The control of the glycosylation of recombinant proteins in mammalian cell culture

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Mammalian cells are used in large-scale culture bioprocesses for the production of commercially valuable compounds used in human health-care. Many biopharmaceuticals are produced as recombinant proteins from such bioprocesses. The most current technology for large-scale production is by fed-batch cultures. However, at a high productivity it is important to ensure that the protein quality for bioactivity and clinical efficacy is not compromised. The quality of a secreted protein may be reduced through many factors that include aggregation or aberrant glycosylation patterns. Recent work in my laboratory has shown that aggregation is a particular problem for hydrophobic protein such as beta-interferon. This phenomenon was minimized by the design of a low-temperature perfusion system. In the present proposal we intend to evaluate the culture conditions that cause variability in glycosylation patterns of selected recombinant proteins that will include immunoglobulin, tissue-type plasminogen activator, interferon-beta and interferon-gamma produced from Chinese hamster ovary (CHO) cells which are used predominantly in industry. The glycosylation profile of the proteins is defined as the type and quantity of attached carbohydrates which can vary even during culture. The profile is important for the bioactivity of the proteins particularly when applied as therapeutics. We will investigate a number of phenomena that may be related to the variability of protein glycosylation. Glycan site occupancy is reduced when media substrates, such as a carbohydrate source, are depleted. This phenomenon will be investigated with nutrient levels shown to prolong viable cell cultures through fed-batch strategies. The frequency of feeding to maintain optimal low level nutrients may affect both site occupancy and the resulting glycan profile. In experiments to simulate large-scale fed-batch culture production the supply of nutrients will be varied in an effort to understand their role in causing any variability of the metabolome that might affect the final glycoprotein product. The objective of the proposed research is to understand and control the critical quality attributes associated with glycosylation of biopharmaceuticals in large-scale bioprocesses.