Pilot Scale Production and Purification of a Soluble E-Selectin-IgG Chimeric Protein
E-selectin is a cell surface protein expressed on activated vascular endothelial cells and involved in the initial process of attachment of circulating leukocytes to endothelial cells in case of inflammation (Vestweber and Blanks, 1999). A recombinant CHO cell line secreting a soluble E-selectin-IgG chimera (Hahne et al., 1993) was cultivated under serum free conditions in DMEM/Ham’s F12 (1∶1) supplemented with transferrin, insulin, and albumin. Data of large scale cultivation of E-selectin-IgG chimera producing CHO cells in a 100 L stirred tank bioreactor and the purification steps of the soluble chimeric protein are presented. Using a repeated batch mode a final cell density of 1.25*E6 cells/ml and a maximum product concentration of 0.65 mg/l were achieved. After removal of the cells by continuous centrifugation and a depth filter clearance step, the supernatant was concentrated via ultra filtration. Purification was performed by affinity chromatography with rProtein A resulting in an overall yield of 72 mg E-selectin-IgG chimera. A following gel filtration step was used to obtain highly purified E-selectin-IgG.
KeywordsSugar Glycerol Filtration Albumin Transferrin
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