cell-based Potency AssayAnalytical cells for automated assays – cryopreserved single-use aliquots
EC50 [ng/mL]
Procedure 1
Medium A 19Cell Culture 24Medium B 18Cell Culture 20
Procedure 3
Medium A 24Cell Culture 23Medium B 19Cell Culture 20
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
| BEBPA 2013 | Markus Wendeler | Novartis Pharma14
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 Pharma15
Acknowledgements
Guillaume Rey• Excel and robot programming,• Performance of experiments
Christian Kaluschke, Cécile Willauer, Bernadette Hauss• Performance of experiments
Olivier Graf and Kamal Egodage• Automation expertise and financial support
Tom Millward, Christoph Bächler, and Irmgard Hofmann• Helpful bioanalytical discussions