Serum-free production of a chimeric E-selectin-IgG protein from 1 to 100 l scale: Repeated batch cultivation versus continuous spin filter perfusion.
On inflamed endothelium the cell surface protein E-selectin isexpressed which supports the initial process of attachment -capturing and rolling of leukocytes. A recombinant CHO cell linesecreting a soluble E-selectin-IgG chimera was cultivated competitively under serum free conditions in three different bioreactor systems: a 1 l Super-Spinner, a 2 l stirred tank bioreactor equipped with a spinfilter, and a 100 l stirred tankbioreactor. In the smallest system 25.4 mg E-selectin-IgG wereproduced in 62 days using a repeated batch process whileachieving a maximal viable cell density of 3.7 x 10(6) cells ml(-1). Using continuous perfusion mode a total amount of35.2 mg were produced with a maximal viable cell density of1.65 x 10(7) cells ml(-1) in the 2 l bioreactor within 29 days. Large scale cultivation in a 100 l stirred tankbioreactor yielded 105.6 mg in three batches with a maximal viable cell density of 9.7 x 10(5) cells ml(-1) within 15 days. After removal of the cells by continuous centrifugation and a depth filter clearance step, the supernatants were concentrated via ultra filtration. Purificationwas performed by affinity chromatography with rProtein A. Integrity of the E-selectin-IgG protein was checked with SDS PAGE. Its activity was verified in a cellular adhesion assay performed with HL-60 cells and a recombinant CHO cell line expressing membrane-anchored E-selectin constitutively, and E-selectin expressing HUVECs, respectively. Soluble E-selectin-IgG was used to block adhesion to these cell layerscompetitively. A concentation of 18.8 and 37.5 mug ml(-1)was sufficient to reduce the amount of adhering HL-60 cells to 50% on CHO and HUVEC layers, respectively.
Werner
Andreas
A
Institute of Cell Culture Technology, University of Bielefeld, Bielefeld, Germany.
Lütkemeyer
Dirk
D
Poggendorf
Iris
I
Lehmann
Jürgen
J
Müthing
Johannes
J