Target tolerance improvement of the immunogenicity assay developed for analysing samples from clinical trials of RGB-14, a proposed biosimilar to Prolia/Xgeva

Abstract

An immunogenicity assay was developed to support the clinical program of RGB-14, a proposed biosimilar to Prolia/Xgeva. The ADA assay was successfully validated following current guidelines and white papers. The validation included an assessment of target (RANKL) interference, and the assay passed the test up to a 30 pg/mL RANKL level. This concentration was anticipated to be the maximum level present in clinical samples. Despite the successful validation, most of the analysed samples from the RGB-14 Phase I clinical trial yielded false-positive results for anti-denosumab antibodies. The investigation concluded that unexpectedly high levels of RANKL were present in the samples and caused interference in the assay. Consequently, the assay was re-developed by adding target-specific reagent, eliminating acid dissociation step and doubling the assay dilution to enhance target tolerance to 10,000 pg/mL RANKL, a requirement for clinical sample testing. The re-developed assay underwent successful validation, and the samples from the clinical trials of RGB-14 were subsequently analysed with the improved assay. The article outlines a strategy for enhancing the target (RANKL) tolerance of the ADA assay. It also underscores the importance of monitoring clinical sample analysis results, even if a validated assay is used for sample testing, as clinical samples may differ from the spiked samples prepared for validation. If unexpected results are obtained, an investigation should be initiated, and the assay should be improved if needed to ensure the reliability of the results.