Control of amino acid transport into Chinese hamster ovary cells

Geoghegan, Darren; Arnall, Claire L.; Hatton, Diane; Noble-Longster, Joanne; Sellick, Christopher A.; Senussi, Tarik; James, David C.

doi:10.1002/bit.26794

Cited by 14 publications

(14 citation statements)

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“…A pathway for oxidative stress was specifically designed so that the impact of high oxygen concentration in cell culture process is more accurately represented. This addition in the model makes (Geoghegan et al, 2018). Prediction of energy utilization and specific productivity could also be improved by considering the level of difficulty associated to the folding step of a specific antibody (Le Fourn et al, 2014), as well as the protein secretory pathway which has been identified as a bottleneck in some cases (Pieper et al, 2017;Rahimpour et al, 2013).…”

Section: Summary Of the Curation Processmentioning

confidence: 99%

Application of a curated genome-scale metabolic model of CHO DG44 to an industrial fed-batch process

Calmels

Cann

Malphettes

et al. 2019

Metabolic Engineering

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CHO cells have become the favorite expression system for large scale production of complex biopharmaceuticals. However, industrial strategies for upstream process development are based on empirical results, due to a lack of fundamental understanding of intracellular activities. Genome scale models of CHO cells have been reconstructed to provide an economical way of analyzing and interpreting large-omics datasets, since they add cellular context to the data. Here the most recently available CHO-DG44 genome-scale specific model was manually curated and tailored to the metabolic profile of cell lines used for industrial protein production, by modifying 601 reactions. Generic changes were applied to simplify the model and cope with missing constraints related to regulatory effects as well as thermodynamic and osmotic forces. Cell line specific changes were related to the metabolism of high-yielding production cell lines. The model was semi-constrained with 24 metabolites measured on a daily basis in n = 4 independent industrial 2L fed batch cell culture processes for a therapeutic antibody production. This study is the first adaptation of a genome scale model for CHO cells to an industrial process, that successfully predicted cell phenotype. The tailored model predicted accurately both the exometabolomics data (r ≥ 0.8 for 96% of the considered metabolites) and growth rate (r = 0.91) of the industrial cell line. Flux distributions at different days of the process were analyzed for validation and suggestion of strategies for medium optimization. This study shows how to adapt a genome scale model to an industrial process and sheds light on the metabolic specificities of a high production process. The curated genome scale model is a great tool to gain insights into intracellular fluxes and to identify possible bottlenecks impacting cell performances during production process. The general use of genome scale models for modeling industrial recombinant cell lines is a long-term investment that will highly benefit process development and speed up time to market.

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Section: Summary Of the Curation Processmentioning

confidence: 99%

Application of a curated genome-scale metabolic model of CHO DG44 to an industrial fed-batch process

Calmels

Cann

Malphettes

et al. 2019

Metabolic Engineering

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“…In addition, Geoghegan et al demonstrated that the mRNA expression of the cysteine transporter, xCT, was upregulated in CHO cells during increased mAb production in the stationary phase of growth. Moreover, this phenotype was sensitive to xCT inhibition by sulfasalazine and linked to oxidative stress induced by high mAb production (Geoghegan et al, 2018). These data taken together with data in this study, suggest that elevated xCT expression in the H 2 O 2 evolved host may be important for boosting BisAb titers and/or responding to oxidative stress.…”

Section: Discussionmentioning

confidence: 99%

A novel hydrogen peroxide evolved CHO host can improve the expression of difficult to express bispecific antibodies

Mistry

Kelsall

Sou

et al. 2021

Biotech & Bioengineering

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The manufacture of bispecific antibodies by Chinese hamster ovary (CHO) cells is often hindered by lower product yields compared to monoclonal antibodies. Recently, reactive oxygen species have been shown to negatively impact antibody production. By contrast, strategies to boost cellular antioxidant capacity appear to be beneficial for recombinant protein expression. With this in mind, we generated a novel hydrogen peroxide evolved host using directed host cell evolution. Here we demonstrate that this host has heritable resistance to hydrogen peroxide over many generations, displays enhanced antioxidant capacity through the upregulation of several, diverse antioxidant defense genes such as those involved in glutathione synthesis and turnover, and has improved glutathione content. Additionally, we show that this host has significantly improved transfection recovery times, improved growth and viability properties in a fed-batch production process, and elevated expression of two industrially relevant difficult to express bispecific antibodies compared to unevolved CHO control host cells. These findings demonstrate that host cell evolution represents a powerful methodology for improving specific host cell characteristics that can positively impact the expression of difficult to express biotherapeutics.bispecific antibody, evolved host, hydrogen peroxide, redox | INTRODUCTIONRecently, an interest in the cellular redox state and its effects on recombinant protein production has emerged (Handlogten et al., 2017(Handlogten et al., , 2020Orellana et al., 2015). Reactive oxygen species (ROS) are partial reduction products of molecular oxygen generated as a result of mitochondrial oxidative phosphorylation and oxidative protein folding within the endoplasmic reticulum (ER; Chevallier

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“…Increasing the transport capacity of the essential amino acids or inserting the complete pathways for their synthesis might be favourable for cell growth and protein production. Geoghegan et al identified amino acid transporters that are likely upregulated in producer cell lines and suggested that overexpression of one or more amino acid transporters might be beneficial for growth and productivity [178].…”

Section: Making Metabolism More Efficientmentioning

confidence: 99%

Key Challenges in Designing CHO Chassis Platforms

et al. 2020

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Following the success of and the high demand for recombinant protein-based therapeutics during the last 25 years, the pharmaceutical industry has invested significantly in the development of novel treatments based on biologics. Mammalian cells are the major production systems for these complex biopharmaceuticals, with Chinese hamster ovary (CHO) cell lines as the most important players. Over the years, various engineering strategies and modeling approaches have been used to improve microbial production platforms, such as bacteria and yeasts, as well as to create pre-optimized chassis host strains. However, the complexity of mammalian cells curtailed the optimization of these host cells by metabolic engineering. Most of the improvements of titer and productivity were achieved by media optimization and large-scale screening of producer clones. The advances made in recent years now open the door to again consider the potential application of systems biology approaches and metabolic engineering also to CHO. The availability of a reference genome sequence, genome-scale metabolic models and the growing number of various “omics” datasets can help overcome the complexity of CHO cells and support design strategies to boost their production performance. Modular design approaches applied to engineer industrially relevant cell lines have evolved to reduce the time and effort needed for the generation of new producer cells and to allow the achievement of desired product titers and quality. Nevertheless, important steps to enable the design of a chassis platform similar to those in use in the microbial world are still missing. In this review, we highlight the importance of mammalian cellular platforms for the production of biopharmaceuticals and compare them to microbial platforms, with an emphasis on describing novel approaches and discussing still open questions that need to be resolved to reach the objective of designing enhanced modular chassis CHO cell lines.

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Control of amino acid transport into Chinese hamster ovary cells

Cited by 14 publications

References 93 publications

Application of a curated genome-scale metabolic model of CHO DG44 to an industrial fed-batch process

Application of a curated genome-scale metabolic model of CHO DG44 to an industrial fed-batch process

A novel hydrogen peroxide evolved CHO host can improve the expression of difficult to express bispecific antibodies

Key Challenges in Designing CHO Chassis Platforms

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