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 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 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 Off-Gas-Based Soft Sensor for Real-Time Monitoring of Biomass and Metabolism in Chinese Hamster Ovary Cell Continuous Processes in Single-Use Bioreactors

Processes ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 2073
Author(s):  
Tobias Wallocha ◽  
Oliver Popp
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Chinese Hamster Ovary ◽  
Mammalian Cell Culture ◽  
Chinese Hamster ◽  
Viable Cell ◽  
Soft Sensor ◽  
Cho Cell ◽  
Continuous Processes ◽  
Single Use

In mammalian cell culture, especially in pharmaceutical manufacturing and research, biomass and metabolic monitoring are mandatory for various cell culture process steps to develop and, finally, control bioprocesses. As a common measure for biomass, the viable cell density (VCD) or the viable cell volume (VCV) is widely used. This study highlights, for the first time, the advantages of using VCV instead of VCD as a biomass depiction in combination with an oxygen-uptake- rate (OUR)-based soft sensor for real-time biomass estimation and process control in single-use bioreactor (SUBs) continuous processes with Chinese hamster ovary (CHO) cell lines. We investigated a series of 14 technically similar continuous SUB processes, where the same process conditions but different expressing CHO cell lines were used, with respect to biomass growth and oxygen demand to calibrate our model. In addition, we analyzed the key metabolism of the CHO cells in SUB perfusion processes by exometabolomic approaches, highlighting the importance of cell-specific substrate and metabolite consumption and production rate qS analysis to identify distinct metabolic phases. Cell-specific rates for classical mammalian cell culture key substrates and metabolites in CHO perfusion processes showed a good correlation to qOUR, yet, unexpectedly, not for qGluc. Here, we present the soft-sensoring methodology we developed for qPyr to allow for the real-time approximation of cellular metabolism and usage for subsequent, in-depth process monitoring, characterization and optimization.

scholarly journals A Metabolomics Approach to Increasing Chinese Hamster Ovary (CHO) Cell Productivity

Metabolites ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 823
Author(s):  
Grace Yao ◽  
Kathryn Aron ◽  
Michael Borys ◽  
Zhengjian Li ◽  
Girish Pendse ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Tca Cycle ◽  
Viable Cell ◽  
Metabolic Model ◽  
Viable Cell Density ◽  
Specific Productivity ◽  
Cho Cell ◽  
High Productivity ◽  
Medium Supplementation

Much progress has been made in improving the viable cell density of bioreactor cultures in monoclonal antibody production from Chinese hamster ovary (CHO) cells; however, specific productivity (qP) has not been increased to the same degree. In this work, we analyzed a library of 24 antibody-expressing CHO cell clones to identify metabolites that positively associate with qP and could be used for clone selection or medium supplementation. An initial library of 12 clones, each producing one of two antibodies, was analyzed using untargeted LC-MS experiments. Metabolic model-based annotation followed by correlation analysis detected 73 metabolites that significantly correlated with growth, qP, or both. Of these, metabolites in the alanine, aspartate, and glutamate metabolism pathway, and the TCA cycle showed the strongest association with qP. To evaluate whether these metabolites could be used as indicators to identify clones with potential for high productivity, we performed targeted LC-MS experiments on a second library of 12 clones expressing a third antibody. These experiments found that aspartate and cystine were positively correlated with qP, confirming the results from untargeted analysis. To investigate whether qP correlated metabolites reflected endogenous metabolic activity beneficial for productivity, several of these metabolites were tested as medium additives during cell culture. Medium supplementation with citrate improved qP by up to 490% and more than doubled the titer. Together, these studies demonstrate the potential for using metabolomics to discover novel metabolite additives that yield higher volumetric productivity in biologics production processes.

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