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Drug Discovery Today. Technologies Dec 2020Recombinant proteins used in biomedical research, diagnostics and different therapies are mostly produced in Chinese hamster ovary cells in the pharmaceutical industry.... (Review)
Review
Recombinant proteins used in biomedical research, diagnostics and different therapies are mostly produced in Chinese hamster ovary cells in the pharmaceutical industry. These biotherapeutics, monoclonal antibodies in particular, have shown remarkable market growth in the past few decades. The increasing demand for high amounts of biologics requires continuous optimization and improvement of production technologies. Research aims at discovering better means and methods for reaching higher volumetric capacity, while maintaining stable product quality. An increasing number of complex novel protein therapeutics, such as viral antigens, vaccines, bi- and tri-specific monoclonal antibodies, are currently entering industrial production pipelines. These biomolecules are, in many cases, difficult to express and require tailored product-specific solutions to improve their transient or stable production. All these requirements boost the development of more efficient expression optimization systems and high-throughput screening platforms to facilitate the design of product-specific cell line engineering and production strategies. In this minireview, we provide an overview on recent advances in CHO cell line development, targeted genome manipulation techniques, selection systems and screening methods currently used in recombinant protein production.
Topics: Animals; Antibodies, Monoclonal; CHO Cells; Cell Differentiation; Cricetinae; Cricetulus; Recombinant Proteins
PubMed: 34895638
DOI: 10.1016/j.ddtec.2021.02.003 -
Biotechnology Journal Mar 2018For over three decades, Chinese hamster ovary (CHO) cells have been the chosen expression platform for the production of therapeutic proteins with complex... (Review)
Review
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.
Topics: Amino Acids; Animals; CHO Cells; Cell Culture Techniques; Cricetinae; Cricetulus; Food; Lactic Acid; Metabolic Networks and Pathways; Metabolomics; Protein Processing, Post-Translational; Recombinant Proteins
PubMed: 29393587
DOI: 10.1002/biot.201700499 -
Applied Microbiology and Biotechnology Feb 2023Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant... (Review)
Review
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.
Topics: Cricetinae; Animals; Humans; Batch Cell Culture Techniques; Cricetulus; CHO Cells; Recombinant Proteins; Bioreactors; Immunoglobulins
PubMed: 36648523
DOI: 10.1007/s00253-022-12342-x -
International Journal of Molecular... Mar 2021The addition of nutrients and accumulation of metabolites in a fed-batch culture of Chinese hamster ovary (CHO) cells leads to an increase in extracellular osmolality in...
The addition of nutrients and accumulation of metabolites in a fed-batch culture of Chinese hamster ovary (CHO) cells leads to an increase in extracellular osmolality in late stage culture. Herein, we explore the effect of osmolality on CHO cell growth, specific monoclonal antibody (mAb) productivity and glycosylation achieved with the addition of NaCl or the supplementation of a commercial feed. Although both methods lead to an increase in specific antibody productivity, they have different effects on cell growth and antibody production. Osmolality modulation using NaCl up to 470 mOsm kg had a consistently positive effect on specific antibody productivity and titre. The addition of the commercial feed achieved variable results: specific mAb productivity was increased, yet cell growth rate was significantly compromised at high osmolality values. As a result, Feed C addition to 410 mOsm kg was the only condition that achieved a significantly higher mAb titre compared to the control. Additionally, Feed C supplementation resulted in a significant reduction in galactosylated antibody structures. Cell volume was found to be positively correlated to osmolality; however, osmolality alone could not account for observed changes in average cell diameter without considering cell cycle variations. These results help delineate the overall effect of osmolality on titre and highlight the potentially negative effect of overfeeding on cell growth.
Topics: Animals; Antibodies, Monoclonal; CHO Cells; Cell Proliferation; Cell Size; Cricetinae; Cricetulus; Glycosylation; Osmolar Concentration; Protein Processing, Post-Translational
PubMed: 33804825
DOI: 10.3390/ijms22073290 -
PloS One 2023Chinese hamster ovary (CHO) cells are widely used for mass production of therapeutic proteins in the pharmaceutical industry. With the growing need in optimizing the...
