Glykosylierung rekombinant erzeugter Pharmazeutika in Abhängigkeit der Kultivierungsbedingungen
Creators: name: Grunwald, Klaudia
Publisher: name: Universität Bielefeld
Year: 2019
Subjects: subject: 660.6

description: The increasing number of approved biopharmaceuticals produced in mammalian cell lines emphasizes the importance of posttranslational modifications. A consistent product quality is a major goal in the production of biotherapeutics, especially recombinant glycoproteins. In contrast to the biosynthesis of the amino acid chain, posttranslational modifications like glycosylation and protein folding may be influenced by changes within the production process. Glycosylation of recombinant proteins is important for their function, half-life and stability in vivo. In this study, glycosylation during a batch cultivation is evaluated. Cells and secreted protein are exposed to continuous change during a cultivation batch process. In early cultivation the supply situation is excellent and a complete glycosylation can be expected. However, the product´s long dwell time may influence product quality. Later in cultivation, substrate supply decreases while metabolites increase, which result in changes in cultivation. As the dwell time of the product produced at later cultivation stages is short, an extracellular degradation has a less significant effect. A time-dependent sampling was established to examine the glycosylation at different cultivation stages. This sampling allows a comparison between product from early cultivation and product resulting from later cultivation stages. In this study, the glycosylation of five recombinant proteins (erythropoietin, an IgG1 antibody, C1-inhibitor, α-1-antitrypsin and antithrombin III) was analysed. The proteins were produced in different cell lines in batch cultivations. Products from three different cultivation periods could be evaluated with the time-dependent sampling. Additional glycoprotein stability was considered after exposure to different cultivation-like scenarios to estimate the influence of extracellular processes. Results of the time-dependent analysis of the glycosylation showed that in early cultivation stages (excellent supply situation) the degree of galactosylation and sialylation (for sialylated products) was higher than in later cultivation stages. Galactosylation and sialylation decreased during cultivation, antennarity however stayed consistent. The antibody showed less core fucosylation but an increase in high mannose structures in later cultivation stages. Antennary fucoses were found in C1-inhibitor and α-1-antitrypsin. The antennary fucosylation of C1-inhibitor decreased with the cultivation time. Glycosylation was dependent on glycosidase activity in the supernatant, but not on chemical processes. This was confirmed via the stability experiments. It is likely that the secreted product is degraded by glycosidases with a decreasing viability of the cell culture due to cell lysis. With the evidence of sialidase activity this assumption was confirmed. Furthermore, it was shown that sialidase activity is dependent on the producing cell line. Changes of intracellular processes due to varying substrate supply and accumulating metabolites were found to be a further reason for changes in glycosylation during a batch cultivation. Mainly glucose supply and metabolism of glutamine appeared to influence the glycosylation process. With the examination of five glycoproteins produced in different cell lines it could be shown that product heterogeneity is cell line dependent as well as product dependent. Such knowledge combined with the awareness of the requirements of the biotherapeutic is essential for optimising a production process.