scholarly journals Glycosylation flux analysis reveals dynamic changes of intracellular glycosylation flux distribution in Chinese hamster ovary fed-batch cultures

2017 ◽  
Author(s):  
Sandro Hutter ◽  
Thomas K. Villiger ◽  
David Brühlmann ◽  
Matthieu Stettler ◽  
Hervé Broly ◽  
...  
Keyword(s):  
Chinese Hamster ◽  
Specific Productivity ◽  
Specific Factor ◽  
Glycan Structure ◽  
Flux Analysis ◽  
Fed Batch ◽  
Dynamic Changes ◽  
Consumption Rates ◽  
The Media ◽  
Mab Production

AbstractN-linked glycosylation of proteins has both functional and structural significance. Importantly, the glycan structure of a therapeutic protein influences its efficacy, pharmacokinetics, pharmacodynamics and immunogenicity. In this work, we developed glycosylation flux analysis (GFA) for predicting intracellular production and consumption rates (fluxes) of glycoforms, and applied this method to CHO fed-batch monoclonal antibody (mAb) production using two different media compositions, with and without additional manganese feeding. The GFA is based on a constraint-based modelling of the glycosylation network, employing a pseudo steady state assumption. While the glycosylation fluxes in the network are balanced at each time point, the GFA allows the fluxes to vary with time by way of two scaling factors: (1) an enzyme-specific factor that captures the temporal changes among glycosylation reactions catalyzed by the same enzyme, and (2) the cell specific productivity factor that accounts for the dynamic changes in the mAb production rate. The GFA of the CHO fed-batch cultivations showed that regardless of the media composition, the fluxes of galactosylation decreased with the cultivation time in comparison to the other glycosylation reactions. Furthermore, the GFA showed that the addition of Mn, a cofactor of galactosyltransferase, has the effect of increasing the galactosylation fluxes but only during the beginning of the cultivation period. The results thus demonstrated the power of the GFA in delineating the dynamic alterations of the glycosylation fluxes by local (enzyme-specific) and global (cell specific productivity) factors.

scholarly journals Glycosylation Flux Analysis of Immunoglobulin G in Chinese Hamster Ovary Perfusion Cell Culture

2018 ◽  
Author(s):  
Sandro Hutter ◽  
Moritz Wolf ◽  
Nan Papili Gao ◽  
Dario Lepori ◽  
Thea Schweigler ◽  
...  
Keyword(s):  
Immunoglobulin G ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Perfusion Culture ◽  
Ammonia Concentration ◽  
Specific Productivity ◽  
Glycan Structure ◽  
Flux Analysis ◽  
Fed Batch ◽  
Clinical Safety

A critical quality attribute of therapeutic monoclonal antibodies (mAbs) is the terminal sugar molecules of the N-linked glycan attached to the fragment crystalizable (Fc) region. There exists naturally-occurring heterogeneity in the N-linked glycan structure of mAbs, and such heterogeneity has a significant influence on the clinical safety and efficacy of mAb drugs. We previously proposed a constraint-based modeling method called glycosylation flux analysis (GFA) to characterize the rates (fluxes) of intracellular glycosylation reactions and applied the method to examine the N-linked glycosylation of immunoglobulin G (IgG) in fed-batch Chinese hamster ovary (CHO) fed-batch cultivations. In this work, we significantly improved the computational efficiency of the GFA, and employed the method to analyze the glycosylation of IgG in continuous perfusion CHO cultivations. Perfusion cell cultures have several advantages over the traditional (fed-)batch operation, including higher productivity per unit volume of reactor and more consistent product quality. The GFA showed that as in the fed-batch cultivation, the dynamical changes of IgG glycan heterogeneity in the perfusion culture are mainly attributed to alterations in the galactosylation flux activity. Furthermore, a regression analysis of the galactosylation flux activity using random forest regression linked the dynamics of galactosylation activity with the cell-specific productivity of IgG and the extracellular ammonia concentration.

scholarly journals Glycosylation Flux Analysis of Immunoglobulin G in Chinese Hamster Ovary Perfusion Cell Culture

