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Mammalian cells are commonly used as hosts in cell culture for biologics production in the pharmaceutical industry. Structured mechanistic models of metabolism have been used to capture complex cellular mechanisms that contribute to varying metabolic shifts in different cell lines. However, little research has focused on the impact of temporal changes in enzyme abundance and activity on the modeling of cell metabolism. In this work, we present a framework for constructing mechanistic models of metabolism that integrate growth‐signaling control of enzyme activity and transcript dynamics. The proposed approach is applied to build models for three Chinese hamster ovary (CHO) cell lines using fed‐batch culture data and time‐series transcript profiles. Leveraging information from the transcriptome data, we develop a parameter estimation approach based on multi‐cell‐line (MCL) learning, which combines data sets from different cell lines and trains the individual cell‐line models jointly to improve model accuracy. The computational results demonstrate the important role of growth signaling and transcript variability in metabolic models as well as the virtue of the MCL approach for constructing cell‐line models with a limited amount of data. The resulting models exhibit a high level of accuracy in predicting distinct metabolic behaviors in the different cell lines; these models can potentially be used to accelerate the process and cell‐line development for the biomanufacturing of new protein therapeutics. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) used in biomedical research and therapeutic applications are primarily produced by Chinese hamster ovary (CHO) cells via fed-batch culture. The growing need for elevated quantities of biologics mandates the continual optimization of the mAb production process. The development of an effective process control method is indispensable for the production of specified mAbs by CHO cells. In this study, the effects of the pH control strategy on CHO cell fed-batch culture to produce an antibody (EA5) were first investigated in a 3 L bioreactor. The results indicate that controlling the culture pH at 7.2 during the fed-batch stage could produce a higher EA5 titer of 6.1 g/L with a lower Man5 ratio of 2.2% by day 14. Based on this, an optimized CHO cell fed-batch culture was conducted in a 15 L bioreactor to verify its effectiveness and stability. In this case, on day 14, an EA5 titer of 6.5 g/L was achieved with productivity of 0.46 g/L/day, which was 1.07-fold higher compared to that of the culture in the 3 L bioreactor. Furthermore, regarding the product quality, a monomer abundance of 96.0%, a main peak of 55.0%, and a Man5 proportion of 2.4% were maintained in the 15 L bioreactor. In addition, different cell clarification processes were evaluated using the CHO cell culture broth from the 3 L and 15 L bioreactors to further improve productivity and economic performance. Overall, this study provides some directions for process intensification and improving the quality of mAbs produced by CHO cells in the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

Hollow fiber filter fouling is a common issue plaguing perfusion production process for biologics therapeutics, but the nature of filter foulant has been elusive. Here we studied cell culture materials especially Chinese hamster ovary (CHO) cell‐derived extracellular vesicles in perfusion process to determine their role in filter fouling. We found that the decrease of CHO‐derived small extracellular vesicles (sEVs) with 50–200 nm in diameter in perfusion permeates always preceded the increase in transmembrane pressure (TMP) and subsequent decrease in product sieving, suggesting that sEVs might have been retained inside filters and contributed to filter fouling. Using scanning electron microscopy and helium ion microscopy, we found sEV‐like structures in pores and on foulant patches of hollow fiber tangential flow filtration filter (HF‐TFF) membranes. We also observed that the Day 28 TMP of perfusion culture correlated positively with the percentage of foulant patch areas. In addition, energy dispersive X‐ray spectroscopy‐based elemental mapping microscopy and spectroscopy analysis suggests that foulant patches had enriched cellular materials but not antifoam. Fluorescent staining results further indicate that these cellular materials could be DNA, proteins, and even adherent CHO cells. Lastly, in a small‐scale HF‐TFF model, addition of CHO‐specific sEVs in CHO culture simulated filter fouling behaviors in a concentration‐dependent manner. Based on these results, we proposed a mechanism of HF‐TFF fouling, in which filter pore constriction by CHO sEVs is followed by cake formation of cellular materials on filter membrane. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are widely used for the industrial production of therapeutic monoclonal antibodies (mAbs). To meet the increasing market demands, high productivity, and quality are required in cell culture. One of the critical attributes of mAbs, from a safety perspective, is mAb fragmentation. However, methods for preventing mAbs fragmentation in CHO cell culture are limited. In this study, we observed that the antibody fragment content increased with increasing titers in fed‐batch cultures for all three cell lines expressing recombinant antibodies. Adding copper sulfate to the culture medium further increased the fragment content, suggesting the involvement of reactive oxygen species (ROS) in the fragmentation process. Though antioxidants may be helpful to scavenge ROS, several antioxidants are reported to decrease the productivity of CHO cells. Among the antioxidants examined, we observed that the addition of catechin or (−)‐epigallocatechin gallate to the culture medium prevented fragmentation content by about 20% and increased viable cell density and titer by 30% and 10%, respectively. Thus, the addition of catechins or compounds of equivalent function would be beneficial for manufacturing therapeutic mAbs with a balance between high titers and good quality. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) used in biomedical research and therapeutic applications are primarily produced by Chinese hamster ovary (CHO) cells via fed-batch culture. The growing need for elevated quantities of biologics mandates the continual optimization of the mAb production process. The development of an effective process control method is indispensable for the production of specified mAbs by CHO cells. In this study, the effects of the pH control strategy on CHO cell fed-batch culture to produce an antibody (EA5) were first investigated in a 3 L bioreactor. The results indicate that controlling the culture pH at 7.2 during the fed-batch stage could produce a higher EA5 titer of 6.1 g/L with a lower Man5 ratio of 2.2% by day 14. Based on this, an optimized CHO cell fed-batch culture was conducted in a 15 L bioreactor to verify its effectiveness and stability. In this case, on day 14, an EA5 titer of 6.5 g/L was achieved with productivity of 0.46 g/L/day, which was 1.07-fold higher compared to that of the culture in the 3 L bioreactor. Furthermore, regarding the product quality, a monomer abundance of 96.0%, a main peak of 55.0%, and a Man5 proportion of 2.4% were maintained in the 15 L bioreactor. In addition, different cell clarification processes were evaluated using the CHO cell culture broth from the 3 L and 15 L bioreactors to further improve productivity and economic performance. Overall, this study provides some directions for process intensification and improving the quality of mAbs produced by CHO cells in the biopharmaceutical industry.

