Validation of the BIACORE 3000 platform for detection of antibodies against erythropoietic agents in human serum samples

SUMMARY Objective: To develop a validated BIACORE immunoassay for the detection and characterization of serum antibodies with specificity for erythropoietic molecules (e.g. darbepoetin alfa). Methods: New Zealand White rabbits (n = 8) were immunized by an intramuscular injection of darbepoetin alfa/adjuvant at 0,4,6, and 8 weeks. Serum was collected for 6 weeks after final injection and pooled for affinity purification. Antibody immunoassay measurements were performed using a BIACORE 3000 with darbepoetin alfa immobilized to the biosensor surface. Human serum samples were spiked with the affinity-purified rabbit antibody to develop and validate the BIACORE immunoassay. Results: The assay was shown to be stable through 180 sample/regeneration cycles and had a threshold of 45.8 response units. The validated limit of detection was 0.40|ig/ml in 100% human serum. The method was robust, with variability not exceeding a 20% coefficient of variation, well within acceptable limits for typical immunoassays. Conclusion: All protein-based therapeutics have a potential for immunogenicity, and antibodies raised against these molecules may have important clinical sequelae. The biotechnology and pharmaceutical industries are challenged to address this potential by developing robust analytical platforms to detect and characterize antibodies directed against therapeutic proteins in clinical specimens. Traditionally, radioimmune precipitation assays and/or enzyme-linked immunoassays (ELISAs) are used for primary detection of host immune response; however, the BIACORE platform may be better suited for this purpose in many instances. This platform represents a robust tool that should be considered for the detection and characterization of antibodies directed against protein-based therapeutics.