Method Validation for Potency Testing of Rituximab Monoclonal Antibody by Complement Dependent Cytotoxicity Assay

Abstract

Rituximab is monoclonal antibody used for the treatment of B-Cell Non-Hodgkin’s Lymphoma and other diseases. At present, there is no monograph for potency assay in the pharmacopeias therefore the manufacturers used various methods for estimating the potency of rituximab. In this study, we developed a potency assay by using complement dependent cytotoxicity (CDC) which could be applied for difference rituximab products. Basically, this method is based on activity of rituximab binding to CD-20 receptors on the surface of WIL2-S cells that activates complement system in serum and leads to cytotoxicity response to concentration of rituximab. The relative potency of rituximab was calculated against its reference drug using 4-parameter logistic equation. The results showed the specificity of the CDC assay to rituximab and WIL2-S cells. The method presented %recovery between 80.0-125.0. Repeatability and intermediate precision of the method showed a geometric coefficient of variation (GCV) values of 8.2 and 5.8% respectively. The relative potency curve had linearity at a range between 50.0-150.0% with a coefficient of determination of 0.998. The data obtained from changing of passage numbers of cells, incubation time, the density of cells and equipment presented that %GCV was less than 25.0. Furthermore, 5 rituximab products were tested by this method, all results were comparable to those of the manufacturers. Taken together, we concluded that the potency test by CDC assay could be used as a standard method for quality control of rituximab.