“One for all”

19-Jul-21 Dr. Hanna Leins, Nuvisan7th YSS, 10-11/06/2021

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“One for all”

Development of a generic ADA Assay on the Gyrolab Platform

Advantages• Detection of all ADA isotypes• Can be used in all species

Disadvantages• Drug inference is a major

challenge - might require cumbersome acid dissociation approaches• Use of drug-specific reagents• Development is effortful


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https://www.mesoscale.com/~/media/files/technical%20notes/bridging%20immunogenicity%20assay.pdf?la=en

Development of a generic ADA Assay: reduction of development effort & shorten time to assay readiness


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https://www.gyrosproteintechnologies.com/gyrolab-technology

Miniaturized Immunoassays• Automated transfer of reagents / samples from a storage microplate onto a disc

containing an affinity capture column at nanoliter scaled filled with streptavidin coated beads

• Requires very small reagent and sample volumes• low sample consumption might allow to assess PK and ADAs from the same

laboratory animal


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Add excessdrug

Spin over disc withimmobilized anti-drugmAb

Detect ADA-drugcomplexes with labeledanti-species antibodies

Sample with ADAsand drug

Alexa Fluor

Sanofi

PRINCIPLE OF THE GENERIC ADA ASSAY

Drug human antibodyADAs mouse anti-human antibodiesCapture reagent anti-human antibodyDetection reagent AlexaFluor648-tagged anti-mouse antibody


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System Suitability Controls• HPC 5000 (5000 ng/mL)• LPC 1000 (1000 ng/mL)• NC

Sensitivity• Ratio LPC 1000 / NC > 1.5

Drug Tolerance• to be determined

No formal cut-point determination. ADA positive samples are to be identified relatively to their pre-dose value (cut-off: >2 x pre-dose).

Starting Conditions (based on DoE)

Capture Antibody 75 µg/mLDetection Antibody 8 nMDrug Spike (Orignator) 30 µg/mLBioaffy 200 CD


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Plasma Serum BufferHPC 5000 4.26 4.46 2.78

LPC 1000 2.32 2.27 0.83

NC 1.65 1.81 0.226

LPC / NC 1.41 1.26 3.69

Optimized Assay ConditionsCapture Antibody 700 nMDetection Antibody 8 nMDrug Spike (Orignator) 30 µg/mLMRD 1:20Bioaffy 1000 CD

Problems• Ratio LPC / NC < 1.5 in matrix• Signal shift from buffer to matrix

0,001,002,003,004,005,00

NC LPC 1000 HPC 5000

Plasma Serum Buffer


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Possible Solutions• Test different detection antibody concentrations• Titrate drug spike• Test different capture antibodies

Capture 1 Capture 2HPC 5000 2.18 37.4

LPC 1000 0.639 10.7

NC 0.173 0.129

LPC / NC 3.69 82.90

10

20

30

40

NC LPC 1000 HPC 5000

Capture 1

Capture 2

Matrix: Buffer


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Serum Plasma BufferCapture 1 1.18 1.16 1.41

Capture 2 2.30 2.35 21.2

Capture 3 1.28 1.45 1.51

LPC / NC Ratio (MRD 1:100)

0,000

0,500

1,000

HPC 5000 LPC 1000 NC

Capture 1

Serum Plasma Buffer

0,00

5,00

10,00


Capture 2

Serum Plasma Buffer

0,00

5,00

10,00

15,00


Capture 3

Serum Plasma Buffer


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Capture Antibody

BiotinStreptavidin Bead

ADA-Drug

complex

Theoretical drug tolerance• Each molecule of capture

antibody can bind two molecules of drug

• Concentration capture antibody: 700 nM

• Saturation should occur starting at 1400 nM (2 x 700 nM) drug spike in; corresponding to a concentration of ≈ 200 µg/mLdrug spike in

Drug

ADA

0,00

1,00

2,00

3,00

4,00

5,00

6,00


50

100

200

300

400

Drug spike [µg/mL]


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50 µg/mL 100 µg/mL 200 µg/mL 300 µg/mL 400 µg/mLHPC 5000 5.91 5.65 4.33 4.05 3.51LPC 1000 2.08 1.79 1.58 1.50 1.22NC 0.758 0.758 0.703 0.681 0.652LPC / NC 2.74 2.37 2.24 2.20 1.87

Drug Tolerance > 400 µg/mL


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Pre-Study Drug Tolerance RunDrug Spike LPC /NC30 µg/mL 2.82

130 µg/mL 2.90230 µg/mL 2.86530 µg/mL 2.12

1030 µg/mL 1.75

Drug Tolerance > 1000 µg/mL

Sample Measurement

Subject Time RU Cut off ( > 2 x pre-dose) ADA Result

1 0 h (pre-dose)504 h

1.813.87 3.62 POS.

2 0 h (pre-dose)504 h

0.1481.63 0.296 POS.

0,00

2,00

4,00

6,00

8,00


30

130

230

530

1030

Drug spike [µg/mL]


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A generic “one for all” ADA Assay on the Gyrolab Platform

was successfully developed• can now be used off-the-shelf to test for ADA in pre-clinical mouse

models• high drug tolerance, > 1000 µg/mL

Be brave enough to change your assay reagents!


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Any questions? Feel free to ask!

Acknowledgement:

Sanofi Aventis Deutschland GmbH

Dr. Katharina Michalik-Gessler

Dr. Daniel Kramer

Nuvisan GmbH, Department Immunoassays

Katja Zeiser

Dr. Uwe Kärcher

Dr. Michaela Golob

“One for all”

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