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Herceptin ADCC & CDC Assays

BioOutsource has the experience, expertise and capabilities to provide the complete ADCC and CDC testing solutions for Herceptin using our off-the-shelf methods for both Herceptin CDC and ADCC. Our ADCC assays can be performed using PBMCs from a variety of internal genotyped donors that are specifically harvested on the day of assay, cultured in advance or from frozen libraries. Our ADCC assay portfolio includes the use of a licensed CD16 positive natural killer cell line to reduce day to day assay variability or engineered natural killer cell lines that are activated following antibody binding. We offer a range of Fc Receptor binding assays and surrogate ADCC assays to complement our functional ADCC assays

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

The principle of both Herceptin (trastuzumab) ADCC and CDC assays is that the relevant target cell line, typically SKBR-3, expressing the Her2 protein are prepared in cell plates using a procedure design to optimise receptor expression. A dilution series of Herceptin is then prepared, added to the plate and allowed to bind to the SKBR-3 cells for period of time. PBMCs (Herceptin (trastuzumab) ADCC bioassay) or complement (Herceptin (trastuzumab) CDC bioassay) are then added; this will bind to Herceptin, bound to the Her2/neu protein; initiating a cell death cascade for ADCC resulting in target cell lysis. The assays are performed with appropriate assay controls, utilising 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, the use of frozen PBMCs (acquired from a single donor, from a single draw) as well as the option to use a stable Natural Killer cell line that expresses ADCC activity. Our standard CDC assay methodologies have been optimised using a specific complement source.