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Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed.Key points• Fed-batch culture in CHO cells is reviewed.• Fed-batch has become a common technology for recombinant protein production.• Fed batch culture promotes recombinant protein production in CHO cells.

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.

Summary Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N‐linked glycans, including the presence of β‐1,2‐xylose and core α‐1,3‐fucose residues in plants, can affect the activity, potency and immunogenicity of plant‐derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N‐glycosylation machinery to allow the synthesis of complex N‐glycans lacking β‐1,2‐xylose and core α‐1,3‐fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant‐specific α‐1,3‐fucosyltransferase and β‐1,2‐xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry‐based N‐glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64‐binding affinity of 2G12 glycovariants produced in wild‐type N. benthamiana, the newly generated FX‐KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco‐engineered antibody performed as well as its CHO‐produced counterpart.

The reliability and methodology of genome-scale metabolic models (GEMs) of Chinese hamster ovary (CHO) cells have advanced.CHO-GEMs have aided in cell line and process development, thus impacting on biomanufacturing efficiency.An integrative model structure can incorporate multiple layers and capture condition-specific cell regulation.Integration of CHO-GEMs with artificial intelligence (AI) and advanced algorithms will enable autonomous bioreactor management for digital biomanufacturing. Genome-scale metabolic models (GEMs) of Chinese hamster ovary (CHO) cells are valuable for gaining mechanistic understanding of mammalian cell metabolism and cultures. We provide a comprehensive overview of past and present developments of CHO-GEMs and in silico methods within the flux balance analysis (FBA) framework, focusing on their practical utility in rational cell line development and bioprocess improvements. There are many opportunities for further augmenting the model coverage and establishing integrative models that account for different cellular processes and data for future applications. With supportive collaborative efforts by the research community, we envisage that CHO-GEMs will be crucial for the increasingly digitized and dynamically controlled bioprocessing pipelines, especially because they can be successfully deployed in conjunction with artificial intelligence (AI) and systems engineering algorithms. Genome-scale metabolic models (GEMs) of Chinese hamster ovary (CHO) cells are valuable for gaining mechanistic understanding of mammalian cell metabolism and cultures. We provide a comprehensive overview of past and present developments of CHO-GEMs and in silico methods within the flux balance analysis (FBA) framework, focusing on their practical utility in rational cell line development and bioprocess improvements. There are many opportunities for further augmenting the model coverage and establishing integrative models that account for different cellular processes and data for future applications. With supportive collaborative efforts by the research community, we envisage that CHO-GEMs will be crucial for the increasingly digitized and dynamically controlled bioprocessing pipelines, especially because they can be successfully deployed in conjunction with artificial intelligence (AI) and systems engineering algorithms.

Mammalian cells are suitable hosts for producing recombinant therapeutic proteins, with Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells being the most commonly used cell lines. Mammalian cell expression system includes stable and transient gene expression (TGE) system, with the TGE system having the advantages of short cycles and simple operation. By optimizing the TGE system, the expression of recombinant proteins has been significantly improved. Here, the TGE system and the detailed and up-to-date improvement strategies of mammalian cells, including cell line, expression vector, culture media, culture processes, transfection conditions, and co-expression of helper genes, are reviewed. Key points • Detailed improvement strategies of transient gene expression system of mammalian cells are reviewed • The composition of transient expression system of mammalian cell are summarized • Proposed optimization prospects for transient gene expression systems

