Development of principle culture medium formulations for chinese hamster ovary (CHO) cells

Abstract

Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for the production of therapeutic proteins. To ensure their maintenance and growth in vitro, a complex combination of nutrients have to be supply through culture medium. Thus, in the last decades, efforts have been made to develop better culture media, free of serum. However, the methods still used for media development are too reliant on empirical knowledge and can be expensive and time-consuming. On the other hand, the method used in this thesis relies on the influence of the environment where a cell is inserted in its metabolic performance, i.e. metabolic engineering by medium manipulation. To study the different physiological states of CHO cells, a metabolic network from the literature was adapted. In that network CHO cells produce Immunoglobulin G (IgG). Using the Metatool 5.1, an initial set of 23240 elementary flux modes (EFMs) were calculated and then divided into: 108 EFMs producing biomass, 16296 EFMs producing IgG, 6658 EFMs carrying out essentially cellular respiration and 178 EFMs assumed as non-feasible. Each group was reduced to 20 EFMs after normalization and subsequent clustering. One medium formulation was purposed for each group of EFMs; however, since the metabolic network had to contain transport reactions of ATP, NADH, NADPH and FADH2 in order to allow the EFMs calculation, the individual result of each group was not realistic because, for example, it was assumed that ATP could be transported from the “exterior” and vice-versa, according to the needs of each cellular physiological state. To consider the pathways where ATP is highly produced and those where it is required, a formulation was purposed regarding a combination of the results from the three groups. The application of these tools may allow media development in a faster and more economical way.