Table 5.

Analytical issues encountered in anti-drug antibody (ADA) measurement

Issue	Definition and explanation	Mitigation or investigational strategies
Drug interference	Alteration in ADA detection (usually impaired detection) in an assay due to the presence of biopharmaceuticals (BP) in the sample	Adjust sample collection time-points to achieve no/lowest BP concentrations (before administration, after washout, during drug holiday); utilize assay formats with a higher degree of drug tolerance (bridge assay, high-density surface, long incubation times); incorporate dissociation step prior to analysis
Target interference	Alteration in ADA detection. In bridge assay formats, multivalent target may cause false positive results (note target levels can increase after BP administration)	Evaluate potential target interference during validation; remove or denature target; add anti-target monoclonal antibodies (mAbs) to block bridging
Pre-existing antibodies	Antibodies reactive with the BP before initiation of treatment	Affinity removal of antibodies or select pool of negative samples to establish negative control. Statistical analysis to identify true negative population for establishing negative control cut-point. Establish individual cut-points using baseline samples. Evaluate increases and decreases from baseline in final population analysis to determine level of BP-induced ADA
Rheumatoid factor (RF) interference	Rheumatoid factor present in sample may elicit positive result	Evaluate potential RF interference during validation; use assay formats that minimize RF interference. Evaluate increases and decreases from baseline in final population analysis to determine level of BP-induced ADA