(52c) Modeling and Modulating N-Linked Glycosylation – Differences and Similarities in Fed-Batch and Continuous Cultures

Mammalian cells represent the state-of-the-art expression system for modern monoclonal antibody (mAb) manufacturing processes due to their ability of posttranslational modifications of the desired product. In particular, N-linked glycosylation plays a crucial role in terms of protein folding, bioavailability and efficacy of antibodies and is therefore considered as one of the major quality attributes. However, the complex and heterogeneous nature of glycosylation has caused problems to identify the most active form(s) and the development of cell culture processes which produce a desired pattern.

It has been shown that cell culture conditions and media components such as pH, trace elements, C-sources and byproducts can have a significant impact on N-linked glycosylation. These parameters inevitably change in modern fed-batch processes with cell densities beyond 2e7 cells/ml. Ammonia accumulation¹, decreasing availability of intracellular nucleotide sugar donors² and changing expression of enzyme transferases³ influence the glycosylation mechanism simultaneously and decrease the degree of glycosylation during a fed-batch process. On the other hand, continuous cultures can prevent the accumulation of byproducts therefore lead to a more consistent product quality throughout the process. Due to less interference factors, the degree of modulating the N-linked glycosylation pattern is different of a fed-batch compared to continuous cultures.

Measurements of basic metabolites and intracellular nucleotide sugars were used to tune a mathematical model to describe the resulting quality attributes based on a dynamic mathematical model of N-linked glycosylation⁴. The combined experimental and modeling approach demonstrates the differences and similarities of the modularity and controllability of glycosylation in these two most common operating systems.

Literature

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2.        Kochanowski, N. et al. Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC. Anal. Biochem. 348, 243–251 (2006).

3.        Wong, D. C. F., Wong, N. S. C., Goh, J. S. Y., May, L. M. & Yap, M. G. S. Profiling of N-glycosylation gene expression in CHO cell fed-batch cultures. Biotechnol. Bioeng. 107, 516–528 (2010).

4.        Jimenez Del Val, I., Nagy, J. M. & Kontoravdi, C. A dynamic mathematical model for monoclonal antibody N-linked glycosylation and nucleotide sugar donor transport within a maturing Golgi apparatus. Biotechnol. Prog. 44, 1–44 (2011).