Researcher(s)
- Rohil Cheepala, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Eleftherios Papoutsakis, Chemical Engineering, University of Delaware
Abstract
Monoclonal antibodies (mAbs) are engineered proteins that bind to specific pathogens/irregularities in the body to help the immune system fight off disease. CHO cells are the industry standard for recombinant protein and mAb manufacturing, producing over 70% of all recombinant proteins. These experiments were conducted to find the ideal culture conditions for CHO cells to produce more mAbs.
Earlier research shows that glutamine and asparagine are heavily consumed essential amino acids compared to others. Glutamine primarily fuels the cells energy production, while asparagine depletion correlates with entering the stationary phase, or antibody production phase. We hypothesize that feeding glutamine in excess to cell consumption will lead to the early buildup of ammonium, which will reduce both cell health and cell viability. Additionally, we hypothesize that asparagine supplementation will lead to an increase in mAb production, as delaying the depletion of asparagine will move the stationary phase farther ahead where there are more healthy cells to produce mAbs.
Two shake-flask fed-batch experiments were conducted with the Kuehner TOM system to measure the oxygen transfer rate (OTR) and carbon dioxide transfer rate (CTR), markers of healthy cellular respiration. The glutamine feeding experiment was conducted for seven days and compares feeding 6 mM glutamine to no glutamine feeding. The asparagine experiment was conducted for thirteen days and compares feeding 4% of culture volume in 150 mM asparagine to no additional asparagine feeding. Apoptosis and mitochondrial flow cytometry assays were used to measure overall cell and mitochondrial health.
The ammonium data for the glutamine feeding experiment shows that excess glutamine feeding leads to inhibitory ammonium build up, which inhibits cell growth. We are currently analyzing the data from asparagine feeding, including the antibody titer kinetics.