Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Database
Language
Publication year range
1.
Enzyme Microb Technol ; 157: 110007, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35219175

ABSTRACT

Cytotoxic T-lymphocyte-associated protein 4-Ig (CTLA4-Ig) produced using Chinese hamster ovary (CHO) cell lines is a fusion protein of CTLA4 and the Fc region of antibody. In the present study, we identified and overexpressed genes capable of increasing sialic acid levels in CTLA4-Ig to develop cell lines using glycoengineering technology. CTLA4-Ig was produced using CHO cells overexpressing N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST). The conditions were wild type (WT), overexpression (GnT-IV, GnT-V, and α2,6-ST), and co-overexpression (GnT-IV and α2,6-ST, and GnT-V and α2,6-ST). GnT-IV and GnT-V were transfected into CHO cells to determine tri-antennary structure formation in CTLA4-Ig. CHOGnT-IV (cells overexpressing GnT-IV) showed the highest tri-antennary structures of glycans. Compared to CHOWT, neutral and mono-sialylated glycans decreased (-10.9% and -18.6%, respectively), while bi- and tri-sialylated N-glycans increased (4.1% and 85.7%, respectively) in CHOGnT-IV∙ST (cells co-overexpressing GnT-IV and α2,6-ST). The sum of the relative quantities of neutral N-glycans decreased from 32.0% to 28.5%, while that of sialylated N-glycans increased from 68.0% to 71.5% in CHOGnT-IV∙ST. These results are the first to demonstrate the co-overexpression of especially GnT-IV and α2,6-ST, which is an effective strategy to increase sialic acid levels and the tri-antennary structure of CTLA4-Ig produced using CHO cell lines.


Subject(s)
Immunoglobulin G , N-Acetylneuraminic Acid , Abatacept , Animals , CHO Cells , CTLA-4 Antigen/genetics , Cricetinae , Cricetulus , N-Acetylneuraminic Acid/metabolism , Polysaccharides/metabolism
2.
J Biotechnol ; 308: 10-20, 2020 Jan 20.
Article in English | MEDLINE | ID: mdl-31756358

ABSTRACT

Mammalian cell culture platform has been successfully implemented for industrial biopharmaceutical production through the advancements in early stage process development including cell-line engineering, media design and process optimization. However, late stage developments such as scale-up, scale-down and large-scale cell cultivation still face many industrial challenges to acquire comparable process performance between different culture scales. One of them is the sparging strategy which significantly affects productivity, quality and comparability. Currently, it is mainly relying on the empirical records due to the lack of theoretical framework and scarcity of available literatures to elucidate intracellular metabolic features. Therefore, it is highly required to characterize the underlying mechanism of physiological changes and metabolic states upon the aeration stress. To this end, initially we cultivated antibody producing CHO cells under mild and harsh sparging conditions and observed that sparging stress leads to the decreased cell growth rate, viability and productivity. Subsequent in silicomodel-driven flux analysis suggested that sparging stress rewires amino acid metabolism towards the enriched H2O2 turnover rate by up-regulated fluxes of amino acid oxidases. Interestingly, many of these H2O2-generating reactions were closely connected with the production of NADH, NADPH and GSH which are typical reducing equivalents. Thus, we can hypothesize that increased amino acid uptake caused by sparging stress contributes to restore redox homeostasis against oxidative stress. The current model-driven systematic data analysis allows us to quickly define distinct metabolic feature under stress condition by using basic cell cultivation datasets.


Subject(s)
Amino Acids/metabolism , Hydrogen Peroxide/pharmacology , Stress, Physiological , Animals , Batch Cell Culture Techniques , Bioreactors , CHO Cells , Cell Proliferation/drug effects , Cell Survival , Computer Simulation , Cricetulus , Culture Media
3.
Biotechnol Lett ; 41(11): 1255-1263, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31541331

