Characterization of IgG4 Monoclonal Antibody Using Various Analytical Techniques for Quality Assessment of Upstream and Downstream Process Sample and Method Development and Validation of Trastuzumab by Hydrophobic Interaction Chromatography

Abstract

Monoclonal antibodies (mAbs) have emerged as pivotal therapeutic agents, with the IgG4 subclass gaining prominence due to their unique properties, such as reduced effectors functions and enhanced stability. This study aims to comprehensively characterize an IgG4 monoclonalantibody using a variety of analytical techniques to evaluate the quality of samples obtained at different stages of upstream and downstream processes. The upstream process involves cellculture, media, feed optimization, and fermentation. Whereas downstream involves purification polishing and final product preparation. Ensuring the quality of mAb at each of its developmental stages is crucial for safe and effective biopharmaceuticals. Various state-of-art analytical techniques are employed for this project. Protein A chromatography technique was used for assessing the titer content of the samples. Size Exclusion Chromatography provides insights about aggregation. Cation Exchange Chromatography was employed to evaluate charge heterogeneity. While N-Glycan Analysis by HILIC chromatography is used to evaluate post-translation modifications (glycosylation). The results obtained from these comprehensive analyses offer a detailed understanding of the IgG4 monoclonal antibody characteristics at different stages of the manufacturing process. The findings indicate that the upstream processes maintain the desired structural and functional attributes of the IgG4 monoclonal antibody. The downstream purification and formulation steps further contribute to the overall product quality, ensuring the removal of impurities and maintaining the integrity of the antibody. Overall, this research underscores the significance of a thorough analytical characterization approach for ensuring the high quality and consistency of monoclonal antibodies throughout the bioprocessing pipeline. Trastuzumab presents several therapeutic advantages and stands as the most widely used drug for breast cancer treatment. However, quantifying trastuzumab has proven challenging due to its complex biochemical and biophysical properties. To address this challenge, a suitable method was developed for its quantification. A hydrophobic interaction chromatography (HIC) method was devised utilizing a TSKgel HIC-ADC Butyl column (4.6mm I.D * 10 cm L, 3.5 μm), 100 mM phosphate buffer at pH 6.2, and an ammonium sulfate gradient. This HIC method exhibited optimal separation capability and precise quantification. Calibration curves demonstrated linearity within the range of 2.5 mg/mL to 2 mg/mL (R2 > 0.99), with accuracy falling between 90% and 110%. System and method precision (%RSD) were calculated as 0.80% and 1.03% respectively. The efficacy of this method was further demonstrated by its application in quantifying samples subjected to oxidative and photolytic stress.