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO–K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp , in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells. • Genome-wide virus-free CRISPR/Cas9 screening was performed in CHO–K1 cells. • Novel and known hyperosmotic stress responsive gene clusters were identified. • Perturbation of identified genes conferred resistance to osmotic stress. • Knockout of Zfr and Pnp improved product titer under hyperosmotic conditions. [ABSTRACT FROM AUTHOR]

Interchain disulfide bonds in monoclonal antibodies may be reduced during large-scale mAb production using Chinese hamster ovary (CHO) cells. This reaction lowers the mAb product yield and purity; however, it may be prevented by screening cell lines that are unsusceptible to reduction and using them in mAb production. Antibody reduction susceptibility may be cell line-dependent. To the best of our knowledge, however, an efficient method of screening reduction-unsusceptible CHO cell lines has not been previously reported. Here, we report a novel screening method that can simultaneously detect and identify mAb reduction susceptibility in lysates containing ≤ 48 CHO cell lines. This evaluation system was equally effective and generated similar results at all culture scales, including 250 mL, 3 L, and 1000 L. Furthermore, we discovered that reduction-susceptible cell lines contained higher total intracellular nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ concentrations than reduction-unsusceptible cell lines, regardless of whether they expressed immunoglobulin (Ig)G4 or IgG1. NADPH or NADP+ supplementation in the lysate of reduction-unsusceptible cells resulted in mAb reduction. Application of the innovative CHO cell line screening approach could mitigate or prevent reductions in large-scale mAb generation from CHO cells. [ABSTRACT FROM AUTHOR]

Background and Aim: The current treatment for hemophilia B is replacement therapy, which involves the intravenous infusion of human coagulation factor IX (hFIX) purified from plasma or a recombinant form produced in mammalian cells. In this study, using a bicistronic expression system, the stable expression of the hFIX in a serum-free and suspension-adapted Chinese hamster ovary cell line (CHO-s) was investigated. Materials and Methods: A DNA fragment consisting of hFIX, Internal Ribosome Entry Site (IRES) and Enhanced Green Fluorescent Protein (EGFP) nucleotide sequences was cloned into pcDNA3.1 expression plasmid under the control of Cytomegalovirus (CMV) promoter. The bicistronic plasmid was then linearized using BglII restriction enzyme and transfected into CHO-s cells. The transfected cells were treated with geneticin for 14 days. The culture medium of the stable cells was then collected and the expression level of the hFIX were examined using western blotting and ELISA. The coagulation activity was also evaluated by the chromogenic method. Results: The recombinant CHO-s cells resistant to geneticin were observed under a fluorescence microscope in green color, which indicated the expression and accumulation of the EGFP in the cytoplasm of the cells. The results of Western blotting confirmed the expression and secretion of the hFIX into the culture medium. The amount of the secreted hFIX was 150 ng/mL/106cells with a coagulation activity of 5.6 ±0.2 mU/mL. Conclusion: Our findings demonstrated that this bicistronic expression system could simultaneously produce EGFP and hFIX in CHO-s cells. This expression system facilitates selection and isolation of hFIX-expressing cells.

