INCREASING GPI-ANCHORED PROTEIN HARVEST CONCENTRATIONS FROM SUSPENSION AND POROUS MICROCARRIER CHO CELL-CULTURES

Chinese hamster ovary (CHO) cells expressing the human melanoma tumour antigen, p97, were used to develop a controlled release process for t he production of recombinant glycosyl-phosphatidylinositol (GPI) ancho red proteins. The cells were cultured either in suspension or immobili zed on porous microcarriers and p97 was selectively cleaved from the c ell surface by the bacterial enzyme, phosphatidylinositol-phospholipas e C (PI-PLC). The kinetics of p97 cleavage from the cell surface by PI -PLC was shown to be approximated by Michaelis-Menten kinetics. The re covered p97 concentrations were increased by reusing the PI-PLC enzyme solution to harvest multiple batches of cells. A convenient PI-PLC as say was developed to monitor the harvesting process and to determine t he stability of PI-PLC under harvesting conditions. Although the PI-PL C was stable under harvesting conditions, it rapidly adsorbed to the c ell surface and was depleted from the reused enzyme solution. In order to maintain PI-PLC activity, it was necessary to add fresh PI-PLC to the reused enzyme solution before harvesting a fresh batch of cells. T he maximum p97 concentration that could be obtained from harvesting CH O cells cultured on porous microcarriers was limited by the dilution e ffects of sample removal, adding fresh PI-PLC and liquid associated wi th settled microcarriers. A model was developed that adequately predic ted the p97 concentration after each harvest and the maximum p97 conce ntration that could be achieved by this harvesting method. The dilutio n effects were minimized by harvesting from centrifuged suspension cul ture cells and the harvested p97 concentration was increased by over s ixfold to 0.64 mg/mL. (C) 1997 John Wiley & Sons, Inc.