Serum-free production of a chimeric E-selectin-IgG protein from 1 to 100 l scale: Repeated batch cultivation versus continuous spin filter perfusion

Abstract

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 × 10⁶ cells ml^-1. Using continuous perfusion mode a total amount of35.2 mg were produced with a maximal viable cell density of1.65 × 10⁷ 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 × 10⁵ 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 μg ml^-1was sufficient to reduce the amount of adhering HL-60 cells to 50% on CHO and HUVEC layers, respectively.