For over three decades, Chinese hamster ovary (CHO) cells have been the chosen expression platform for the production of therapeutic proteins with complex post‐translational modifications. However, ...the metabolism of these cells is far from perfect and optimized, and requires substantial know how and process optimization and monitoring to perform efficiently. One of the main reasons for this is the production and accumulation of toxic and growth‐inhibiting metabolites during culture. Lactate and ammonium are the most known, but many more have been identified. In this review, an overview of metabolites that deplete and accumulate throughout the course of cultivations with toxic and growth inhibitory effects to the cells is presented. Further, an overview of the CHO metabolism with emphasis to metabolic pathways of amino acids, glutathione (GSH), and related compounds which have growth‐inhibiting and/or toxic effect on the cells is provided. Additionally, relevant publications which describe the applications of metabolomics as a powerful tool for revealing which reactions occur in the cell under certain conditions are surveyed and growth‐inhibiting and toxic metabolites are identified. Also, a number of resources that describe the cellular mechanisms of CHO and are available on‐line are presented. Finally, the application of this knowledge for bioprocess and medium development and cell line engineering is discussed.
Chinese hamster ovary (CHO) cells are the chosen expression platform for the production of therapeutic proteins. ‘Omics data sets can aid and guide the rational design and generation of mammalian cells factories free of unwanted metabolic products and therefore capable of achieving higher cell densities and productivities. In this review, a number of metabolites depleting and accumulating throughout the course of cultivations, that have toxic and growth inhibitory effects to the cells are presented. Additionally, the potential application of this knowledge along with the use of analytical methods and on‐line metabolic resources for enhancing CHO cell lines by combining media development and cell line engineering approaches is discussed.
The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of ...biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality. With the growing toolbox of genetic engineering technologies, it is now possible to address general as well as host‐ or biopharmaceutical‐specific product quality obstacles.
In this review, we present diverse expression systems derived from mammalians, bacteria, yeast, plants, and insects as well as available genetic engineering tools. We focus on genes for knockout/knockdown and overexpression for meaningful approaches to improve biopharmaceutical PTMs and discuss their applicability as well as future trends in the field.
The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality.
In mammalian cells, >25% of synthesized proteins are exported through the secretory pathway. The pathway complexity, however, obfuscates its impact on the secretion of different proteins. Unraveling ...its impact on diverse proteins is particularly important for biopharmaceutical production. Here we delineate the core secretory pathway functions and integrate them with genome-scale metabolic reconstructions of human, mouse, and Chinese hamster ovary cells. The resulting reconstructions enable the computation of energetic costs and machinery demands of each secreted protein. By integrating additional omics data, we find that highly secretory cells have adapted to reduce expression and secretion of other expensive host cell proteins. Furthermore, we predict metabolic costs and maximum productivities of biotherapeutic proteins and identify protein features that most significantly impact protein secretion. Finally, the model successfully predicts the increase in secretion of a monoclonal antibody after silencing a highly expressed selection marker. This work represents a knowledgebase of the mammalian secretory pathway that serves as a novel tool for systems biotechnology.
Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and ...variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.
Chinese hamster ovary (CHO) cells are the most widely used production host for therapeutic proteins. With the recent emergence of CHO genome sequences, CHO cell line engineering has taken on a new ...aspect through targeted genome editing. The bacterial clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9) system enables rapid, easy and efficient engineering of mammalian genomes. It has a wide range of applications from modification of individual genes to genome‐wide screening or regulation of genes. Facile genome editing using CRISPR/Cas9 empowers researchers in the CHO community to elucidate the mechanistic basis behind high level production of proteins and product quality attributes of interest. In this review, we describe the basis of CRISPR/Cas9‐mediated genome editing and its application for development of next generation CHO cell factories while highlighting both future perspectives and challenges. As one of the main drivers for the CHO systems biology era, genome engineering with CRISPR/Cas9 will pave the way for rational design of CHO cell factories.
With the recent emergence of CHO genomic sequences and the RNA‐guided CRISPR/Cas9 technology, CHO cell line engineering has entered a new era with efficient targeted genome engineering. In this review the authors describe the basis for CRISPR/Cas9‐mediated genome engineering and its applications, which will pave the way for rational design of the next generation of CHO cell factories.
Chinese hamster ovary (CHO) cells are the preferred cell factory for the production of therapeutic glycoproteins. Although efforts primarily within bioprocess optimization have led to increased ...product titers of recombinant proteins (r-proteins) expressed in CHO cells, post-transcriptional bottlenecks in the biosynthetic pathway of r-proteins remain to be solved. To this end, the ectopic expression of transgenes (effector genes) offers great engineering potential. However, studies on effector genes have in some cases led to inconsistent results. Whereas this can in part be attributed to product specificity, other experimental and cellular factors are likely important contributors to these conflicting results. Here, these factors are reviewed and discussed with the objective of guiding future studies on effector genes.
Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product ...quality, or contaminate the final product. Here, we present an effort to create a "clean" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.
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.
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