High Intracellular Seed Train BiP Levels Correlate With Poor Production Culture Performance in CHO Cells

2018 ◽  
Vol 13 (10) ◽  
pp. 1700746 ◽  
Author(s):  
Meg Tung ◽  
Danming Tang ◽  
Szu-Han Wang ◽  
Dejin Zhan ◽  
Karen Kiplinger ◽  
...  
Keyword(s):  
Cho Cells ◽  
Production Culture ◽  
Culture Performance ◽  
Seed Train

scholarly journals Amino acid feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells

2021 ◽  
Author(s):  
Iman Shahidi Pour Savizi ◽  
Nader Maghsoudi ◽  
Ehsan Motamedian ◽  
Nathan E. Lewis ◽  
Seyed Abbas Shojaosadati
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Recombinant Protein ◽  
Cho Cells ◽  
Lactate Production ◽  
Tca Cycle ◽  
Control Culture ◽  
Ammonium Concentration ◽  
Cho Cell ◽  
Culture Performance

Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5 mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity.

scholarly journals Amino acid feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells

2021 ◽  
Author(s):  
Iman Shahidi Pour Savizi ◽  
Nader Maghsoudi ◽  
Ehsan Motamedian ◽  
Nathan E. Lewis ◽  
seyed abbas shojaosadati
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Recombinant Protein ◽  
Cho Cells ◽  
Lactate Production ◽  
Tca Cycle ◽  
Control Culture ◽  
Ammonium Concentration ◽  
Cho Cell ◽  
Culture Performance

Abstract Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5 mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The measurement of TCA cycle activity supported this finding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity.

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.

Cell-specific regulation of IRS-1 gene expression: role of E box and C/EBP binding site in HepG2 cells and CHO cells

Diabetes ◽  
1997 ◽  
Vol 46 (3) ◽  
pp. 354-362 ◽  
Author(s):  
K. Matsuda ◽  
E. Araki ◽  
R. Yoshimura ◽  
K. Tsuruzoe ◽  
N. Furukawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Cho Cells ◽  
Hepg2 Cells ◽  
E Box ◽  
Specific Regulation

Optimization of Perfusion Rate Acceleration is a Key Stage in Development of Highly Productive Perfusion Cultivation of CHO Cells

2016 ◽  
Vol 32 (4) ◽  
pp. 60-67
Author(s):  
A. N. MOROZOV ◽  
Z. V. ZAKHAROV ◽  
R. A. KOCHELABOV ◽  
D. V. TYUPA ◽  
A. V. ISERKAPOV ◽  
...  
Keyword(s):  
Cho Cells ◽  
Perfusion Rate ◽  
Rate Acceleration ◽  
Perfusion Cultivation
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