Identification and elimination of target-related matrix interference in a neutralizing anti-drug antibody assay.

Biopharmaceuticals administered to the human body have the potential to trigger the production of anti-drug (also called anti-therapeutic) antibodies (ADA) that can neutralize the therapeutic activity. For antibody therapeutics, cell-based neutralizing ADA assays are frequently used to evaluate ADA in clinical studies. We developed a method to detect neutralizing antibodies against MEDI-575, a fully human IgG2κ antagonistic antibody against PDGFR-α. We evaluated three assay formats, two of which measured late responses, cell proliferation and apoptosis, whereas the third assay detected an early signaling event, phosphorylation of PDGFR-α. Measuring phosphorylation provided a superior assay window and therefore was developed as a neutralizing ADA (NAb) assay. Matrix interference, however, was significant, and could be identified to be caused by PDGF-AA and PDGF-AB, apparently the two most abundant ligands of PDGFR-α present in human serum samples. A simple pre-treatment step, addition of an inhibitory antibody to PDGF-A, a subunit present in PDGF-AA and PDGF-AB, was found to eliminate matrix interference, increasing assay reliability and sensitivity. We integrated the pre-treatment step into assay development and qualified a robust NAb assay.