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Glycolipid membrane anchored recombinant protein production from CHO cells cultGred on porous microcarriers
Author(s) -
Kennard M. L.,
Piret J. M.
Publication year - 1994
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260440108
Subject(s) - chinese hamster ovary cell , microcarrier , recombinant dna , phosphatidylinositol , phospholipase c , bioreactor , cell culture , glycolipid , chemistry , biochemistry , biology , microbiology and biotechnology , cell , receptor , botany , signal transduction , genetics , gene
Recombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano‐transferrin (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 culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate‐buffered saline (PBS) containing 10 mU/mL phosphatidylinositol‐phospholipase C (PI‐PLC). The GPI anchor was selectively cleaved by PI‐PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 μg/mL due to difficulties in maintaining stable confluent T‐flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 10 7 cell/mL. The p97 was harvested at up to 100 μg/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum. © 1994 John Wiley & Sons, Inc.