Detection of anti-drug antibodies to aflibercept by enzyme-linked immunosorbent assay: Development and validation of the method

INTRODUCTION. Aflibercept is used to treat neovascular age-related macular degeneration, a leading cause of vision loss. Immunogenicity is one of the significant adverse effects that results in the production of anti-drug antibodies (ADAs). The development of a method for detecting ADAs to aflibercept is essential for immunogenicity assessment. AIM. This study aimed to develop and validate an analytical method for detection and stepwise characterisation of total anti-drug antibodies to aflibercept in human serum utilising the enzyme-linked immunosorbent assay. MATERIALS AND METHODS. The method was developed using a solid-phase ELISA. Aflibercept (GNR-098, JSC GENERIUM, Russia) and a conjugate of GNR-098 with horseradish peroxidase were employed. Polyclonal antibodies against GNR-098 were obtained by immunising rats, followed by serum collection and antibody purification via affinity chromatography. ADA detection was performed using labelled GNR-098 in a bridging ELISA. Model samples were prepared by spiking pooled sera from healthy donors with rat polyclonal antibodies. Method validation included the following parameters: cut-off points; precision; limit of detection (LOD); drug tolerance; specificity; matrix effect; minimum required dilution; robustness; reagent stability; hook effect. RESULTS. An analytical method includes a screening assay for the presumptive detection of ADAs; a confirmatory assay to establish ADA specificity and assay for ADA titer determination and IgE isotyping. The cut-off points for the screening assay and IgE-specific assay were 1.3 and 1.2, respectively; for the confirmatory assay, 15.2%. The LOD of the screening assay was 185 ng/mL. The method demonstrated drug tolerance (interference from soluble target, hrVEGF 165 ) at concentrations up to 2,000 pg/mL. No matrix effects were observed in the screening or confirmatory assays across 13 test samples containing ADAs at 280 ng/mL. The absence of a hook effect was confirmed in both assays using samples with ADA concentrations five times higher than the high-positive control. CONCLUSIONS. An analytical method was developed for the ELISA-based detection and stepwise characterisation of total anti-drug antibodies to aflibercept (GNR-098) in human serum. The method’s suitability was confirmed through validation. This analytical procedure will facilitate the immunogenicity assessment of aflibercept.