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all‐in‐one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site‐specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double‐cut and double‐nick donor formats significantly improved targeting accuracy by 2.3–8.3‐fold and 19–22‐fold, respectively, compared to standard circular donors. Notably, Cas9‐mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low‐fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR‐based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications. [ABSTRACT FROM AUTHOR]

Immunoglobulin A (IgA) has been showing potential as a new therapeutic antibody. However, recombinant IgA suffers from low yield. Supplementation of the medium is an effective approach to improving the production and quality of recombinant proteins. In this study, we adapted IgA1-producing CHO-K1 suspension cells to a high concentration (150 mM) of different disaccharides, namely sucrose, maltose, lactose, and trehalose, to improve the production and quality of recombinant IgA1. The disaccharide-adapted cell lines had slower cell growth rates, but their cell viability was extended compared to the nonadapted IgA1-producing cell line. Glucose consumption was exhausted in all cell lines except for the maltose-adapted one, which still contained glucose even after the 9th day of culturing. Lactate production was higher among the disaccharide-adapted cell lines. The specific productivity of the maltose-adapted IgA1-producing line was 4.5-fold that of the nonadapted line. In addition, this specific productivity was higher than in previous productions of recombinant IgA1 with a lambda chain. Lastly, secreted IgA1 aggregated in all cell lines, which may have been caused by self-aggregation. This aggregation was also found to begin inside the cells for maltose-adapted cell line. These results suggest that a high concentration of disaccharide-supplemented induced hyperosmolarity in the IgA1-producing CHO-K1 cell lines. In addition, the maltose-adapted CHO-K1 cell line benefited from having an additional source of carbohydrate. [ABSTRACT FROM AUTHOR]

Technologies for efficiently expanding Chinese hamster ovary (CHO) cells, the primary host cells for antibody production, are of growing industrial importance. Various processes for the use of microcarriers in CHO suspension cultures have been developed, but there have been very few studies on cell-adhesive microcarriers that are similar in size to cells. In this study, we proposed a new approach to suspension cultures of CHO cells using cell-sized condensed and crosslinked gelatin microparticles (GMPs) as carriers. Unlike commercially available carriers with sizes typically greater than 100 μm, each cell can adhere to the surface of multiple particles and form loose clusters with voids. We prepared GMPs of different average diameters (27 and 48 μm) and investigated their effects on cell adhesion and cluster formation. In particular, small GMPs promoted cell proliferation and increased IgG4 production by the antibody-producing CHO cell line. The data obtained in this study suggest that cell-sized particles, rather than larger ones, enhance cell proliferation and function, providing useful insights for improving suspension-culture-based cell expansion and cell-based biologics production for a wide range of applications. [ABSTRACT FROM AUTHOR]

Appropriate treatment of Hemophilia B is vital for patients' quality of life. Historically, the treatment used was the administration of coagulation Factor IX derived from human plasma. Advancements in recombinant technologies allowed Factor IX to be produced recombinantly. Successful recombinant production has triggered a gradual shift from the plasma derived origins of Factor IX, as it provides extended half-life and expanded production capacity. However, the complex post-translational modifications of Factor IX have made recombinant production at scale difficult. Considerable research has therefore been invested into understanding and optimizing the recombinant production of Factor IX. Here, we review the evolution of recombinant Factor IX production, focusing on recent developments in bioprocessing and cell engineering to control its post-translational modifications in its expression from Chinese Hamster Ovary (CHO) cells. [ABSTRACT FROM AUTHOR]

Abstract Animal‐derived anti‐IgG secondary antibodies are currently employed to stain and screen of human monoclonal antibody(mAb)‐producing cells, but using animal‐derived antibodies may raise the concerns of high cost, complicated operations and biological safety issues in biopharmaceutical manufacturing. Nanobodies(VHHs) are attractive forms of antibodies for their straightforward engineering and expression in both eukaryotic and prokaryotic systems. Using phage‐displayed immune llama VHH library, we identified new anti‐Fc VHHs that could bind to human Fc with high affinity. In GFP fusion format, the anti‐Fc VHH‐GFP generated dramatically stronger FACS signals than AF488 conjugated anti‐IgG antibodies when used for staining mAb‐producing CHO cells. Furthermore, preparative sorting of CHO cells based on anti‐Fc VHH‐GFP staining resulted in the enrichment of cell lines capable of synthesizing mAb at high productivity. This safe and cost‐efficient anti‐Fc VHH‐GFP may optimize the process of generating highly productive cell lines for therapeutic mAb production compared to conventional animal‐derived fluorescent antibodies.