Polysorbate (PS) is routinely used in biopharmaceutical formulation to stabilize the active pharmaceutical ingredient.Trace amounts of hydrolytic host cell proteins (HCPs) persist in the downstream purification process and can degrade PS in the final drug product over time, compromising its stability.Genomic removal of PS-degrading enzymes in the Chinese hamster ovary (CHO) host cell line allowed their traceless removal from the bioprocess.The combined knockout (KO) of the genes encoding nine previously confirmed PS-degrading HCPs resulted in a viable CHO host cell line with strongly reduced PS degradation potential.This is the first report of successful excision of a gene cluster >1 Mb in size from the CHO genome.The final multi-hydrolase KO cell line yielded competitive monoclonal antibody titers with high product quality and significantly reduced hydrolytic activity. Enzymatic degradation of polysorbates (PS) in biologic drug formulations is often caused by hydrolytic host cell proteins (HCPs) and can lead to particle formation and reduced shelf-life. Here, we present a host cell line-engineering approach by removing nine Chinese hamster ovary (CHO) host cell hydrolases, which have previously been confirmed to degrade PS. Strikingly, the sequential genomic knockout (KO) of these hydrolases, including the genetic removal of two entire gene clusters of unprecedented size, yielded viable CHO host cell line variants. This novel host cell line was further optimized using the additional KO of the two key proapoptotic genes, Bax and Bak1. Thus, we generated a competitive multi-hydrolase KO CHO host cell line that was further shown to be highly suitable for the generation of recombinant therapeutic glycoproteins. Most importantly, PS degradation and hydrolytic activity were drastically reduced, providing an avenue toward future PS degradation-free biologics-manufacturing processes. Enzymatic degradation of polysorbates (PS) in biologic drug formulations is often caused by hydrolytic host cell proteins (HCPs) and can lead to particle formation and reduced shelf-life. Here, we present a host cell line-engineering approach by removing nine Chinese hamster ovary (CHO) host cell hydrolases, which have previously been confirmed to degrade PS. Strikingly, the sequential genomic knockout (KO) of these hydrolases, including the genetic removal of two entire gene clusters of unprecedented size, yielded viable CHO host cell line variants. This novel host cell line was further optimized using the additional KO of the two key proapoptotic genes, Bax and Bak1. Thus, we generated a competitive multi-hydrolase KO CHO host cell line that was further shown to be highly suitable for the generation of recombinant therapeutic glycoproteins. Most importantly, PS degradation and hydrolytic activity were drastically reduced, providing an avenue toward future PS degradation-free biologics-manufacturing processes. [Display omitted] In the opinion of the authors, the presented technology of knocking out multiple host cell hydrolases from the Chinese hamster ovary (CHO) host cell genome to reduce polysorbate (PS) degradation is ready for implementation. We proved key considerable aspects when generating a novel CHO host cell line, such as transfectability, metabolic selection, ability to endure a conventional fed-batch process without compromising productivity, and consistent product quality attributes compared with the parental host cell line. Given that we performed cultivations in small-scale bioreactors mimicking the real-world process, we suggest a technology readiness level (TRL) of 5 out of 9 (based on NASA’s TRL system). In terms of the recently published Biomanufacturing Readiness Level (BRL) developed by the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), we would classify our technology at BLR 4 out of 9, as we have performed initial tests with industry-relevant feedstocks. Nevertheless, full-scale implementation might harbor further implications. In contrast to the parental CHO host cell line, detailed characterization data on the multi-hydrolase KO host cell line are not available. To ensure its seamless application in an industrial bioprocess, further characterization experiments should test media fit, long-term stability of clonal production cell lines, suitability to express a variety of biologics (including complex antibody formats), and capability for large-scale bioreactor cultivations. In addition, we can only predict that the presented reduction of hydrolytic activity translates all the way down to the final drug product. A complete bioprocess including the final formulation steps followed by stability studies will ultimately demonstrate whether our technology has the ability to solve the PS degradation challenge entirely. Enzymatic degradation of polysorbates (PS) in biologic formulations is often caused by hydrolytic host cell proteins (HCPs) and can lead to particle formation and reduced shelf-life. Combining hydrolase knockouts in a single Chinese hamster ovary (CHO) host cell line enabled traceless enzyme removal and represented a big step toward the production of PS degradation-free biologics.

The occurrence of autophagy in recombinant Chinese hamster ovary (rCHO) cell culture has attracted attention due to its effects on therapeutic protein production. Given the significance of glycosylation in therapeutic proteins, this study examined the effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoproteins in rCHO cells. Three chemical autophagy inhibitors known to inhibit different stages were separately treated with two rCHO cell lines that produce the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44. All autophagy inhibitors significantly decreased the sialylation of Fc-fusion glycoprotein in both cell lines. The decrease in sialylation of Fc-fusion glycoprotein is unlikely to be attributed to the release of intracellular enzymes, given the high cell viability and low activity of extracellular sialidases. Interestingly, the five intracellular nucleotide sugars remained abundant in cells treated with autophagy inhibitors. In the mRNA expression profiles of 27 N -glycosylation-related genes using the NanoString nCounter system, no significant differences in gene expression were noted. With the positive effect of supplementing nucleotide sugar precursors on sialylation, attempts were made to enhance the levels of intracellular nucleotide sugars by supplying these precursors. The addition of nucleotide sugar precursors to cultures treated with inhibitors successfully enhanced the sialylation of Fc-fusion glycoproteins compared to the control culture. This was particularly evident under mild stress conditions and not under relatively severe stress conditions, which were characterized by a high decrease in sialylation. These results suggest that inhibiting autophagy in rCHO cell culture decreases sialylation of Fc-fusion glycoprotein by constraining the availability of intracellular nucleotide sugars. Key points •  The autophagy inhibition in rCHO cell culture leads to a significant reduction in the sialylation of Fc-fusion glycoprotein. •  The pool of five intracellular nucleotide sugars remained highly abundant in cells treated with autophagy inhibitors. •  Supplementation of nucleotide sugar precursors effectively restores decreased sialylation, particularly under mild stress conditions but not in relatively severe stress conditions. Graphical Abstract

