Module 9 2024

03/09/2024

Challenges in Immunogenicity Assessment Assays

• Cut-point determination – influenced by sample size, data distributions/transformation, methods used for determining analytical & biologicals outliers, matrix (pre- vs in-study cut points), pre- existing reactivity, …

• Pre-existing antibodies (e.g., anti-PEG, rheumatoid factors, HAMA)

• Assay sensitivity – influenced by the choice and performance of analytical platform & controls

• Assay controls (PC, NC, acceptance criteria)

• Matrix effects (complement components,…)

• Drug interference - often the main bioanalytical challenge for therapeutic mAbs. Minimum Required Dilution (MRD), incubation time and ratio of reagents need to be optimised; consider dissociation protocols

sulfo-Tag

•

Target interference

monomeric soluble target can bind therapeutic and prevent ADA binding → false negative; membrane-bound target or multimeric soluble target may form bridge with therapeutic → false positive

TmAb TmAb

biotin

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Drug-Tolerant Assay - Acid Dissociation

Drug interference is an issue for therapeutic mAbs: • long half-life (typically 10-20 days) •

often administered chronically at high dose, without a wash-out period.

sulfo-Tag

ADA

TmAb TmAb

TmAb TmAb

TmAb TmAb

+ acid ADA

+ base

ADA

+ assay reagents

biotin

Depletion of therapeutic Ab e.g., Affinity capture elution (ACE) Solid-phase extraction (SPEAD) pH shift anti-idiotype (PIA)

Impact of acid treatment on ADA integrity? Development of milder dissociation methods e.g., High ionic strength dissociation with MgCl 2 (HISDA)

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