Improvement of Sialylation in Chinese Hamster Ovary (CHO) Cell Culture by Feeding N-Acetylmannosamine

Chemically defined (CD) media are routinely used in the production of biologics in Chinese Hamster Ovary (CHO) cell culture and provide enhanced raw material control. Nutrient optimized CD media is an important path to increase cell growth and monoclonal antibody (mAb) productivity in recombinant CHO cell lines. However, nutrient optimization efforts for CD media typically rely on multi-factorial and experimental design of experiment (DoE) approaches or complex mathematical models of cellular metabolism or gene expression systems. Moreover, the majority of these efforts are aimed at amino acids since they constitute essential nutrients in CD media as they directly contribute to biomass and protein production. In this study, we demonstrate the utilization of multi-variate data analytics (MVDA) coupled with amino acid stoichiometric balances (SBs) to increased cell growth and mAb productivity in efforts to reduce CD media development efforts. SBs measure the difference between theoretical demand of amino acids and the empirically measured fluxes to identify metabolic states of the cell. When coupled with MVDA, the statistical models were not only able to highlight key amino acids towards cell growth or productivity, but also provided direction on metabolic favorability of the amino acid. Experimental validation of our approach resulted in a 55% increase in total cell growth and about an 80% increase in total mAb productivity. Increased specific consumption of stoichiometrically balanced amino acids and decreased specific consumption of glucose was also observed in optimized CD media suggesting favorable consumption of desired nutrients and a potential for energy redistribution towards increased cellular growth or mAb productivity.

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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 ◽

10.3390/pr9112073 ◽

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.

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Recombinant Human Erythropoietin Production in Chinese Hamster Ovary Cells Is Enhanced by Supplementation of α-Helix Domain of 30Kc19 Protein

Applied Sciences ◽

10.3390/app112211009 ◽

2021 ◽

Vol 11 (22) ◽

pp. 11009

Author(s):

Hyeonjin Cha ◽

Ju-Hyun Park

Keyword(s):

Cell Culture ◽

Recombinant Protein ◽

Protein Production ◽

Chinese Hamster ◽

Therapeutic Protein ◽

Cho Cell ◽

Human Erythropoietin ◽

30Kc19 Protein ◽

Cho Cell Culture ◽

Α Helix

The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an Escherichia coli expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP+ ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins.

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

Processes ◽

10.3390/pr6100176 ◽

2018 ◽

Vol 6 (10) ◽

pp. 176 ◽

Cited By ~ 2

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.

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