Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium

Abstract

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine—the selection medium for Gs‐based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co‐transfection of Gs‐Enbrel and Aspg‐Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5‐fold higher specific productivity and ~3‐fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.

Advances in serum‐free media for CHO cells: From traditional serum substitutes to microbial‐derived substances

“…[ 20 ] Lastly, CHO cells have the unique characteristic of growing both adherently and in suspension, making them suitable for serum‐free and large‐scale cell cultivation in bioreactors. [ 18 ]…”

“…This characteristic breaks through the limitations of low efficiency and industrial‐scale production in other mammalian expression systems for synthesizing target proteins. [ 17,18 ] Thirdly, CHO cells have a relatively low risk of viral infection. Genomic and transcriptomic analysis have shown that necessary genes for viral infection such as viral entry receptor genes are absent in CHO cell lines, and the application experience of CHO cells has also confirmed its safety.…”

Effects of the COVID-19 pandemic: new approaches for accelerated delivery of gene to first-in-human CMC data for recombinant proteins

“… 42 New generations of engineered CHO cell lines, such as the CHO double knocked-out for glutamine synthetase (CHOGSKO), may be considered because they combine the advantages of higher expression levels and lower risk of instability of expression through more stringent selective pressure. 43 , 44 …”

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells

“…The mammalian expression system mainly includes expression vector, serum-free medium, and cell line. Among them, the mammalian cell lines include non-human cell lines and human cell lines, and the commonly used non-human mammalian cell is Chinese hamster ovary (CHO) cell (Ha et al 2023 ). Although non-human mammalian cells have safety and efficacy, RTPs expressed are easy to cause immunity in the human body, resulting in the elimination of foreign RTPs by the circulatory system, due to the different PTMs, especially the difference in glycosylation from human cells.…”