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Betenbaugh, Michael J.

Kraft, Lauren

Ruiz-Márquez, Kevin J.

Sacco, Sarah A.

Smith, Kevin D.

Young, Jamey

English

Engineering Conferences International

Tuesday, April 25, 2023           Session 3: Systems and Synthetic Biology for Improved Cell Culture Performance METABOLIC ENGINEERING OF HIGH-PRODUCTIVITY CHO HOST LINES FOR BIOMANUFACTURING  Jamey D. Young, Department of Chemical and Biomolecular Engineering; Vanderbilt University, USA  j.d.young@vanderbilt.edu Sarah A. Sacco, Department of Chemical and Biomolecular Engineering  Kevin J. Ruiz-Márquez, Department of Chemical and Biomolecular Engineering Lauren Kraft, BioTherapeutics Development & Supply, Janssen Research and Development, USA  Kevin D. Smith, BioTherapeutics Development & Supply, Janssen Research and Development, USA  Michael J. Betenbaugh, Johns Hopkins University, USA   Key Words: Chinese hamster ovary; Metabolic flux analysis; Monoclonal antibody; Mitochondrial metabolism; Glutamine synthetase  Industrial bioprocesses place extraordinary demands on the intermediary metabolism of host cells to meet the biosynthetic requirements for maximal growth and product formation. Therefore, engineering cell metabolism to promote high yield and specific productivity (qP) is a major goal of biomanufacturing research. 13C metabolic flux analysis (MFA) provides a comprehensive approach to quantify host metabolic phenotypes by applying stable isotopes to trace the flow of carbon through intracellular biochemical pathways. Our previous 13C MFA studies have discovered that high-producing CHO cell cultures possess a different metabolic phenotype compared to low-producing cultures. In particular, it was found that high mAb production is correlated to lactate consumption and elevated mitochondrial TCA cycle flux during stationary growth phase. Based on these findings, we have engineered multiple gene targets to enhance mitochondrial metabolism and overall bioenergetics of CHO host lines. First, we overexpressed a mitochondrial regulator to enhance TCA cycle flux and oxidative phosphorylation, which significantly improved final titers and qP compared to the parental line. Second, we engineered a cell line with reduced glutamine overflow and increased mAb production by attenuating the promoter driving expression of the glutamine synthetase (GS) selection marker. We have applied 13C MFA to understand how these gene alterations impact intracellular metabolic fluxes and mAb biosynthesis. Finally, we expanded our metabolic flux models to include pathways for synthesis of nucleotide-sugars that supply precursors for product glycosylation, and have validated these models in cultures fed 13C-glucose or 13C-galactose. This presentation will highlight how host cell engineering guided by 13C MFA can be applied to improve metabolic efficiency and product biosynthesis, which has broad implications for biomanufacturing research.    

Metabolic engineering of high-productivity CHO host lines for biomanufacturing

https://dc.engconfintl.org/cgi/viewcontent.cgi?filename=0&article=1021&context=ccexviii&type=additional

Metabolic engineering of high-productivity CHO host lines for biomanufacturing

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Engineering Conferences International