UBC Theses and Dissertations
Bioprocess optimization for recombinant protein production from mammalian cells Goudar, Chetan T.
Mammalian cells are being increasingly used to manufacture complex therapeutic proteins given their ability to properly fold and glycosylate these proteins. However, protein yields are low and process enhancements are necessary to ensure economically viable processes. Methods for yield improvement, bioprocess development acceleration and rapid quantification and monitoring of cell metabolism were investigated in this study. Recognizing the adverse effect of high PCO₂ on cell growth, metabolism and protein productivity, a novel PCO₂ reduction strategy based on NaHCO₃ elimination was investigated that decreased PCO₂ by 65-72%. This was accompanied by 68-123% increases in growth rate and 58-92% increases in productivity. To enable rapid and robust data analysis from early stage process development experiments, logistic equations were used to effectively describe the kinetics of batch and fed-batch cultures. Substantially improved specific rate estimates were obtained from the logistic equations when compared with current modeling approaches. Metabolic flux analysis was used to obtain quantitative information on cellular metabolism and the validity of using the balancing method for flux estimation was verified with data from isotope tracer studies. Error propagation from prime variables into specific rates and metabolic fluxes was quantified using Monte-Carlo analysis which indicated 8-22% specific rate error for 5-15% error in prime variable measurement. While errors in greater metabolic fluxes were similar to those in the corresponding specific rates, errors in the lesser metabolic fluxes were extremely sensitive to greater specific rate errors such that lesser fluxes were no longer representative of cellular metabolism. The specific rate to metabolic flux error relationship could be accurately described by the corresponding normalized sensitivity coefficient. A framework for quasi-real-time estimation of metabolic fluxes was proposed and implemented to serve as a bioprocess monitoring and early warning system. Methods for real-time oxygen uptake and carbon dioxide production rate estimation were developed that enabled rapid flux estimation. This framework was used to characterize cellular response to pH and dissolved oxygen changes in a process development experiment and can readily be applied to a manufacturing bioreactor. Overall, the approaches for protein productivity enhancement and rapid metabolism monitoring developed in this study are general with potential for widespread application to laboratory and manufacturing-scale perfusion and fed-batch mammalian cell cultivations.
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