Enbrel ADCC & CDC Assays

BioOutsource has the experience, expertise and capabilities to provide the complete ADCC and CDC testing solutions for Enbrel using our off-the-shelf methods for both Enbrel CDC and ADCC. Enbrel CDC and ADCC assays require TNF-Alpha to be present as the membrane-bound form at sufficiently high levels in order for the methods to be successful; a cell line, engineered to express membrane bound TNF-Alpha is required for these assays. Our classical ADCC assays are performed using PBMCs from a variety of genotyped donors that are specifically harvested on the day of assay. Our ADCC assay portfolio also includes a surrogate ADCC assay which uses an engineered cell line stably expressing the FcgRIIIa receptor to negate donor-specific variability. We offer a range of Fc Receptor binding assays to complement our ADCC assays, and C1q binding assays to complement our CDC assay.

The Enbrel CDC and ADCC bioassays measure the combined activity of the Fab binding and effector functions of Enbrel, mediated through an initial interaction with C1q (CDC) or the Fc-Gamma Receptor IIIa (FcRIIIa) (ADCC). BioOutsource are industry experts in the development, optimization and qualification/validation of bioassays and have applied this expertise to Enbrel (etanercept) to offer ADCC and CDC assays to our customers. We have provided services to support clone selection, characterization and biosimilarity activities with assays performed using our existing methodology, adapted to incorporate the customer’s reference standard of choice. Where required, rapid assay qualification may be achieved using the customer’s material.

The principle of both Enbrel (etanercept) ADCC and CDC assays is that the target cell line, expressing the membrane bound form of TNF-Alpha, is prepared in cell plates. A dilution series of Enbrel is then prepared, added to the plate and allowed to bind to the target cells for period of time. PBMCs (Enbrel ADCC bioassay) or complement (Enbrel CDC bioassay) are then added; this will bind to Enbrel/membrane-bound TNF-Alpha complex to initiate a cell death cascade resulting in target cell lysis. The assays are performed with appropriate assay controls, utilizing a non-radioactive assay endpoint. Sample results can be reported as a per cent of total cell death or as a relative potency measurement against the reference standard.

ADCC assays are well known for day to day variability arising from the donor PBMCs that are used in the methodology and BioOutsource have implemented a number of different strategies to control this variation. These include donor screening programmes so that assays are only performed using qualified individuals, and a standardized cell culture regime. Our standard CDC assay methodologies have been optimized using a specific complement source.