(10b) Effects Of Microcarrier Culture And Temperature Shift On Productivity And Glycosylation Of Recombinant Proteins Produced In Chinese Hamster Ovary Cells

A large number of the recently developed biopharmaceuticals are complex glycoproteins including thromobolytics, cytokines, blood-clotting factors, growth factors, and immunoglobulins. The need for post-translational processing and glycosylation of these proteins for biological activity has focused attention on the use of mammalian cells as hosts for recombinant protein production. Chinese hamster ovary (CHO) cells have emerged as one of the most widely used cell lines in the biotechnology industry, in part because they are robust and well characterized. As demand for recombinant proteins has increased, a variety of approaches have been employed to increase production scale. The situation has become particularly acute in the last several years as many recently approved and pipeline biopharmaceuticals require doses hundreds or even thousands of times greater than previous biopharmaceuticals. One approach that has been increasingly employed is the use of perfusion culture, particularly for cells grown on microcarriers. Mild hypothermia has also been reported as a simple way to improve the production of recombinant therapeutic proteins in mammalian cell cultures.

However, it is unclear when employing these strategies what effects they will have on growth, specific productivity and glycosylation of the recombinant proteins. We have investigated the effects of microcarrier culture combined with a temperature shift on two CHO cells lines producing different recombinant protein (secreted alkaline phosphatase and tissue-plasminogen activator). In controlled, agitated bioreactors, suspension cultures reached higher cell densities and product titers than microcarrier cultures for both cell lines. Growth and metabolic activities showed similar trends in suspension and microcarrier culture regardless of cell line. However, the responses of the specific productivities to both microcarrier culture and hypothermia differed significantly between the cell lines.

For the secreted alkaline phosphatase produced, culture conditions affected the degree of fucosylation and the amount of sialic acid present on the glycans. Growth on microcarriers and reduced temperature cultures both showed an increase in sialic acid when compared to the suspension cultures at 37 C. While the increase in sialylation could be advantageous, it must be weighed against the possibility of reduced specific productivities. The results of our studies indicate that it is difficult to generalize the effects of culture conditions on protein production and glycosylation as the cellular responses are often both cell-line and protein-product dependent.