Increasing GPI-anchored protein harvest concentrations from suspension and porous microcarrier CHO cell cultures

Abstract

Chinese hamster ovary (CHO) cells expressing the human melanoma tumour antigen, p97, were used to develop a controlled release process for the production of recombinant glycosyl‐phosphatidylinositol (GPI) anchored proteins. The cells were cultured either in suspension or immobilized on porous microcarriers and p97 was selectively cleaved from the cell surface by the bacterial enzyme, phosphatidylinositol‐phospholipase 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 recovered p97 concentrations were increased by reusing the PI‐PLC enzyme solution to harvest multiple batches of cells. A convenient PI‐PLC assay was developed to monitor the harvesting process and to determine the stability of PI‐PLC under harvesting conditions. Although the Pl‐PLC was stable under harvesting conditions, it rapidly adsorbed to the cell 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. The maximum p97 concentration that could be obtained from harvesting CHO cells cultured on porous microcarriers was limited by the dilution effects of sample removal, adding fresh PI‐PLC and liquid associated with settled microcarriers. A model was developed that adequately predicted the p97 concentration after each harvest and the maximum p97 concentration that could be achieved by this harvesting method. The dilution effects were minimized by harvesting from centrifuged suspension culture cells and the harvested p97 concentration was increased by over sixfold to 0.64 mg/mL. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 136–147, 1997.

Modification of a recombinant GPI-anchored metalloproteinase for secretion alters the protein glycosylation

“…Proteins can then be harvested from the cell surface using a bacterial enzyme phosphatidylinositol-phospholipase C (PI-PLC) specific for GPI anchors (Low et al, 1986). This approach has many advantages including a high degree of product purity after the initial harvest (Kennard et al, 1993;Sunderji et al, 1997).…”