Event

Title: Exploiting redox for sensing and controlling antibody-based biomanufacturing

Committee members:
Dr. William E. Bentley, Chair
Dr. Gregory F. Payne
Dr. Yang Tao
Dr. Ian M. White
Dr. Amy J. Karlsson, Dean's Representative

Abstract:
Monoclonal antibodies (mAbs) are an important class of therapeutics that are safe, effective, and made in large quantities. These proteins are comprised of the assembly of two large chains and two smaller chains, pieced together by disulfide bonds. They are predominantly synthesized by engineered Chinese Hamster Ovary cells (CHO) and secreted into large, 2000L+ bioreactors. Cells grown in these reactors often experience stress from their manufacturing environments and these stresses can influence antibody yield, quality, and integrity.

 
One key stressor to mAbs during production is oxidative damage, wherein specific amino acid residues of mAbs can be irreversibly oxidized leading to diminished function. In other cases, the disulfide bonds of mAbs can be reduced, also leading to dysfunctional fragments and decreased titer. Maintaining the appropriate redox (oxidation and reduction) balance is key to maintaining the quality of the antibody products and the growth of the antibody-producing CHO cells. It is therefore very important to monitor and control the redox state of the cell cultures. Process analytical technologies are employed to monitor critical redox influencing parameters enabling their adjustment as needed. A major influencer of redox state and mAb integrity is cysteine and its dimer cystine, regulating disulfide formation between antibody chains. Typical cysteine monitoring occurs off-line through highly specialized equipment such as liquid chromatography and mass spectrometry, which are not well suited for monitoring bioreactors. Even when extra effort is placed on real-time monitoring and adjusting for redox balance, up to 12% of antibodies can become fragmented. In the absence of tight control, complete fragmentation can occur.

Recently, we discovered that we can use a simple protocol and a relatively inexpensive potentiostat, electrodes, and sulfhydryl-specific mediator Ferrocene dimethanol (Fcn) to detect and quantify free cysteine as well as cysteine thiols of reduced fragmented mAb. We referred to this as Fcn mediated electrochemical probing (Fcn MEP). Fcn MEP was developed to rapidly quantify cysteine and mAb fragments in phosphate buffered saline (PBS) and Dulbecco’s modified eagle medium (DMEM).

 
In this dissertation, we build upon this work to explore the development of novel, low-cost, and rapid technologies to better control antibody quality and yield. Firstly, Fcn MEP is evaluated in the biologically relevant environments to understand its applicability in monitoring cysteine in CHO medium. Secondly, a new technology leveraging Fcn access to thiol residues is investigated for reformation of mAb fragment disulfide bonds. Specifically, for the first time, the use of Fcn for electrochemical regeneration (ECR) of intact mAb from reduced mAb fragments is quantitatively demonstrated. This methodology could be applied in a variety of production stages, including in bioreactors, purification, and perhaps even post formulation. We believe this establishes a basic understanding of MEP and ECR that can be integrated into biomanufacturing environments as a means for (i) ensuring the mAb critical quality attributes (CQA) needed for FDA clearance and use and (ii) correcting dysfunctional mAb fragmentation thereby maintaining high product titer.