GLYCOLIPID MEMBRANE-ANCHORED RECOMBINANT PROTEIN-PRODUCTION FROM CHO CELLS CULTURED ON POROUS MICROCARRIERS

Recombinant proteins were harvested from Chinese hamster ovary (CHO) c ells by a controlled release process, which increased the purity and c oncentration of the harvested protein. Recombinant human melanotransfe rrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl-phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T-flask cultures, and the p97 harvested by replacing the growth medium for 30 min with phosphate-buffered saline (PBS) con taining 10 mU/mL phosphatidylinositol-phospholipase C (PI-PLC). The GP I anchor was selectively cleaved by PI-PLC. In fresh medium, the CHO c ells regained over 95% of their p97 expression within 40 h. The proces s was repeated for eight harvests. Harvested protein concentrations va ried from 1.5 to 3.8 mu g/mL due to difficulties in maintaining stable confluent T-flask cultures. Harvesting from cells growing on porous m icrocarriers was investigated to increase p97 product concentrations a nd to overcome culture stability problems. Semicontinuous cultures wer e maintained in spinners for up to 76 days with average bioreactor cel l densities of over 10(7) cell/mt. The p97 was harvested:at up to 100 mu g/mL and 30% purity with protein production remaining stable for 14 harvest cycles. Production of high levels of p97 from CHO cells was m aintained at 0.5% serum. (C) 1994 John Wiley and Sons, Inc.