Biopharmaceuticals, such as antibody therapeutics, are produced by culturing mammalian cells with chemically defined media that consist of more than 50 synthesized components. The screening of medium components related to culture performance and the subsequent optimization of the composition are required in the development of new modalities, host cells, and culture methods. Screening the components to be optimized is typically labor-intensive. The easiest approach is media blending, which creates variations in the concentrations of the components with only liquid mixing. However, a workflow for systematically determining experimental conditions (i.e., how to blend media) has not been established. Therefore, we reassessed media blending from a mathematical perspective and proposed a workflow for the first time. In the workflow, we evaluated the use of a commercially available chemically defined media to maximize simplicity and applicability. From a mathematical perspective, we clarified that multicollinearity is an inevitable challenge in both experimental design and its analysis. Under the constraint, we showed that one of the most appropriate experimental conditions could be systematically calculated and selected by applying D-optimal design focusing on the principal components. We performed a case study of cell culture to screen medium components under 120 experimental conditions using 11 chemically defined media designed for Chinese Hamster ovary cells. The case study provided a reasonable set of components that explained the variance in viable cell concentrations, which range from 5.8 to 19.4 (× 10 6 ) cells/mL. Finally, our mathematical redefinition also enabled the design of a dedicated media set for media blending. Key points • The constraints in media blending were clearly explained. • A systematic workflow from blending design to analysis was proposed. • The workflow also enabled the design of a dedicated media set for media blending.

There is a growing interest in perfusion or continuous processes to achieve higher productivity of biopharmaceuticals in mammalian cell culture, specifically Chinese hamster ovary (CHO) cells, towards advanced biomanufacturing. These intensified bioprocesses highly require concentrated feed media in order to counteract their dilution effects. However, designing such condensed media formulation poses several challenges, particularly regarding the stability and solubility of specific amino acids. To address the difficulty and complexity in relevant media development, the biopharmaceutical industry has recently suggested forming dipeptides by combining one from problematic amino acids with selected pairs to compensate for limitations. In this study, we combined one of the lead amino acids, L-tyrosine, which is known for its poor solubility in water due to its aromatic ring and hydroxyl group, with glycine as the partner, thus forming glycyl-L-tyrosine (GY) dipeptide. Subsequently, we investigated the utilization of GY dipeptide during fed-batch cultures of IgG-producing CHO cells, by changing its concentrations (0.125 × , 0.25 × , 0.5 × , 1.0 × , and 2.0 ×). Multivariate statistical analysis of culture profiles was then conducted to identify and correlate the most significant nutrients with the production, followed by in silico model-guided analysis to systematically evaluate their effects on the culture performance, and elucidate metabolic states and cellular behaviors. As such, it allowed us to explain how the cells can more efficiently utilize GY dipeptide with respect to the balance of cofactor regeneration and energy distribution for the required biomass and protein synthesis. For example, our analysis results uncovered specific amino acids (Asn and Gln) and the 0.5 × GY dipeptide in the feed medium synergistically alleviated the metabolic bottleneck, resulting in enhanced IgG titer and productivity. In the validation experiments, we tested and observed that lower levels of Asn and Gln led to decreased secretion of toxic metabolites, enhanced longevity, and elevated specific cell growth and titer. Key points • Explored the optimal Tyr dipeptide for the enhanced CHO cell culture performance • Systematically analyzed effects of dipeptide media by model-guided approach • Uncovered synergistic metabolic utilization of amino acids with dipeptide Graphical abstract

Chinese Hamster Ovary (CHO) cells, employed primarily for manufacturing monoclonal antibodies and other recombinant protein (r-protein) therapeutics, are emerging as a promising host for vaccine antigen production. This is exemplified by the recently approved CHO cell-derived subunit vaccines (SUV) against respiratory syncytial virus (RSV) and varicella-zoster virus (VZV), as well as the enveloped virus-like particle (eVLP) vaccine against hepatitis B virus (HBV). Here, we summarize the design, production, and immunogenicity features of these vaccine and review the most recent progress of other CHO-derived vaccines in pre-clinical and clinical development. We also discuss the challenges associated with vaccine production in CHO cells, with a focus on ensuring viral clearance for eVLP products.