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

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Presentation on theme: "Markus Wendeler, Novartis Pharma – Technical R&D Biologics BEBPA 2013, Basel Automated Potency Assays: Platforms for Binding ELISAs and Cell-Based Assays."— Presentation transcript:

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

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 Pharma2

3 Automated Platforms for Analytics | BEBPA 2013 | Markus Wendeler | Novartis Pharma3 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)

4 Automated Bioanalytics Platforms | BEBPA 2013 | Markus Wendeler | Novartis Pharma4 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

5 Robotic system: Hamilton ® and Tecan ® Systems | BEBPA 2013 | Markus Wendeler | Novartis Pharma5 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/

6 Generic automated Binding ELISA Plate layout and assay setup | BEBPA 2013 | Markus Wendeler | Novartis Pharma6 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

7 Automated Binding ELISA: Capacity + Flexibility | BEBPA 2013 | Markus Wendeler | Novartis Pharma7 Washing + pipetting steps Incubation stepsPreparation of samplesStop solution + data capture blockingsamples detection blockingsamples detection blockingsamples detection Plate 1 Plate 2 Plate 3 Plate 1 4 samples + Ref project A Plate 1 4 samples + Ref project A Plate 2 4 samples + Ref project B Plate 2 4 samples + Ref project B Plate 3 4 samples + Ref project C Plate 3 4 samples + Ref project C 3 plates per robot; 4 samples per plate 12 samples + 3x Ref project X, Y or Z 3 plates per robot; 4 samples per plate 12 samples + 3x Ref project X, Y or Z ~6 h Source of image: http://www.hamiltonrobotics.com/

8 Automated Binding ELISA: Capacity + Flexibility | BEBPA 2013 | Markus Wendeler | Novartis Pharma8 First run, 2 robots in parallel Second run, 2 robots in parallel Run1 : Plate 1 -6 24 samples of project A Run1 : Plate 1 -6 24 samples of project A Maximum capacity per day Run2 : Plate 1 -6 24 samples of project A Run2 : Plate 1 -6 24 samples of project A 48 samples of the same project Run 1: Plate 1-6 4 sample each of 6 different projects (A-F) 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) 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: http://www.hamiltonrobotics.com/

9 Automated Binding ELISA Automated vs. manual ELISA performance | BEBPA 2013 | Markus Wendeler | Novartis Pharma9 Binding ELISA 1, (n=4) 50%100%200% manualautomatedmanualautomatedmanualautomated Accuracy (%)951109399 96 Precisison; GRSD (%)6193131 Binding ELISA 2, (n=4) 50%100%200% manualautomatedmanualautomatedmanualautomated Accuracy (%)100 103 Precision; GRSD (%)444244 Comparison of binding ELISA performed manually and on a robotic system 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.

10 Automated Binding ELISA: Qualification of the robotic system and validation of the generic ELISA | BEBPA 2013 | Markus Wendeler | Novartis Pharma10 Binding ELISA 1 200% 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.

11 Automated Binding ELISA: Homogeneity on plate | BEBPA 2013 | Markus Wendeler | Novartis Pharma11 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

12 Automated Cell-based Potency Assay Concept: Automated assay performance + cell culture maintenance | BEBPA 2013 | Markus Wendeler | Novartis Pharma12 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 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 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/

13 Automated Cell-based Potency Assay Analytical cells for automated assays – cryopreserved single-use aliquots | BEBPA 2013 | Markus Wendeler | Novartis Pharma13 EC50 [ng/mL] Procedure 1 Medium A19 Cell Culture24 Medium B18 Cell Culture20 Procedure 3 Medium A24 Cell Culture23 Medium B19 Cell Culture20 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

14 Summary | BEBPA 2013 | Markus Wendeler | Novartis Pharma14 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

15 Acknowledgements | BEBPA 2013 | Markus Wendeler | Novartis Pharma15 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


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