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Immunogenicity assays are required to evaluate anti-drug antibody (ADA) responses that can be generated against biotherapeutic modalities. Regulatory guidelines focus on clinical requirements, yet it has become apparent that industry has applied these clinical recommendations for immunogenicity assessment to nonclinical studies in varying degrees. ADAs are an anticipated outcome of dosing a humanized or fully human biotherapeutic into an animal. However, a nonclinical ADA response is rarely predictive of the immunogenic potential in humans. The addendum to ICH S6 recommends that immunogenicity should be explicitly examined where there is: evidence of altered pharmacodynamic activity; unexpected changes in exposure in the absence of a pharmacodynamic marker or evidence of immuno-mediated reactions. The European Bioanalytical Forum has extensively discussed and reached a consensus on a minimal strategic approach of when and what to include for nonclinical immunogenicity assessments. Additionally, this paper recommends a strategy for ADA assay validation and sample analysis for those cases when it is considered necessary to include an immunogenicity assessment in nonclinical toxicology studies.

The analysis of biomarkers by ligand-binding assays offers significant challenges compared with the bioanalysis of small and large molecule drugs. The presence of endogenous analyte is a commonly cited issue. Also the sourcing and application of appropriate calibration or reference standards can present many issues. One of the main challenges is ensuring the continuity and validity of biomarker data when the source or lot number of calibration standard changes within or between studies. Several strategies exist in attempting to deal with this and standardize the biomarker data through the assay life or looking for ways to compare and normalize biomarker data. In this manuscript, the European Bioanalysis Forum view on dealing with calibration standards in biomarker assays is described.

Aim: Long-term stability testing of drug candidates in biological matrix is a key parameter in bioanalytical method validation. The European Bioanalysis Forum formed a Topic Team to evaluate the use of isochronic design for long-term stability testing of large molecules., Method: Isochronic design is based on storage of samples at a reference temperature (below -130°C) where the samples are considered stable. The stability samples are stored at the intended storage temperature and then transferred to the reference temperature, while a set of reference samples is stored the entire storage period at the reference temperature. Stability and reference samples will then be analyzed in one run at the end of the storage period. The mean concentrations of the stability samples are compared either to their nominal concentrations or to the mean concentrations of the reference samples., Conclusion: The design minimizes day-to-day variation, reduces workload and adds to the flexibility in the laboratory.