Monoclonal antibodies (mAbs) and other protein therapeutics are among the most expensive of all drugs to manufacture. Making these therapies more affordable and available to the public will improve both the health and quality of life of millions of patients in the U.S. and around the globe. The proposed research aims to identify strategies for improving mAb production by engineering the metabolism of Chinese hamster ovary (CHO) cells. CHO cells are used to produce 60-70% of all protein therapeutics in the US. The studies will use a CHO cell line provided by Janssen R&D that is capable of high mAb productivity. High-producing cell lines of this kind are not typically available to academic labs, and therefore this collaboration provides a unique opportunity to test the proposed metabolic engineering strategies in an industrially relevant host line. This work is significant because it will enable novel approaches for enhancing the productivity and consistency of mammalian cell bioprocesses, thus lowering drug development and manufacturing costs of therapeutic antibodies. This project will also provide the unique educational opportunity for a graduate student and post-doctoral researcher to engage in collaborative research with industry scientists, culminating in a 3-month internship in which the graduate student will perform experiments in a Janssen R&D facility. Such an experience will provide these trainees with ideal preparation for a career in the biotechnology industry or in a government or academic lab.

The accelerating trend toward highly targeted monoclonal antibody (mAb) therapeutics has led to a critical need for enhanced productivity in mammalian cell bioprocesses. Previous NSF-sponsored research has found that high-producing CHO cell lines consistently exhibit enhanced citric acid cycle (CAC) activity compared to low- or non-producing hosts. However, the extent to which this metabolic phenotype is required to drive high-yield protein expression is still unclear, and it is unknown whether CAC flux can be rationally engineered to promote increased mAb production. The long-term goal of this research is to identify metabolic engineering strategies that promote a high-productivity metabolic phenotype in mammalian hosts leading to increased product yield and quality. Because this phenotype is expected to involve up-regulation of mitochondrial oxidative metabolism, the overall objective of the current application is to engineer CHO cells to enhance CAC flux while assessing the impacts on mAb titer, cell specific production rate (CSPR), and glycan profile. First, an industrial CHO host line provided by Janssen R&D will be engineered to constitutively up-regulate oxidative CAC metabolism. A specific mitochondrial regulatory protein will be overexpressed and 13C metabolic flux analysis (MFA) will be applied to guide local pathway engineering to further enhance CAC flux. Second, an inducible expression vector will be used to dynamically redirect carbon flux into CAC during stationary phase. The working hypothesis is that induction of mitochondrial metabolism at the onset of stationary phase will enhance CSPR while enabling the culture to reach peak cell density during exponential phase, thus maximizing final mAb titer. Third, IgG glycan profiles will be assessed to determine how manipulating CHO central metabolism impacts product glycosylation. The rationale for the proposed research is that it will determine whether central carbon metabolism of CHO cells can be engineered to drive increased mAb production while maintaining consistent product quality. This project is co-funded by the Biotechnology and Biochemical Engineering Program of the CBET Division, by the GOALI Program of the Division of Industrial Innovation and Partnerships and by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biosciences.

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Vanderbilt University Medical Center
United States
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