Immunoglobulin A (IgA) of sows is critically important for assessing piglets' protective capacity against porcine epidemic diarrhea virus (PEDV). Here, we report a therapeutic chimeric anti-PEDV IgG/IgA expressed by Chinese hamster ovary (CHO) cells for oral treatment of PED. The chimeric anti-PEDV IgG/IgA was produced by the CHO cell lines, in which the heavy chain was constructed by combining the VH, Cγ1 and hinge regions of PEDV IgG mAb 8A3, and the Cα2 and Cα3 domains of a Mus musculus immunoglobulin alpha chain. The chimeric anti-PEDV IgG/IgA could neutralize the strains of CV777 (G1), P014 (G2) and HN1303 (G2) in vitro effectively, showing broad-spectrum neutralization activity. The in vivo challenge experiments demonstrated that chimeric anti-PEDV IgG/IgA (9C4) produced in the CHO cell supernatant could alleviate clinical diarrhea symptoms of the PEDV infection in piglets. In general, our study showed that chimeric anti-PEDV IgG/IgA produced from CHO cell line supernatants effectively alleviates PEDV infection in piglets, which also gives the foundation for the construction of fully functional secretory IgA by the J chain introduction to maximize the antibody therapeutic effect. [ABSTRACT FROM AUTHOR]

Animal‐derived anti‐IgG secondary antibodies are currently employed to stain and screen of human monoclonal antibody(mAb)‐producing cells, but using animal‐derived antibodies may raise the concerns of high cost, complicated operations and biological safety issues in biopharmaceutical manufacturing. Nanobodies(VHHs) are attractive forms of antibodies for their straightforward engineering and expression in both eukaryotic and prokaryotic systems. Using phage‐displayed immune llama VHH library, we identified new anti‐Fc VHHs that could bind to human Fc with high affinity. In GFP fusion format, the anti‐Fc VHH‐GFP generated dramatically stronger FACS signals than AF488 conjugated anti‐IgG antibodies when used for staining mAb‐producing CHO cells. Furthermore, preparative sorting of CHO cells based on anti‐Fc VHH‐GFP staining resulted in the enrichment of cell lines capable of synthesizing mAb at high productivity. This safe and cost‐efficient anti‐Fc VHH‐GFP may optimize the process of generating highly productive cell lines for therapeutic mAb production compared to conventional animal‐derived fluorescent antibodies. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are currently the most widely used host cells for recombinant therapeutic protein (RTP) production. Currently, the RTP yields need to increase further to meet the market needs and reduce costs. In this study, three stabilizing and anti-repressor (SAR) elements from the human genome were selected, including human SAR7, SAR40, and SAR44 elements. SAR elements were cloned upstream of the promoter in the eukaryotic vector, followed by transfection into CHO cells, and were screened under G418 pressure. Flow cytometry was used to detect enhanced green fluorescent protein (eGFP) expression levels. The gene copy numbers and mRNA expression levels were determined through quantitative real-time PCR. Furthermore, the effect of the stronger SAR elements on adalimumab was investigated. The results showed that transgene expression levels in the SAR-containing vectors were higher than that of the control vector, and SAR7 and SAR40 significantly increased and maintained the long-term expression of the transgene in CHO cells. In addition, the transgene expression level increase was related with gene copy numbers and mRNA expression levels. Collectively, SAR elements can enhance the transgene expression and maintain the long-term expression of a transgene in transfected CHO cells, which may be used to increase recombinant protein production in CHO cells.

