Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

scholarly journals Metabolic engineering of CHO cells for the development of a robust protein production platform

PLoS ONE ◽  
2017 ◽  
Vol 12 (8) ◽  
pp. e0181455 ◽  
Author(s):  
Sanjeev Kumar Gupta ◽  
Santosh K. Srivastava ◽  
Ankit Sharma ◽  
Vaibhav H. H. Nalage ◽  
Darshita Salvi ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Protein Production ◽  
Production Platform

scholarly journals Comparison of three glycoproteomic methods for the analysis of CHO cells treated with 1,3,4-O-Bu3ManNAc

2020 ◽  
Author(s):  
Joseph L. Mertz ◽  
Shisheng Sun ◽  
Bojiao Yin ◽  
Michael J. Betenbaugh ◽  
Kevin J. Yarema ◽  
...  
Keyword(s):  
Cho Cells ◽  
Protein Function ◽  
Solid Phase ◽  
Chinese Hamster ◽  
Comprehensive Analysis ◽  
The Other ◽  
Post Translational Modification ◽  
Hydrophilic Interaction ◽  
Analysis Methods ◽  
Human Proteins

AbstractComprehensive analysis of the glycoproteome is critical due to the widespread importance of this post-translational modification to protein function, and difficult because of the tremendous complexity it exhibits. Here we compared three glycoproteomic analysis methods, a recently described chemoenzymatic glycoproteome analysis methods, N-linked glycans and glycosite containing peptides (NGAG), Solid-phase extraction of N-linked glycoproteins (SPEG), and hydrophilic interaction liquid chromatography (HILIC), for the analysis of N-linked glycosites of Chinese hamster ovarian (CHO) cells treated with 1,3,4-O-Bu3ManNAc. The NGAG protocol resulted in substantially increased glycosite identifications over both SPEG and HILIC. Interestingly, while the glycosites identified by SPEG and HILIC overlapped strongly, NGAG identified many glycosites not observed in either of the other two methods. Further, utilizing the enhanced intact glycopeptide identification afforded by the NGAG workflow, we also found that of the sugar analog 1,3,4-O-Bu3ManNAc increases sialylation of proteins secreted by CHO cells, including an ectopically expressed human proteins.

Metabolic Engineering of N-Linked Glycoform Synthesis Systems in Chinese Hamster Ovary (CHO) Cells

1997 ◽  
pp. 489-494 ◽  
Author(s):  
James E. Bailey ◽  
Pablo Umaña ◽  
Sherrill Minch ◽  
Michael Harrington ◽  
Martin Page ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster

scholarly journals Comparison of Three Glycoproteomic Methods for the Analysis of the Secretome of CHO Cells Treated with 1,3,4-O-Bu3ManNAc

2020 ◽  
Vol 7 (4) ◽  
pp. 144
Author(s):  
Joseph L. Mertz ◽  
Shisheng Sun ◽  
Bojiao Yin ◽  
Yingwei Hu ◽  
Rahul Bhattacharya ◽  
...  
Keyword(s):  
Cho Cells ◽  
Protein Function ◽  
Solid Phase ◽  
Chinese Hamster ◽  
Comprehensive Analysis ◽  
Post Translational Modification ◽  
Hydrophilic Interaction ◽  
Acid Biosynthesis ◽  
Human Erythropoietin ◽  
Single Method

Comprehensive analysis of the glycoproteome is critical due to the importance of glycosylation to many aspects of protein function. The tremendous complexity of this post-translational modification, however, makes it difficult to adequately characterize the glycoproteome using any single method. To overcome this pitfall, in this report we compared three glycoproteomic analysis methods; first the recently developed N-linked glycans and glycosite-containing peptides (NGAG) chemoenzymatic method, second, solid-phase extraction of N-linked glycoproteins (SPEG), and third, hydrophilic interaction liquid chromatography (HILIC) by characterizing N-linked glycosites in the secretome of Chinese hamster ovary (CHO) cells. Interestingly, the glycosites identified by SPEG and HILIC overlapped considerably whereas NGAG identified many glycosites not observed in the other two methods. Further, utilizing enhanced intact glycopeptide identification afforded by the NGAG workflow, we found that the sugar analog 1,3,4-O-Bu3ManNAc, a “high flux” metabolic precursor for sialic acid biosynthesis, increased sialylation of secreted proteins including recombinant human erythropoietin (rhEPO).

scholarly journals Advances of Glycometabolism Engineering in Chinese Hamster Ovary Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Huan-Yu Zhang ◽  
Zhen-Lin Fan ◽  
Tian-Yun Wang
Keyword(s):  
Protein Expression ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Recombinant Protein Expression ◽  
Cell Metabolism ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Production Costs ◽  
Post Translational Modification ◽  
Ovary Cells

As the most widely used mammalian cell line, Chinese hamster ovary (CHO) cells can express various recombinant proteins with a post translational modification pattern similar to that of the proteins from human cells. During industrial production, cells need large amounts of ATP to support growth and protein expression, and since glycometabolism is the main source of ATP for cells, protein production partly depends on the efficiency of glycometabolism. And efficient glycometabolism allows less glucose uptake by cells, reducing production costs, and providing a better mammalian production platform for recombinant protein expression. In the present study, a series of progresses on the comprehensive optimization in CHO cells by glycometabolism strategy were reviewed, including carbohydrate intake, pyruvate metabolism and mitochondrial metabolism. We analyzed the effects of gene regulation in the upstream and downstream of the glucose metabolism pathway on cell’s growth and protein expression. And we also pointed out the latest metabolic studies that are potentially applicable on CHO cells. In the end, we elaborated the application of metabolic models in the study of CHO cell metabolism.

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

scholarly journals Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia

2021 ◽  
Vol 22 (10) ◽  
pp. 5218
Author(s):  
Tomu Kamijo ◽  
Takahiro Kaido ◽  
Masahiro Yoda ◽  
Shinpei Arai ◽  
Kazuyoshi Yamauchi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cho Cells ◽  
Fibrinogen Level ◽  
Culture Media ◽  
Chinese Hamster ◽  
Plasma Fibrinogen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cho Cell ◽  
Accelerated Degradation ◽  
Heterozygous Patient

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients’ plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and “D:D” interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient’s hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles

2021 ◽  
pp. 2100098
Author(s):  
Benjamin F. Synoground ◽  
Claire E. McGraw ◽  
Kathryn S. Elliott ◽  
Christina Leuze ◽  
Jada R. Roth ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Ammonia Stress ◽  
Igg Glycosylation
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