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Chinese hamster ovary (CHO) cells are used extensively to produce protein therapeutics, such as monoclonal antibodies (mAbs), in the biopharmaceutical industry. MAbs are large proteins that are energetically demanding to synthesize and secrete; therefore, high-producing CHO cell lines that are engineered for maximum metabolic efficiency are needed to meet increasing demands for mAb production. Previous studies have identified that high-producing cell lines possess a distinct metabolic phenotype when compared to low-producing cell lines. In particular, it was found that high mAb production is correlated to lactate consumption and elevated TCA cycle flux. We hypothesized that enhancing flux through the mitochondrial TCA cycle and oxidative phosphorylation would lead to increased mAb productivities and final titers. To test this hypothesis, we overexpressed peroxisome proliferator-activated receptor 𝛾 co-activator-1⍺ (PGC-1⍺), a gene that promotes mitochondrial metabolism, in an IgG-producing parental CHO cell line. Stable cell pools overexpressing PGC-1⍺ exhibited increased oxygen consumption, indicating increased mitochondrial metabolism, as well as increased mAb specific productivity compared to the parental line. We also performed 13C metabolic flux analysis (MFA) to quantify how PGC-1⍺ overexpression alters intracellular metabolic fluxes, revealing not only increased TCA cycle flux, but global upregulation of cellular metabolic activity. This study demonstrates the potential of rationally engineering the metabolism of industrial cell lines to improve overall mAb productivity and to increase the abundance of high-producing clones in stable cell pools.
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Creation of monoclonal antibody expressing CHO cell lines grown with sodium butyrate and characterization of resulting antibody glycosylation
Chinese hamster ovary (CHO) cells are the primary mammalian cell lines utilized to produce monoclonal antibodies (mAbs). The upsurge in biosimilar development and the proven health benefits of mAb treatments reinforces the need for innovative methods to generate robust CHO clones and enhance production, while maintaining desired product quality attributes. Among various product titer-enhancing approaches, the use of histone deacetylase inhibitors (HDACis) such as sodium butyrate (NaBu) has yielded promising results. The titer-enhancing effect of HDACi treatment has generally been observed in lower producer cell lines but those studies are typically done on individual clones. Here, we describe a cell line development (CLD) platform approach for creating clones with varying productivities. We then describe a method for selecting an optimal NaBu concentration to evaluate potential titer-enhancing capabilities in a fed-batch study. Finally, a method for purifying the mAb using protein A chromatography, followed by glycosylation analysis using mass spectrometry, is described. The proposed workflow can be applied for a robust CLD process optimization to generate robust clones, enhance product expression, and improve product quality attributes.
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- PAR ID:
- 10379140
- Date Published:
- Journal Name:
- Methods in enzymology
- Volume:
- 660
- ISSN:
- 0076-6879
- Page Range / eLocation ID:
- 267-295
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/bs.mie.2021.06.039