Invasive Aspergillosis is a high-risk illness with a high death rate in immunocompromised people due to a lack of early detection and timely treatment. Based on immunology study, we achieved an efficient production of anti-galactomannan antibody by Chinese hamster ovary (CHO) cells and applied it to time-resolved fluoroimmunoassay for Aspergillus galactomannan detection. We first introduced dual promoter expression vector into CHO host cells, and then applied a two-step screening strategy to screen the stable cell line by methionine sulfoximine pressurization. After amplification and fermentation, antibody yield reached 4500 mg/L. Then we conjugated the antibodies with fluorescent microspheres to establish a double antibody sandwich time-resolved fluoroimmunoassay, which was compared with the commercial Platelia™ Aspergillus Ag by clinical serum samples. The preformed assay could obtain the results in less than 25 min, with a limit of detection for galactomannan of approximately 1 ng/mL. Clinical results of the two methods showed that the overall percent agreement was 97.7% (95% CI: 96.6%–98.4%) and Cohen's kappa coefficient was 0.94. Overall, the assay is highly consistent with commercial detection, providing a more sensitive and effective method for the rapid diagnosis of invasive aspergillosis. [Display omitted] • An efficient and stable cell line expression system was constructed to produce of anti-galactomannan monoclonal antibodies. • The antibody yield reached 4500 mg/L by scaling up fermentation, a 1.3-fold increase. • A time-resolved fluorescence immunoassay was established by conjugating the antibodies to detect aspergillosis. • The assay is highly consistent with commercial Platelia™ Aspergillus Ag according to clinical serum results. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4. Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of >3 g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003 cells were evaluated by fed-batch culture. The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003 cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5 g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production. [ABSTRACT FROM AUTHOR]

Although antibodies have attracted attention as next-generation biopharmaceuticals, the costs of purifying the products and of arranging the environment for cell cultivation are high. Therefore, there is a need to increase antibody efficacy and improve product quality as much as possible. Since antibodies are glycoproteins, their glycan structures have been found to affect the function of antibodies. Especially, afucosylation of the N-linked glycan in the Fc region is known to significantly increase antibody-dependent cellular cytotoxicity. In this study, we established a double-mutant ΔGMDΔGFT in which GDP-mannose 4,6-dehydratase and GDP-fucose transporter were knocked out in Chinese hamster ovary cells, a platform for biopharmaceutical protein production. By adapting ΔGMDΔGFT cells to serum-free medium and constructing suspension-cultured cells, we established host CHO cells with no detected fucosylated glycans and succeeded in production of afucosylated antibodies. We also demonstrated that, in culture in the presence of serum, fucosylation occurs due to contamination from serum components. Furthermore, we found that afucosylation of glycans does not affect cell growth after adaptation to serum-free medium as compared to wild-type CHO cells growth and does not significantly affect the expression levels of other endogenous fucose metabolism-related enzyme genes. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cell cultures are widely used due to their high productivity, industrial track record, safety record, and correct post-translational modifications. Fed-batch and perfusion modes have been mostly adopted as traditional commercial manufacturing process using CHO cells for the production of biopharmaceuticals. Thus, we compared fed-batch and perfusion culture processes for the commercial-scale production of monoclonal antibody. Main production process was performed after confirming the comparable culture performance in seed train of different operation modes, and then perfusion cultures showed 1.6-fold higher peak viable cell density (VCD) with a longer culture duration than that of fed-batch cultures. Also, as the result of main batches, total product amount increased over 450% in perfusion cultures. Importantly, perfusion process allowed to improve the higher purification yield while maintaining acceptable product quality, because the level of process impurity could be reduced by using retention device. When economic comparison of fed-batch versus perfusion processes was carried out under the equal scale of upstream and downstream, perfusion mode was more a cost-effective bioprocess that could reduce the cost of goods. In conclusion, although comparison data of triplicate commercial batches are of course specific case for a given antibody production, this study verified the possibility of using perfusion mode as an efficient system for commercial producing the desired antibody and is a meaningful report in the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

Abstract Recent cell culture media for mammalian cells can be abundantly formulated with nutrients supporting production, but such media can be limited to use in host cell culture, transfection, cell cloning, and cell growth under the low cell density conditions. In many cases, appropriate platform media are used for cell line development, and then replaced with rich media for production. In this study, we demonstrate rich chemically defined media for Chinese hamster ovary (CHO) cells that are suitable as basal media both for cell line development and for final production of culture process. Set up for transfection, semi-solid media optimization, mini-pool screening, and single cell cloning media development were performed, and final clones were obtained with higher productivity in fed-batch culture mode using rich formulated media comparing with lean formulated media. Developed methods may remove the requirements for cell adaptation to production media after cell line development, and relieve the clonality issues associated with changing the culture media. Furthermore, established methods have advantages over traditional approaches, including saving resources and decreasing the time and the effort required to optimize the production process.

