The administration of biotherapeutics has the potential to induce potent immune responses. Among these responses, the production of anti-drug antibodies (ADA), including a subset of ADA referred to as neutralizing antibodies (NAb), is of heightened concern. Aside from their capacity to alter the pharmacological profile of a given biotherapeutic, NAb can also pose significant safety risks, especially in instances where an endogenous counterpart to the drug exists. As such, the inclusion of an assay to detect NAb in clinical samples is critical to the effectiveness of a tiered approach to immunogenicity assessment. PF-06730512 is a biotherapeutic protein being developed for the treatment of primary Focal Segmental Glomerulosclerosis (FSGS). To support the immunogenicity assessment of PF-06730512, a cell-based assay was developed for the detection of NAb in FSGS serum samples. Herein, we describe the development of the assay with a focus on the challenges faced, including drug and blood collection tube interferences in NAb detection. The outcome of our efforts was a robust assay capable of detecting 1 μg/mL of a NAb positive control in the presence of clinically relevant drug concentrations up to 30 μg/mL.
Keywords: ROBO2 and SLIT2; anti-drug antibody (ADA); blood collection tube interference; homogenous time-resolved fluorescence (HTRF); neutralizing antibody (NAb).