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

TLDR: The glycosylation flux analysis (GFA) for predicting intracellular production and consumption rates (fluxes) of glycoforms is developed and applied to CHO fed-batch immunoglobulin G (IgG) production using two different media compositions, with and without additional manganese feeding.

About: This article is published in Metabolic Engineering.The article was published on 2017-09-01 and is currently open access. It has received 31 citations till now. The article focuses on the topics: Glycosylation & N-linked glycosylation.

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Frequently asked questions

Q1. What have the authors contributed in "Glycosylation flux analysis reveals dynamic changes of intracellular glycosylation flux distribution in chinese hamster ovary fed-batch cultures" ?

In this paper, the authors proposed a constraint-based approach to predict the reaction fluxes in the N-linked glycan. 

Q2. What is the important quality attribute of recombinant therapeutic proteins?

One of the most important quality attributes of recombinant therapeutic proteins is the structure of the asparagine-linked oligosaccharide sugar or N-linked glycan attached to the proteins. 

Q3. Why is a decrease in GalT activity a common phenomenon in fed-batch processes?

A decrease in GalT activity is a frequently observed phenomena in industrial fed-batch processes of CHO-S culture (Gawlitzek et al., 2000), because of the adverse environmental conditions to which the cells are exposed (e.g., by-products, nutrient limitation and overfeeding, osmotic stress). 

Q4. How does the GFA predict the intracellular flux?

The GFA predicts the intracellular fluxes by optimizing over the values of the enzyme specific factors 𝛼𝐽(𝑡𝑘) and the reference flux values 𝑣𝐼,𝑗 𝑟𝑒𝑓 . 

Q5. What is the reason for the drop in the GFA?

The authors attributed this drop to the sensitivity of GalT activity to environmental changes, particularly the accumulation of ammoniain the cell culture. 

Q6. What is the role of the N-linked glycan in the biopharmaceutic?

This glycan structure can influence protein folding (Aebi, 2013), secretion (Zhang and Kaufman, 2006) and stability (Arosio et al., 2013), and has an impact on bio-activity (Umaña et al., 1999), efficacy (Goh et al., 2014) and immunogenicity (Harding et al., 2010). 

Q7. What is the cell specific productivity of process A and B?

The cell specific productivity in the two processes generally increased with the cultivation time, with process A having a slightly higher fold-increase. 

Q8. What was the pH of the cells?

Cells were first expanded in shake bottles and the cellseeding density was set to cells/mL. A temperature shift from 36.5 °C to 33.0 °C and a pH shift from 7.1 to 6.9 were conducted on the fifth culture day. 

Q9. What is the effect of the accumulation of ammonia in the Golgi?

In this regard, the accumulation of ammonia during the course of the cell culture (see SI Figure 2) could cause a pH increase in the Golgi, leading to a decrease in the GalT activity. 

Q10. What is the effect of the drop in the enzyme-specific factor?

the drop in the GalT-specific factor explained the increase of FA2 and the decrease of galactosylated glycan structures towards the end of the two fed-batch cultivations. 

Q11. What is the relative deviation between the two processes?

The relative deviation between the two processes is computed as follows:𝑅𝐵/𝐴 = 𝑧𝐵 − 𝑧𝐴0.5(𝑧𝐵 + 𝑧𝐴)(11)where zA and zB denote the variable of interest (i.e. the viable cell density and specific productivities) of process A and B, respectively.