Chinese hamster ovary (CHO) cells are a ubiquitous tool for industrial therapeutic recombinant protein production. However, consistently generating high‐producing clones remains a major challenge during the cell line development process. The glutamine synthetase (GS) and dihydrofolate reductase (DHFR) selection systems are commonly used CHO expression platforms based on controlling the balance of expression between the transgenic and endogenous GS or DHFR genes. Since the expression of the endogenous selection gene in CHO hosts can interfere with selection, generating a corresponding null CHO cell line is required to improve selection stringency, productivity, and stability. However, the efficiency of generating bi‐allelic genetic knockouts using conventional protocols is very low (<5%). This significantly affects clone screening efficiency and reduces the chance of identifying robust knockout host cell lines. In this study, we use the GS expression system as an example to improve the genome editing process with zinc finger nucleases (ZFNs), resulting in improved GS‐knockout efficiency of up to 46.8%. Furthermore, we demonstrate a process capable of enriching knockout CHO hosts with robust bioprocess traits. This integrated host development process yields a larger number of GS‐knockout hosts with desired growth and recombinant protein expression characteristics. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the improvement of mAb production in Chinese hamster ovary (CHO) cells due to the increasing demand for these products. In this regard, various chromatin-modifying elements such as insulators have been incorporated in the expression vectors to augment mAb expression. In this study, human gamma-satellite insulator containing vectors were utilized for the expression of an anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) mAb in CHO-K1 cells. To this aim, dual expression vectors encoding the antibody light chain (LC) and heavy chain (HC) with or without the insulator element were constructed, and mAb expression was evaluated in transient and stable expression. Based on the results, mAb expression significantly increased in the stable cell pool, and clonal cells developed using the human gamma-satellite insulator. In contrast, transient antibody expression was not affected by the insulator element. Finally, the enhancement of LC and HC mRNA levels was found in the insulator containing stable cell pools using the quantitative real-time-polymerase chain reaction (qRT-PCR). Our findings showed the positive effect of the human gamma-satellite insulator on the stable expression of an anti-PCSK9 immunoglobulin G1 (IgG1) mAb in CHO-K1 cells using dual expression vectors. [ABSTRACT FROM AUTHOR]

Rapidly expanding pipelines in biopharmaceutical industry and increasingly stringent requirements from regulatory agencies call for a more efficient and rational study design at early-stage cell culture process development. Recent developments in high-throughput bioreactor system and design of experiments (DoE) enable cell culture process development sector to meet such demand by integrating both technological advances. In this study, we compared varied forms of fractional factorial designs (FFDs) and definitive screening design (DSD) covering key process parameters such as temperature shift target, temperature shift timing, feed strategy, pH, dissolved oxygen, harvest time and additives in Ambr® 15 bioreactors to better understand the initial cell culture process and thus optimize it. Results showed that DSD was more effective and efficient in detecting both main and quadratic effects comparing to FFDs. Comparison of varied available model development strategies revealed that two-stage forward regression combined with corrected Akaike's information criterion (AICc) and least absolute shrinkage and selection operator (LASSO) regression combined with self-validated ensembled models (SVEM) offered superior predictive capability. Moreover, key DoE regularities was evaluated for their validity. In conclusion, DSD is a suitable experimental design for the early-stage cell culture process development if quadratic effects might exist. Statistical models derived based on DSD-based dataset could be applied for predictive optimization of antibody producing bioprocess while regularities such as effect heredity and effect hierarchy might be relaxed considering higher-order interaction effects are more likely to existed for a cell-based biological system. • Utilizing high-throughput bioreactors and DoE to streamline early-stage cell culture optimization. • DSD was more effective and efficient in detecting both main and quadratic effects comparing to FFDs. • AICc-based two-stage forward stepwise regression models and SVEM-Lasso offered superior predictive capability. • Relaxing effect heredity and hierarchy while still adhering to the principles of effect sparsity and model parsimony. [ABSTRACT FROM AUTHOR]

• A cleaved trimeric pre-fusion F protein was needed for RSV vaccine development • Vector design and clone selection ensured the pre-fusion conformation and trimeric assembly • Seeding density optimization and a biphasic low temperature shift further increased its yield • The process was scalable and manufacturable for clinical manufacturing • Preliminary Phase I data indicate the pre-fusion antigen has a promising efficacy Respiratory syncytial virus (RSV) is a highly contagious virus causing severe infection in infants and the elderly. Various approaches are being used to develop an effective RSV vaccine. The RSV fusion (F) subunit, particularly the cleaved trimeric pre-fusion F, is one of the most promising vaccine candidates under development. The pre-fusion conformation elicits the majority of neutralizing antibodies during natural infection. However, this pre-fusion conformation is metastable and prone to conversion to a post-fusion conformation, thus hindering the potential of this construct as a vaccine antigen. The Vaccine Research Center (VRC) at the National Institutes of Health (NIH) designed a structurally stabilized pre-fusion F glycoprotein, DS-Cav1, that showed high immunogenicity and induced a neutralizing response in animal studies. To advance this candidate to clinical manufacturing, a production process that maintained product quality (i.e. a cleaved trimer with pre-fusion conformation) and delivered high protein expression levels was required. This report describes the development of the vaccine candidate including vector design and cell culture process development to meet these challenges. Co-transfection of individual plasmids to express DS-Cav1 and furin (for DS-Cav1 cleavage and activation) demonstrated a superior protein product expression and pre-fusion conformation compared to co-expression with a double gene vector. A top clone was selected based on these measurements. Protein expression levels were further increased by seeding density optimization and a biphasic hypothermia temperature downshift. The combined efforts led to a high-yield fed-batch production of approximately 1,500 mg/L (or up to 15,000 doses per liter) at harvest. The process was scaled up and demonstrated to be reproducible at 50 L-scale for toxicity and Phase I clinical trial use. Preliminary phase I data indicate the pre-fusion antigen has a promising efficacy (Crank et al., 2019). [ABSTRACT FROM AUTHOR]

