High ionic strength dissociation assay reduces dimeric target interference in immunogenicity testing.

Aim: The presence of di-/multi-meric forms of soluble target in biological samples can interfere in anti-drug antibody (ADA) assays, leading to increased background values and potentially false positivity. The authors investigated the use of the high ionic strength dissociation assay (HISDA) to reduce target interference in two different ADA assays. Results: Interference caused by homodimeric FAP was successfully eliminated to enable cut point determination after applying HISDA. Biochemical experiments confirmed the dissociation of homodimeric FAP after treatment with high ionic strength conditions. Conclusion: HISDA is a promising approach to simultaneously achieve high drug tolerance and reduced interference by noncovalently bound dimeric target molecules in ADA assays without extensive optimization, which is particularly advantageous in routine use.

Comparison of assay formats used for the detection of pre-existing anti-drug antibodies against monoclonal antibodies.

Aim: Assessment of pre-existing anti-drug antibody (preADA) reactivity at early drug development stages can be beneficial for candidate selection. We investigated the applicability of a generic immune-complex anti-drug antibody (ADA) assay for early preADA assessment as an easily available alternative to the commonly used ADA bridging assay. Results: The results confirmed the expected assay difference regarding isotype detectability. Tested drug candidates were identified as preADA-reactive using the immune-complex ADA assay despite its limitation of not being able to detect IgM-type preADAs. Conclusion: We recommend a purpose-driven use of the two assay formats. For the purpose of ranking different Pro329Gly mutation-bearing drug candidates, the immune-complex ADA assay is preferred in the phase before selecting a drug for clinical development.

High ionic strength dissociation assay (HISDA) for high drug tolerant immunogenicity testing.

Aim: Antidrug antibody (ADA) assessment may be challenged in studies that involve the administration of high doses of biotherapeutics and/or with long half-lives. In such cases, ADA assays with optimized drug tolerance are desired. Material & Methods: We evaluated the use of MgCl2 to develop high ionic strength dissociation assays in two investigational examples (bridging enzyme-linked immunosorbent ADA assays) to attain high drug tolerance while maintaining best possible structural integrity of ADAs. Results: Both ADA-bridging assays treated with MgCl2 showed improved drug tolerance and higher signal-to-blank values compared with overnight incubation or acid treatment. Conclusion: The use of MgCl2 treatment in ADA-bridging assays provides a sensitive, drug tolerant and easy-to-use alternative in cases where acid dissociation is not possible or unwanted.

Evaluation of potential biotin interference in immunogenicity testing.

Aim: One-step bridging assays typically used for immunogenicity testing may be challenged by biotin interference (BI) caused by widely available dietary supplementation or medically prescribed high-dose therapies. We investigated BI in two one-step antidrug antibody assays. Results: Both assays showed biotin-related interference, with the peptide-based assay being less affected than the antibody-based assay. BI was reduced by minimum required dilution adaption from 10 to 1% and eliminated by a depletion-based sample pretreatment. Conclusion: Increased biotin levels have the potential to interfere with immunogenicity testing methods that use biotin technology. Since the extent of interference differs from assay to assay, assessment during development phase is recommended. Minimum required dilution adjustment or sample pretreatment are options to reduce or eliminate BI.