A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

Abstract

Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation.

Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality

“…For further improvement, we suggest a univariate analysis to enrich the so far derived effective media supplement toolkit by specific drivers among the array of 17 components such as manganese for further fine‐tuning of the glycans still out‐of‐specification. This specific glycan modeling can be refined with further process information (such as viable cell density, productivity) to increase the mechanistic knowledge by integrating further important characteristics besides the media composition in the framework of glycosylation modulation (del Val et al, ; Fan et al, ; Grainger and James, ; Jimenez del Val et al, ). At later stages of process development, such additional (dynamic) characteristics are likely to play a key role in building predictive process models.…”

Model‐based optimization of antibody galactosylation in CHO cell culture

“…The same solver was used to design the independent feeding strategy used for validation. The model framework comprises three submodels: an unstructured cell growth, death, and metabolism model, a model describing the NSD synthesis and an N‐linked glycosylation model (Jimenez del val et al, ; del Val et al ). The cell culture model estimates the specific cell growth and specific mAb productivity that are used as inputs for the NSD synthesis model.…”

“…The measured NSD concentration is the total intracellular NSD concentration that accounts both the concentration of NSDs in the cytosolic and Golgi environment, respectively. As proposed by del Val, Polizzi, and Kontoravdi (), the current model assumes a constant Golgi volume and a variable linear velocity through Golgi (), to describe the different rates at which secretory cargo (the mAb) traverses the Golgi apparatus (Presley et al, ).…”

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

“…Following q IgG and Amm in the ranking are the glucose uptake rate (q GLC ) and bleed rate (B), indicating that the specific growth rate has a moderate contribution to the changes in the IgG galactosylation activity. The influence of growth rate on IgG galactosylation is somewhat expected, as the nucleotide sugar UDP-Galactose is used for different forms of cellular glycosylation, not solely for IgG [43]. on IgG galactosylation is somewhat expected, as the nucleotide sugar UDP-Galactose is used for different forms of cellular glycosylation, not solely for IgG [43].…”

“…The influence of growth rate on IgG galactosylation is somewhat expected, as the nucleotide sugar UDP-Galactose is used for different forms of cellular glycosylation, not solely for IgG [43]. on IgG galactosylation is somewhat expected, as the nucleotide sugar UDP-Galactose is used for different forms of cellular glycosylation, not solely for IgG [43].…”