• UCOE element was associated with increased yield of antibody. • UCOE pools had greater number of positive clones and more stable expression. • Clones that stably co-express mAb and its specific SINEUP had more mAb titer. • Stably expressed SINEUP clones had stable long-term expression. • Combined UCOE and SINEUP provided more optimized expression conditions in CHO cells. The growing need for monoclonal antibodies (mAbs) necessitates the development of novel and efficient production approaches. Regulatory elements like ubiquitous chromatin-opening elements (UCOEs) have been employed for improvement of the mAb expression in the Chinese hamster ovary (CHO) cells. SINEUPs are a class of long non-coding RNAs, which can improve the translation of partly overlapping mRNAs. A combination of these two elements might lead to higher production of mAbs. Therefore, the current study was conducted to investigate the effects of SINEUPs and A2UCOE on the expression of an IgG1 in the CHO-K1 cells. Hence, after constructing the mAb, mAb-SINEUP, and mAb-UCOE vectors, four stable cell pools were generated through combining the above vectors. According to the expression analysis, antibody yields were higher in the mAb-SINEUP and mAb-UCOE cell pools compared to the mAb cells. In addition, the cells possessing both SINEUP and UCOE elements provided the best expression. Persistent mAb expression was observed for over 2 months in these cells, whilst the expression was decreased in the mAb pool. SINEUP and UCOE positively influenced the stable mAb expression. It can be concluded that the SINEUP and UCOE enhance the antibody stability and expression level separately and their combination improves the mAb production in the CHO cells. [ABSTRACT FROM AUTHOR]

A major goal for process and cell engineering in the biopharmaceutical industry is enhancing production through increasing volumetric and cell‐specific productivities (CSP). Here, we present 5′‐deoxy‐5′‐(methylthio)adenosine (MTA), the degradation product of S‐(5′‐adenosyl)‐L‐methionine (SAM), as a highly attractive native additive which can boost CSP by 79% when added to exponentially growing cells at a concentration of 250–300 μm. Notably, cell viability and cell size remain higher than in non‐treated cultures. In addition, cell cycle arrests first in S‐, then in G2‐phase before levelling out compared to non‐treated cultivations. Intensive differential gene analysis reveals that expression of genes for cytoskeleton mediated proteins and vesicle transport is amplified by treatment. Furthermore, the interaction of MTA with cell proliferation additionally stimulated recombinant protein formation. The results may serve as a promising starting point for further developments in process and cell engineering to boost productivity. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells. [ABSTRACT FROM AUTHOR]

Recent cell culture media for mammalian cells can be abundantly formulated with nutrients supporting production, but such media can be limited to use in host cell culture, transfection, cell cloning, and cell growth under the low cell density conditions. In many cases, appropriate platform media are used for cell line development, and then replaced with rich media for production. In this study, we demonstrate rich chemically defined media for Chinese hamster ovary (CHO) cells that are suitable as basal media both for cell line development and for final production of culture process. Set up for transfection, semi-solid media optimization, mini-pool screening, and single cell cloning media development were performed, and final clones were obtained with higher productivity in fed-batch culture mode using rich formulated media comparing with lean formulated media. Developed methods may remove the requirements for cell adaptation to production media after cell line development, and relieve the clonality issues associated with changing the culture media. Furthermore, established methods have advantages over traditional approaches, including saving resources and decreasing the time and the effort required to optimize the production process. [ABSTRACT FROM AUTHOR]

