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Duong, Tam Thi Ngoc

University of British Columbia

The majority of monoclonal antibodies (mAbs) are produced by fed-batch processes using Chinese Hamster Ovary (CHO) cells. Although glutamine has been typically provided in excess, its metabolism and decay cause ammonium accumulation, a toxic byproduct that can inhibit cell growth and reduce culture productivity. To understand how best to reduce ammonium accumulation, both the partial and total replacement of glutamine by asparagine were investigated. The CHO-K1 cells grew at the highest rate in batch cultures when both asparagine and glutamine were supplied, indicating that asparagine can contribute to maximal cell growth rates. Without glutamine in the medium, we confirmed that cultures with asparagine were able to grow at intermediate rates and without increasing cell death. However, without both asparagine and glutamine, or with asparagine and an inhibitor of the glutamine synthesis pathway, the CHO-K1 cells did not grow, and cell death was increased. As a result, it was concluded that the growth of CHO-K1 cells in this study required both glutamine synthetase activity and asparagine when glutamine was not included in the medium. These findings were then tested in industrially relevant fed-batch cultures. When glutamine was either not fed or also not supplied in the initial medium, culture growth was unaffected, and the culture viability was increased. Furthermore, the specific productivity of monoclonal antibody was 1.5-fold higher. These improvements in culture physiology were also studied by metabolomics and transcriptomic analyses. The changes in metabolome levels were more notable than changes in transcript levels. The improvements in the cultures without glutamine in the medium could be attributed to substrate-level control of metabolic reactions rather than significant changes in transcriptomic gene expression. This study advanced our understanding of the physiology of CHO-K1 cells in batch and fed-batch conditions and substantiated a promising prospect for “glutamine-free” fed-batch cultures that increased the mAb productivity of CHO cells. It also revealed challenges in setting up small scale experimental cultures at pre-specified pH levels in bicarbonate-buffered cell culture medium formulations, such that a model was developed and tested for the a priori formulation of these widely used media.

Text

2022-12-21

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/83461

Chemical and Biological Engineering

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/