Processes ◽  
2018 ◽  
Vol 6 (10) ◽  
pp. 176 ◽  
Author(s):  
Sandro Hutter ◽  
Moritz Wolf ◽  
Nan Papili Gao ◽  
Dario Lepori ◽  
Thea Schweigler ◽  
...  
Keyword(s):  
Cell Culture ◽  
Immunoglobulin G ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Specific Productivity ◽  
Glycan Structure ◽  
Flux Analysis ◽  
Cho Cell ◽  
Clinical Safety ◽  
Perfusion Cell Culture

The terminal sugar molecules of the N-linked glycan attached to the fragment crystalizable (Fc) region is a critical quality attribute of therapeutic monoclonal antibodies (mAbs) such as immunoglobulin G (IgG). There exists naturally-occurring heterogeneity in the N-linked glycan structure of mAbs, and such heterogeneity has a significant influence on the clinical safety and efficacy of mAb drugs. We previously proposed a constraint-based modeling method called glycosylation flux analysis (GFA) to characterize the rates (fluxes) of intracellular glycosylation reactions. One contribution of this work is a significant improvement in the computational efficiency of the GFA, which is beneficial for analyzing large datasets. Another contribution of our study is the analysis of IgG glycosylation in continuous perfusion Chinese Hamster Ovary (CHO) cell cultures. The GFA of the perfusion cell culture data indicated that the dynamical changes of IgG glycan heterogeneity are mostly attributed to alterations in the galactosylation flux activity. By using a random forest regression analysis of the IgG galactosylation flux activity, we were further able to link the dynamics of galactosylation with two process parameters: cell-specific productivity of IgG and extracellular ammonia concentration. The characteristics of IgG galactosylation dynamics agree well with what we previously reported for fed-batch cultivations of the same CHO cell strain.

scholarly journals Glycosylation flux analysis reveals dynamic changes of intracellular glycosylation flux distribution in Chinese hamster ovary fed-batch cultures

2017 ◽  
Vol 43 ◽  
pp. 9-20 ◽  
Author(s):  
Sandro Hutter ◽  
Thomas K. Villiger ◽  
David Brühlmann ◽  
Matthieu Stettler ◽  
Hervé Broly ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Flux Distribution ◽  
Chinese Hamster ◽  
Flux Analysis ◽  
Fed Batch ◽  
Dynamic Changes ◽  
Batch Cultures

scholarly journals Metabolic Analysis of the Asparagine and Glutamine Dynamics in an Industrial CHO Fed-Batch Process

2021 ◽  
Author(s):  
Brian Kirsch ◽  
Sandra Bennun ◽  
Adam Mendez ◽  
Amy Johnson ◽  
Hongxia Wang ◽  
...  
Keyword(s):  
Metabolic Flux ◽  
Chinese Hamster ◽  
Tca Cycle ◽  
Batch Process ◽  
Exponential Phase ◽  
Flux Analysis ◽  
Fed Batch ◽  
Cho Cell ◽  
Feed Composition ◽  
Trade Offs

Chinese Hamster Ovary (CHO) cell lines are grown in cultures with varying asparagine and glutamine concentrations, but further study is needed to characterize the interplay between these amino acids. By following 13C-glucose, 13C-glutamine, and 13C-asparagine tracers using metabolic flux analysis (MFA), CHO cell metabolism was characterized in an industrially relevant fed-batch process under glutamine supplemented and low glutamine conditions during early and late exponential growth. For both conditions MFA revealed glucose as the primary carbon source to the tricarboxylic acid (TCA) cycle followed by glutamine and asparagine as secondary sources. Early exponential phase CHO cells prefer glutamine over asparagine to support the TCA cycle under the glutamine supplemented condition, while asparagine was critical for TCA activity for the low glutamine condition. Overall TCA fluxes were similar for both conditions due to the trade-offs associated with reliance on glutamine and/or asparagine. However, glutamine supplementation increased fluxes to alanine, lactate and enrichment of glutathione, N-Acetyl-Glucosamine (NAG) and pyrimidine-containing-molecules. The late exponential phase exhibited reduced central carbon metabolism dominated by glucose, while lactate reincorporation and aspartate uptake were preferred over glutamine and asparagine. These 13C studies demonstrate that metabolic flux is process time dependent and can be modulated by varying feed composition.

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.

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