Module 9 2024

03/09/2024

Neutralisation Assay

Determination of the neutralising potential of induced ADA is required by regulators - deviation needs a strong justification. Principle : presence of neutralising antibodies (NAb) directed against the therapeutic → biological activity induced by a known concentration of the therapeutic will be reduced or abolished.

Neutralisation is therefore assessed by measuring the degree of therapeutic activity inhibition induced by the sample.

Two types of assays: cell-based (bioassay) and non-cell-based (competitive ligand binding assay)

MoA of the therapeutic will dictate the format to use, for example: • Non-cell-based assay relevant when a therapeutic mAb acts by binding to a soluble ligand and blocking it from interacting with its receptor • Cell-based assays recommended for mAb where effector functions important for the clinical effect, or for ADC

NAb assay format selection also depends on the assay performance characteristics and the risk of immunogenicity.

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Cell-based Neutralisation Assay - Example

Principle: Reporter gene assay (RGA) Cell line expressing the Firefly luciferase (FL) under a promoter responsive to TNF 

(A) TNF → expression of FL (B) TNF + TNF antagonist → reduced expression of FL (C) TNF + TNF antagonist + neutralising ADA → expression of FL

A

B

C

TNF antagonist dose-response curve

NAb dose-response curve

TNF dose-response curve

50000

60000

0 10000 20000 30000 40000 50000 60000 70000 80000

1 ng/ml

50000

40000

10 ng/ml

40000

30000

30000

RLU (FL)

20000

20000

10000

10000

0.25 0.5 1 2 4 8 16 32 64 0

0.5 1 2 4 8 16 32 64 128 0

8

16

32

64

128

256

512

Adalimumab, ng/ml

1024

2048

4096

NAb, ng/ml

TNF, pg/ml

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