Chinese hamster ovary (CHO) cells are widely used for mass production of therapeutic proteins in the pharmaceutical industry. With the growing need in optimizing the performance of producer CHO cell lines, research on CHO cell line development and bioprocess continues to increase in recent decades. Bibliographic mapping and classification of relevant research studies will be essential for identifying research gaps and trends in literature. To qualitatively and quantitatively understand the CHO literature, we have conducted topic modeling using a CHO bioprocess bibliome manually compiled in 2016, and compared the topics uncovered by the Latent Dirichlet Allocation (LDA) models with the human labels of the CHO bibliome. The results show a significant overlap between the manually selected categories and computationally generated topics, and reveal the machine-generated topic-specific characteristics. To identify relevant CHO bioprocessing papers from new scientific literature, we have developed supervized models using Logistic Regression to identify specific article topics and evaluated the results using three CHO bibliome datasets, Bioprocessing set, Glycosylation set, and Phenotype set. The use of top terms as features supports the explainability of document classification results to yield insights on new CHO bioprocessing papers.
Topics: Cricetinae; Animals; Humans; CHO Cells; Cricetulus; Data Mining; Phenotype; Glycosylation
PubMed: 37022994
DOI: 10.1371/journal.pone.0274042 -
Cells May 2022Hyperosmolality can occur during industrial fed-batch cultivation processes of Chinese hamster ovary (CHO) cells as highly concentrated feed and base solutions are added...
Hyperosmolality can occur during industrial fed-batch cultivation processes of Chinese hamster ovary (CHO) cells as highly concentrated feed and base solutions are added to replenish nutrients and regulate pH values. Some effects of hyperosmolality, such as increased cell size and growth inhibition, have been elucidated by previous research, but the impact of hyperosmolality and the specific effects of the added osmotic-active reagents have rarely been disentangled. In this study, CHO cells were exposed to four osmotic conditions between 300 mOsm/kg (physiologic condition) and 530 mOsm/kg (extreme hyperosmolality) caused by the addition of either high-glucose-supplemented industrial feed or mannitol as an osmotic control. We present novel single-cell cultivation data revealing heterogeneity in mass gain and cell division in response to these treatments. Exposure to extreme mannitol-induced hyperosmolality and to high-glucose-oversupplemented feed causes cell cycle termination, mtDNA damage, and mitochondrial membrane depolarization, which hints at the onset of premature stress-induced senescence. Thus, this study shows that both mannitol-induced hyperosmolality (530 mOsm/kg) and glucose overfeeding induce severe negative effects on cell growth and mitochondrial activity; therefore, they need to be considered during process development for commercial production.
Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Glucose; Mannitol; Single-Cell Analysis
PubMed: 35681457
DOI: 10.3390/cells11111763 -
Applied Microbiology and Biotechnology Apr 2022Chinese hamster ovary (CHO) cells are the most commonly used host cell lines for therapeutic protein production. Exposure of these cells to highly concentrated feed...
Chinese hamster ovary (CHO) cells are the most commonly used host cell lines for therapeutic protein production. Exposure of these cells to highly concentrated feed solution during fed-batch cultivation can lead to a non-physiological increase in osmolality (> 300 mOsm/kg) that affects cell physiology, morphology, and proteome. As addressed in previous studies (and indeed, as recently addressed in our research), hyperosmolalities of up to 545 mOsm/kg force cells to abort proliferation and gradually increase their volume-almost tripling it. At the same time, CHO cells also show a significant hyperosmolality-dependent increase in mitochondrial activity. To gain deeper insight into the molecular mechanisms that are involved in these processes, as detailed in this paper, we performed a comparative quantitative label-free proteome study of hyperosmolality-exposed CHO cells compared with control cells. Our analysis revealed differentially expressed key proteins that mediate mitochondrial activation, oxidative stress amelioration, and cell cycle progression. Our studies also demonstrate a previously unknown effect: the strong regulation of proteins can alter both cell membrane stiffness and permeability. For example, we observed that three types of septins (filamentous proteins that form diffusion barriers in the cell) became strongly up-regulated in response to hyperosmolality in the experimental setup. Overall, these new observations correlate well with recent CHO-based fluxome and transcriptome studies, and reveal additional unknown proteins involved in the response to hyperosmotic pressure by over-concentrated feed in mammalian cells.Key points• First-time comparative proteome analysis of CHO cells exposed to over-concentrated feed.• Discovery of membrane barrier-forming proteins up-regulation under hyperosmolality.• Description of mitochondrial and protein chaperones activation in treated cells.