ABSTRACT

OBJECTIVE: To examine the effects of suramin in CHO cell cultures in terms of the cell culture performance and quality of the Fc-fusion protein. RESULTS: Suramin had positive effects on the CHO cell cultures. The addition of suramin caused an increase in the viable cell density, cell viability, and titer of the Fc-fusion protein. Moreover, suramin had no impact on protein aggregation and enhanced the sialic acid contents of Fc-fusion protein by 1.18-fold. The enhanced sialylation was not caused by the increased nucleotide sugar level but by the inhibition of sialidase activity. The results showed that suramin inhibited apoptosis and had positive impacts on the productivity and quality of Fc-fusion protein. CONCLUSION: The addition of suramin increased the production of Fc-fusion protein and enhanced sialylation when added as a supplement to the media component in CHO cell cultures. This study suggested that suramin could be a beneficial additive during the biological production in terms of the productivity and quality of Fc-fusion protein.


Subject(s)
Cell Culture Techniques/methods , Immunoglobulin Fc Fragments/metabolism , Recombinant Proteins/metabolism , Suramin/pharmacology , Animals , CHO Cells , Cell Death/drug effects , Cricetinae , Cricetulus , Glycosylation , Immunoglobulin Fc Fragments/genetics , N-Acetylneuraminic Acid/metabolism , Recombinant Proteins/genetics
4.
Enzyme Microb Technol ; 103: 53-58, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28554385

ABSTRACT

Terminal sialic acids on N-glycan of recombinant human erythropoietin are very important for in vivo half-life, as this glycoprotein has three N-glycosylation sites. N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) catalyze the formation of a glycan antennary structure. These enzymes display different reaction kinetics for a common substrate and generally show low expression in Chinese hamster ovary (CHO) cells. Therefore, genetic control of Mgat expression is an effective method to increase sialic acid contents by enhancing glycan antennarity. To produce highly sialylated albumin-erythropoietin (Alb-EPO), we co-overexpressed the Mgat1 and Mgat4 genes in CHO cells and determined the optimal ratio of Mgat1:Mgat4 gene expression. All transfected cell lines showed increased gene expression of Mgat4, including Mgat1 overexpressing cell line. Sialic acid content of Alb-EPO was highest in co-transfected cells with excess Mgat4 gene, and these cells showed a higher tri- and tetra-antennary structure than control cells. Based on these results, we suggest that co-transfection of the Mgat1 and Mgat4 genes at a ratio of 2:8 is optimal for extension of antennary structures. Also, regulation of Mgat gene expression in the glycan biosynthesis pathway can be a novel approach to increase the terminal sialic acids of N-glycans.


Subject(s)
Erythropoietin/biosynthesis , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Serum Albumin, Human/biosynthesis , Animals , CHO Cells , Cricetulus , Erythropoietin/chemistry , Erythropoietin/genetics , Genetic Vectors , Glycosylation , Humans , Protein Engineering , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Serum Albumin, Human/chemistry , Serum Albumin, Human/genetics , Sialic Acids/chemistry , Transfection , Up-Regulation
5.
J Biosci Bioeng ; 120(2): 218-22, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25641580

ABSTRACT

Mesenchymal stem cells (MSCs) are capable of self-renewal and can differentiate into various types of cells for therapeutic purposes. MSCs are frequently cultured in a two-dimensional (2D) system. However, MSCs can lose their differentiation capacity over time in this culture system. In addition, the available surface area for the propagation of cells is limited. Therefore, various three-dimensional (3D) culture systems have been developed. In this study, we developed the scaffold-free 3D culture systems for the expansion of periosteum-derived progenitor cells (PDPCs) as spheres. The spheroid formation of PDPCs was induced using a rotation platform. The spheres maintained their viability and proliferation ability. Moreover, expression levels of the stemness marker genes and proteins were higher in cells grown on 3D culture system than in 2D culture system. In conclusion, a simple and economical 3D culture system has been developed that can increase the potential of PDPCs for clinical use.


Subject(s)
Cell Culture Techniques/methods , Mesenchymal Stem Cells/cytology , Periosteum/cytology , Biomarkers/analysis , Cell Differentiation , Cell Proliferation , Cell Separation , Cell Survival , Cells, Cultured , Humans , Mesenchymal Stem Cells/metabolism , Rotation , Spheroids, Cellular/cytology , Tissue Scaffolds
SELECTION OF CITATIONS
SEARCH DETAIL
...