Chromosomes in Chinese hamster ovary (CHO) cells are labile. We have shown that high-chromosome-number CHO cells have greater potential to become robust producers of recombinant proteins. One explanation being the increase in transgene integration sites. However, high-chromosome-number cell clones produce more IgG3 following culture of single-cell clones, even under conditions that yield the same number of integrations as cells with normal chromosome numbers. Here, we characterized high-chromosome-number cells by transcriptome analysis. RNA standards were used to normalize transcriptomes of cells that had different chromosome numbers. Our results demonstrate that the mRNA ratio of β-actin and many other genes in high-chromosome-number cells to that in normal-chromosome-number cells per cell (normalized to RNA standards) was smaller than the equivalent genomic size and cell volume ratios. Many genes encoding membrane proteins are more highly expressed in high-chromosome-number cells, probably due to differences in cell size caused by the increase in chromosomes. In addition, genes related to histone modification and lipid metabolism are differentially expressed. The reduced transcript level required per protein produced in total and the different intracellular signal transductions might be key factors for antibody production. [ABSTRACT FROM AUTHOR]

Abstract The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.

The endoplasmic reticulum (ER) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about how this vital ATP transport occurs across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER). Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca2+ gradient across the ER membrane is necessary for ATP transport into the ER, through SLC35B1/AXER. We propose that under physiological conditions, increases in cytosolic Ca2+ inhibit ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation (OxPhos) inhibitors and that ER protein misfolding increases ATP uptake from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e. an ‘anti-Warburg’ effect.

The Chinese hamster ovary (CHO) cell line is the most widely used host cell for therapeutic antibody production. Although its productivity has been improved by various strategies to satisfy the growing global demand, some difficult-to-express (DTE) antibodies remain at low secretion levels. To improve the production of various therapeutic antibodies, it is necessary to determine possible rate-limiting steps in DTE antibody secretion in comparison with other high IgG producers. Here, we analyzed the protein secretion process in CHO cells producing the DTE immunoglobulin G (IgG) infliximab. The results from chase assays using a translation inhibitor revealed that infliximab secretion could be nearly completed within 2 h, at which time the cells still retained about 40% of heavy chains and 65% of light chains. Using fluorescent microscopy, we observed that these IgG chains remained in the endoplasmic reticulum and Golgi apparatus. The cells inefficiently form fully assembled heterodimer IgG by making LC aggregates, which may be the most serious bottleneck in the production of DTE infliximab compared with other IgG high producers. Our study could contribute to establish the common strategy for constructing DTE high-producer cells on the basis of rate-limiting step analysis. [ABSTRACT FROM AUTHOR]

Biopharmaceuticals represented by immunoglobulin G (IgG) are produced by the cultivation of recombinant animal cells, especially Chinese hamster ovary (CHO) cells. It is thought that the intracellular secretion process of IgG is a bottleneck in the production of biopharmaceuticals. Many studies on the regulation of endogenous secretory protein expression levels have shown improved productivity. However, these strategies have not universally improved the productivity of various proteins. A more rational and efficient establishment of high producer cells is required based on an understanding of the secretory processes in IgG producing CHO cells. In this study, a CHO cell line producing humanized IgG1, which was genetically fused with fluorescent proteins, was established to directly analyze intracellular secretion. The relationship between the amount of intracellular and secreted IgG was analyzed at the single cell level by an automated single-cell analysis and isolation system equipped with dual color fluorescent filters. The amounts of intracellular and secreted IgG showed a weak positive correlation. The amount of secreted IgG analyzed by the system showed a weak negative linear correlation with the specific growth of isolated clones. An immunofluorescent microscopy study showed that the established clones could be used to analyze the intracellular secretion bottleneck. This is the first study to report the use of fluorescent protein fusion IgG as a tool to analyze the secretion of recombinant CHO cells. [ABSTRACT FROM AUTHOR]

Objective: To explore the impact of taurine on monoclonal antibody (mAb) basic charge variants in Chinese hamster ovary (CHO) cell culture.Results: In fed-batch culture, adding taurine in the feed medium slightly increased the maximum viable cell density and mAb titers in CHO cells. What’s more, taurine significantly decreased the lysine variant and oxidized variant levels, which further decreased basic variant contents from 32 to 27%. The lysine variant content in the taurine culture was approximately 4% lower than that in control condition, which was the main reason for the decrease in basic variants. Real-time PCR and cell-free assay revealed that taurine played a critical role in the upregulation of relative basic carboxypeptidase and stimulating extracellular basic carboxypeptidase activities.Conclusion: Taurine exhibits noticeable impact on lower basic charge variants, which are mainly due to the decrease of lysine variant and oxidized protein variants. [ABSTRACT FROM AUTHOR]