Date of Award
Semester of Degree
Restricted Access Dissertation
Ph.D. in Paper and Bioprocess Engineering
Paper & Bioprocess Engineering
Steering Committee Member
Steering Committee Member
Monoclonal antibodies (mAbs) are the dominant biotherapeutics currently on the market. A typical mAb has a molecular weight of approximately 150 kDa and consists of two identical light chains and heavy chains, linked by interchain disulfide bonds. Disulfide bonds play a crucial role in protein folding and structural stabilization. However, during mAb manufacturing, disulfide bond reduction may occur, causing low-molecular-weight species and impacting protein quality and stability. Producing high quality mAb with complete disulfide bonds is integral to ensure protein structure and activity. Therefore, studies are necessary for better understanding of disulfide bond reduction and development of strategies to produce high-purity antibodies.
This dissertation is composed of five chapters. First chapter provided a review of mAb disulfide reduction and oxidation, covering structural perspective of antibody and disulfide bonds, root causes of disulfide reduction and current industrial mitigation strategies, historical studies of in-vitro disulfide reoxidation and analytical techniques for monitoring and characterizing antibody disulfide reduction and integrity. Second, a systematic study was performed to examine factors that influence disulfide formation kinetics. A kinetic model was constructed to predict disulfide formation rates and to help optimize the disulfide reoxidation condition. Third, the optimal condition for in-vitro disulfide reoxidation was applied to downstream Protein A chromatography using a redox wash to promote on-column disulfide formation, thus to produce intact antibodies from the reduced form. Fourth, factors that affect antibody stability were holistically investigated. By applying the fundamental redox reactions, a redox system (cysteine/cystine) was introduced into drug substance formulation. Acting as a scavenger of reactive oxygen species (ROS) generated from light exposure, the redox system stabilizes the protein by inhibiting aggregation, suppressing oxidation and diminishing product coloration. Lastly, the dissertation was concluded with a summary and outlook.
Overall, the results indicate that redox reaction, a fundamental phenomenon that occurs in our daily life, can be readily applied in antibody manufacturing to ensure product quality and efficacy, and ultimately to affect patients’ safety. This study offers a good example of out-of-box thinking for biopharmaceutical industry so that these seemingly complex issues can be solved through a simplified solution occurring in our daily life.
Tan, Zhijun, "DISULFIDE BOND REDUCTION AND REOXIDATION OF MONOCLONAL ANTIBODIES IN BIOLOGICS THERAPEUTICS PROCESSES - KINETICS UNDERSTANDING AND APPLICATIONS" (2020). Dissertations and Theses. 171.