Method Development for Quantification and Validation of Host Cell Protein Impurities Using Elisa and Molecular Weight Determination of Therapeutic Monoclonal Antibody Using Sds-Page

Abstract

Therapeutic monoclonal antibodies (mAbs) are one of the fastest growing classes of biotherapeutic agents in India as well as the world. The mAbs are used in the treatment of wide range of diseases, including cancer, inflammatory and autoimmune. The mAb of interest in present study is called Sunmab (therapeutic monoclonal antibody developed using E.coli bacteria and marketed by SUN Pharmaceuticals). The manufacturing process of mAbs using host cell/ organisms generates impurities known as Host cell protein (HCP) impurities. These are the process related impurities and are present in low levels (ng/ml or PPM) along with the mAb. The analysis of these HCPs is important because if present in large amount they can reduce the shelf life, efficiency and stability of therapeutic mAb formulations. They also generate proteolytic immune response known as cytokines storms within the body. According to ICH Q6b guidelines the accepted level of HCP in formulation is less than 1000 μg/ mL. The presence of HCP can be analysed by different analytical techniques like ELISA, SDS-PAGE, Western blotting, odyssey Infrared image systems and mass spectrometry. ELISA is commonly used technique to analyse the HCP content in biopharmaceuticals. In present study ELISA method was developed to quantify the HCPs content in SunmAb. The solubility of SunmAb was in 25mM Tris buffer (pH-8.6). SunmAb having concentration of 0.5mg/ml was prepared and quantified for the HCP content. Method quantification was performed according to the ICH Q2 (R2) guidelines. The method was linear, having concentration range of 1-100ng/ml of reference standard concentration with correlation coefficient of 1.0. The method was able to quantify the concentration of HCP upto 1ng/ml limit. The developed method was accurate and precise with observed RSD less than 20 %. In minor project, we have determined the molecular weight of Adalimumab using SDS-PAGE technique. The concentration of polyacrylamide gel used was 4-10% and was selected to analyze the intact and reduced molecular weight of Adalimumab sample having concentration of 4ug/ml. The applied current was 120 V. The beta-mercaptoethanol reagent was used in the reduction study to cleave the disulfide bonds in the hinge region and thus separating the heavy and light chains of the antibody. The detection of bands was done using silver staining technique at around 150 kDa for intact antibody and around 50kDa and 30kDa for heavy and light chains respectively