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


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)


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


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/


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


Automated Binding ELISA: Capacity + Flexibility

Washing + pipetting steps Incubation stepsPreparation of samples Stop solution + data capture

blocking samples detection



Plate 1

Plate 2

Plate 3

Plate 14 samples + Ref

project A


project B


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/


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.

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/


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.


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.


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


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




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


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


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

Markus Wendeler, Novartis Pharma – Technical R&D Biologics BEBPA 2013, Basel Automated Potency...

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