Topics: Animals; CHO Cells; Cell Culture Techniques; Cricetinae; Cricetulus; Osmolar Concentration; Proteome
PubMed: 35312825
DOI: 10.1007/s00253-022-11861-x -
Proceedings of the Japan Academy.... 2016Peroxisome is a single-membrane-bounded ubiquitous organelle containing a hundred different enzymes that catalyze various metabolic pathways such as β-oxidation of very... (Review)
Review
Peroxisome is a single-membrane-bounded ubiquitous organelle containing a hundred different enzymes that catalyze various metabolic pathways such as β-oxidation of very long-chain fatty acids and synthesis of plasmalogens. To investigate peroxisome biogenesis and human peroxisome biogenesis disorders (PBDs) including Zellweger syndrome, more than a dozen different complementation groups of Chinese hamster ovary (CHO) cell mutants impaired in peroxisome biogenesis are isolated as a model experimental system. By taking advantage of rapid functional complementation assay of the CHO cell mutants, successful cloning of PEX genes encoding peroxins required for peroxisome assembly invaluably contributed to the accomplishment of cloning of pathogenic genes responsible for PBDs. Peroxins are divided into three groups: 1) peroxins including Pex3p, Pex16p and Pex19p, are responsible for peroxisome membrane biogenesis via Pex19p- and Pex3p-dependent class I and Pex19p- and Pex16p-dependent class II pathways; 2) peroxins that function in matrix protein import; 3) those such as Pex11pβ are involved in peroxisome division where DLP1, Mff, and Fis1 coordinately function.
Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Humans; Peroxisomal Disorders; Peroxisomes
PubMed: 27941306
DOI: 10.2183/pjab.92.463 -
Biotechnology Journal Jan 2017As a possible viable and non-invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and...
As a possible viable and non-invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non-invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones.
Topics: Adalimumab; Amine Oxidase (Copper-Containing); Animals; CHO Cells; Cluster Analysis; Cricetulus; Endoplasmic Reticulum; Humans; Lipids; Metals; Microscopy, Confocal; Molecular Imaging; Principal Component Analysis; Protein Engineering; Proteins; Recombinant Proteins; Spectrum Analysis, Raman
PubMed: 27440252
DOI: 10.1002/biot.201600037 -
Iranian Biomedical Journal Sep 2023CD20 is a differentiation-related antigen exclusively expressed on the membrane of B lymphocytes. CD20 amplification is observed in numerous immune-related disorders,...
BACKGROUND
CD20 is a differentiation-related antigen exclusively expressed on the membrane of B lymphocytes. CD20 amplification is observed in numerous immune-related disorders, making it an ideal target for immunotherapy of hematological malignancies and autoimmune diseases. MAb-based therapies targeting CD20 have a principal role in the treatment of several immune-related disordes and cancers, including CLL. Fc gamma receptors mediate CD20 internalization in hematopoietic cells; therefore, this study aimed to establish non-hematopoietic stable cell lines overexpressing full-length human CD20 antigen as an in vitro model for CD20-related studies.
METHODS
CD20 gene was cloned into the transfer vector. The lentivirus system was transfected to packaging HEK 293T cells, and the supernatants were harvested. CHO-K1 cells were transduced using recombinant viruses, and a stable cell pool was developed by the antibiotic selection. CD20 expression was confirmed at the mRNA and protein levels.
RESULTS
Simultaneous expression of GFP protein facilitated the detection of CD20-expressing cells. Immunophenotyping analysis of stable clones demonstrated expression of CD20 antigen. In addition, the mean fluorescence intensity was significantly higher in the CD20-CHO-K1 clones than the wild-type CHO-K1 cells.
CONCLUSION
This study is the first report on using second-generation lentiviral vectors for the establishment of a non-hematopoietic cell-based system, which stably expresses full-length human CD20 antigen. Results of stable CHO cell lines with different levels of CD20 antigen are well suited to be used for CD20-based investigations, including binding and functional assays.
Topics: Cricetinae; Animals; Humans; Antigens, CD20; CHO Cells; Cricetulus; Genetic Vectors
PubMed: 37873643
DOI: 10.61186/ibj.27.5.269