Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

Animal cell culture technology for therapeutic protein production has shown significant improvement over the last few decades. Chinese hamster ovary (CHO) cells have been widely adapted for the production of biopharmaceutical drugs. In the biopharmaceutical industry, it is crucial to develop cell culture media and culturing conditions to achieve the highest productivity and quality. However, CHO cells are significantly affected by apoptosis in the bioreactors, resulting in a substantial decrease in product quantity and quality. Thus, to overcome the obstacle of apoptosis in CHO cell culture, it is critical to develop a novel method that does not have minimal concern of safety or cost. Herein, we showed for the first time that exosomes, which are nano-sized extracellular vesicles, derived from CHO cells inhibited apoptosis in CHO cell culture when supplemented to the culture medium. Flow cytometric and microscopic analyses revealed that substantial amounts of exosomes were delivered to CHO cells. Higher cell viability after staurosporine treatment was observed by exosome supplementation (67.3%) as compared to control (41.1%). Furthermore, exosomes prevented the mitochondrial membrane potential loss and caspase-3 activation, meaning that the exosomes enhanced cellular activities under pro-apoptotic condition. As the exosomes supplements are derived from CHO cells themselves, it is not only beneficial for the biopharmaceutical productivity of CHO cell culture to inhibit apoptosis, but also from a regulatory standpoint to diminish any safety concerns. Thus, we conclude that the method developed in this research may contribute to the biopharmaceutical industry where minimizing apoptosis in CHO cell culture is beneficial.

mentioning

confidence: 99%

Inhibition of apoptosis using exosomes in Chinese hamster ovary cell culture

Han

Rhee

2018

“…Indeed, the capacity for this cellular process is commonly found to be limiting in protein biomanufacturing systems (Baeshen et al, 2015;Delic et al, 2014;Zhou, Raju, Alves, & Gilbert, 2018). These problems can be overcome by genetically engineering the cellular protein folding and secretion machinery; however, the relative benefits of over-expressing discrete effector genes are context-specific, dependent on product folding pathways, chassis macromolecular compositions, and recombinant protein expression levels (Delic et al, 2014;Hansen et al, 2017). These problems can be overcome by genetically engineering the cellular protein folding and secretion machinery; however, the relative benefits of over-expressing discrete effector genes are context-specific, dependent on product folding pathways, chassis macromolecular compositions, and recombinant protein expression levels (Delic et al, 2014;Hansen et al, 2017).…”

Section: Protein Folding and Secretionmentioning

confidence: 99%

“…Further, negative feedback loops containing stress-responsive promoters can be designed to automatically tune product expression levels down when burden causes deleterious effects to chassis performance (Ceroni et al, 2018;Dragosits, Nicklas, & Tagkopoulos, 2012). Although cell factory capacities can be increased via genetic engineering, the performance of the designed chassis tends to be highly context-specific, dependent on the product, cell strain, expression cassette, and production process (Delic et al, 2014;Fischer, Handrick, & Otte, 2015;Hansen, Pristovšek, Kildegaard, & Lee, 2017;Waegeman & Soetaert, 2011). Accordingly, expression cassette design, and therefore product yields, are constrained by the limitations of the chassis.…”

mentioning

confidence: 99%

Whole synthetic pathway engineering of recombinant protein production

Brown

Gibson

Hatton

et al. 2018

The output from protein biomanufacturing systems is a function of total host cell biomass synthetic capacity and recombinant protein production per unit cell biomass.In this study, we describe how these two properties can be simultaneously optimized via design of a product-specific combination of synthetic DNA parts to maximize flux through the protein synthetic pathway and the use of a host cell chassis with an increased capability to synthesize both cell and product biomass. Using secreted alkaline phosphatase (SEAP) production in Chinese hamster ovary cells as our example, we demonstrate how an optimal composition of input components can be assembled from a minimal toolbox containing rationally designed promoters, untranslated regions, signal peptides, product coding sequences, cell chassis, and genetic effectors. Product titer was increased 10-fold, compared with a standard reference system by (a) identifying genetic components that acted in concert to maximize the rates of SEAP transcription, translation, and translocation, (b) selection of a cell chassis with increased biomass synthetic capacity, and (c) engineering the host cell factory's capacity for protein folding and secretion. This whole synthetic pathway engineering process to design optimal expression cassette-chassis combinations should be applicable to diverse recombinant protein and host cell-type contexts.

“…They present several advantages, including their capacity to produce human‐like posttranslational modifications and to grow at high density in suspension in chemically defined culture media. Moreover, CHO cells are considered to be a safe host for the production of therapeutic proteins (Hansen, Pristovsek, Kildegaard, & Lee, 2017).…”

Section: Introductionmentioning

confidence: 99%

Overexpression of transcription factor Foxa1 and target genes remediate therapeutic protein production bottlenecks in Chinese hamster ovary cells

Berger

Fourn²,

Masternak

et al. 2020

Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy‐to‐express antibody, whereas Erp27 and Erp57 co‐overexpression increased cell density, viability, and the yield of difficult‐to‐express proteins. Foxa1 overexpression increased cell density, cell viability, and easy‐ and difficult‐to‐express protein yields, whereas it decreased reactive oxygen species late in fed‐batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult‐ and/or easy‐to‐express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields.