Treatment With Anti-human T Lymphocytes Cd25 Monoclonal Antibody (wutac) Process And Its Impurity Identification

The therapeutic murine monoclonal antibody (mAb) of WuTac against human lymphocyte antigen CD25 which was proprietarily developed by Wuhan Institute of Biological Products is mainly used to clinically prevent renal graft rejection. And presently, the clinical trial phaseⅠhas been accomplished and the phaseⅡis under way. To further improve its safety and effectivity, this paper was focused on the optimization and validation of pilot experiment, and analysis and identification of this product.According to the purification process of WuTac, the mobile phase of Hydrophobic Interaction Chromatography (HIC) was investigated, and the type and concentration of the salt and the chromatographic gradient conditions were explored and preliminarily determined for pilot experiment. The absorption and elution conditions of HIC for WuTac were 1.0 M sodium chloride + 20 mM PB (pH 7.4) (10 ml/min) and 20 mM PB (pH 7.4) (20 ml/min), respectively. After purification of the ascitic fluids with caprylic acid– ammonium sulfate combined with HIC technique, the SDS– PAGE and High Performance Liquid Chromatography (HPLC) were used to determine the purity of this product, the Flow Cytometry was hired to measure the titer, the SDS– PAGE in associated with Mass Spectrometry (MS) was employed to identify the impurity band between 130 and 170 kDa, the two-dimensional electrophoresis combined with MS was used to validate the impurity protein after HIC, the Isoelectrofocusing (IEF) was harnessed to measure the isoelectric point (PI), and the Biosensor (BIAcore 3000) was hired to determine the absolute affinity of this kind of mAb. The results indicate that the HIC technique can remove the dimmers, polymers, and murine ceruloplasmin from the intermediate product precipitated by caprylic acid– ammonium sulfate, and the rate of monomers can be achieved 100% without any murine impurity proteins. It also shows that the isoelectric point of WuTac is 5.65-5.94, and the KA of WuTac before and after HIC is 5.85×1010 L/mol and 3.49×1011 L/mol, separately.In conclusion, the HIC technique can polish this kind of product, and the data obtained lay the basis for the establishment and improvement of quality control system.