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![Screening of stable cell lines from different GS-KO CHO cell lines. (A) Ab concentration in culture supernatants in 24-well plates of individual single-cell clones derived from different GS-KO CHO cells. Ab productivity [Ab titer (bar) and Qp (black circle)] (B) and IVCD (C) of the top two highest-producing clones derived from each GS-KO CHO cell for a 14-day simple fed-batch culture are illustrated.](publication/371944479/figure/fig7/AS:11431281171082729@1688009971873/Screening-of-stable-cell-lines-from-different-GS-KO-CHO-cell-lines-A-Ab-concentration.png)
Screening of stable cell lines from different GS-KO CHO cell lines. (A) Ab concentration in culture supernatants in 24-well plates of individual single-cell clones derived from different GS-KO CHO cells. Ab productivity [Ab titer (bar) and Qp (black circle)] (B) and IVCD (C) of the top two highest-producing clones derived from each GS-KO CHO cell for a 14-day simple fed-batch culture are illustrated.
Source publication
The glutamine synthetase (GS)-based Chinese hamster ovary (CHO) selection system is an attractive approach to efficiently identify suitable clones in the cell line generation process for biologics manufacture, for which GS-knockout (GS-KO) CHO cell lines are commonly used. Since genome analysis indicated that there are two GS genes in CHO cells, de...
Citations
... Engineering of cell lines and optimization of transfection and selection processes have been the main areas of emphasis for improving the selection strength of the GS gene. It was demonstrated that knockout of the endogenous GS genes in CHO cells, using the zinc finger nuclease (ZFN) technology, resulted in multiple cell lines with higher sensitivity to MSX selection and resulted in a six-fold increase in the frequency of high producers for a recombinant mAb, increasing the effectiveness of the cell line development and clone screening procedure [61,62]. The strength of selection can be effectively increased while using the same concentration of MSX by suppressing the endogenous GS gene expression and by boosting the glutamine content in the cell culture medium before transfection [63]. ...
The increasing demand for biosimilar monoclonal antibodies (mAbs) has prompted the development of stable high-producing cell lines while simultaneously decreasing the time required for screening. Existing platforms have proven inefficient, resulting in inconsistencies in yields, growth characteristics, and quality features in the final mAb products. Selecting a suitable expression host, designing an effective gene expression system, developing a streamlined cell line generation approach, optimizing culture conditions, and defining scaling-up and purification strategies are all critical steps in the production of recombinant proteins, particularly monoclonal antibodies, in mammalian cells. As a result, an active area of study is dedicated to expression and optimizing recombinant protein production. This review explores recent breakthroughs and approaches targeted at accelerating cell line development to attain efficiency and consistency in the synthesis of therapeutic proteins, specifically monoclonal antibodies. The primary goal is to bridge the gap between rising demand and consistent, high-quality mAb production, thereby benefiting the healthcare and pharmaceutical industries.
Graphical Abstract
... Meanwhile, the single clone of GS-KO CHO was cultured using Dynamis™ medium supplemented with anticlumping agent (1:1000 dilution) as per the optimal culture medium described in a previous report. 18 Adenosine stock solution and cordycepin stock solution were prepared by dissolving powder in PBS buffer (pH 7.4) to obtain the concentration of 20 mM, and each stock solution was filtered (PES syringe filter, 0.2 μm) before adding into cell cultures. ...
In this study, we investigated the effect of adenosine and its derivative cordycepin on the production yield of a recombinant human monoclonal antibody (adalimumab) in two commonly used Chinese Hamster ovary (CHO) cell lines that have different gene amplification systems, namely CHO‐DHFR⁻ and GS‐CHO knockout (GS‐KO CHO) cells and that were grown in batch culture, with and without glucose feeding. The results showed that adenosine suppressed the cell growth rate and increased the fraction of cells in S phase of the cell cycle for both CHO cell lines. Different concentrations and exposure times of adenosine feeding were tested. The optimal yield of adalimumab production was achieved with the addition of 1 mM adenosine on day 2 after start of the batch culture. Adenosine could significantly improve antibody titers and productivity in both CHO cell lines in cultures without glucose feeding. However, upon glucose feeding, adenosine did not improve antibody titers in CHO‐DHFR⁻ cells but extended culture duration and significantly increased antibody titers in GS‐KO CHO cells. Therefore, adenosine supplementation might be useful for antibody production in GS‐KO CHO cells in medium‐ to large‐scale batches. In case of cordycepin, a derivative of adenosine, CHO‐DHFR⁻ cells required higher concentration of cordycepin than GS‐KO CHO cells around 10 times to display the changes in cell growth and cell cycle. Moreover, cordycepin could significantly increase antibody titers only in CHO‐DHFR⁻ cell cultures without glucose feeding.
... In this step, cell lines that have the potential to produce high-yield antibodies are selected from a pool of transfected cells. This process involves screening and identifying stable clones that produce high quantities of mAbs and ensuring that they maintain their productivity over long-term cultivation 52 . ...
Chinese Hamster Ovary (CHO) cells have proven to be a remarkable tool in the production of monoclonal antibodies, offering numerous advantages such as high expression levels and post-translational modifications. Through the utilization of advanced techniques, researchers have been able to optimize CHO cell cultures for enhanced antibody production. However, it is crucial to consider various factors such as media composition, cell line engineering, and bioprocess optimization to achieve optimal results. By carefully addressing these considerations, researchers can harness the full potential of CHO cells for the production of high-quality monoclonal antibodies. Thus, the field of biotechnology continues to advance at an astonishing pace, the production of monoclonal antibodies holds immense promise for revolutionizing medicine. These highly specific molecules have become indispensable tools in diagnostics, therapeutics, and research. However, their production requires careful consideration of various factors to ensure optimal yield and quality. In this article, we delve into the world of Chinese Hamster Ovary (CHO) cells and explore the techniques and considerations involved in harnessing their potential for monoclonal antibody production.
Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation.
Key points
• The recombinant therapeutic protein degradation in CHO cell systems is reviewed.
• Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation.
• Reducing the degradation can improve the quality of recombinant therapeutic proteins.
