Inclusion of S-sepharose beads in the culture medium significantly improves recovery of secreted rBPI(21) from transfected CHO-K1 cells

rBPI(23) a recombinant N-terminal fragment of human bactericidal/permeabili ty-increasing protein (BPI), kills gram-negative bacteria and binds endotox in, rBPI(21), a variant, in which cysteine 132 is changed to alanine, retai ns the activities of rBPI(23). Initial attempts using conventional ion-exch ange chromatography to purify rBPI(23) from culture supernatants of transfe cted CHO-K1 cells resulted in lower than expected yields. Also, ELISA of su pernatants from CHO-K1 transfectants expressing rBPI(23) or rBPI(21) yielde d variable signals. Results from pulse-chase experiments using [S-35]methio nine had indicated that rBPI(23) could not be detected in the culture mediu m by 7 h of chase, suggesting that these proteins were degraded and/or boun d to cells, media components, or vessel surfaces. To address these issues, we developed a novel process whereby sterile S-Sepharose beads were added d irectly to the cell culture medium, For attached cells, the beads were adde d to confluent cultures with serum-free medium for the expression phase, wh ile for suspension-adapted cells, beads were added at the beginning of cult ure growth. The S-Sepharose was then separated from cells and media and was hed, and BPI was eluted with high-salt buffer. This approach yielded up to a 50-fold improvement in recovery of rBPI(23) and rBPI(21) from roller bott les, shake flasks, and 2-liter fermenters. It also resulted in improved det ection and quantitation of secreted rBPI(23) and rBPI(21) by ELISA. Results of competition binding studies with iodinated rBPI(21) in conjunction with unlabeled rBPI(21) and rBPI(23) or with heparin demonstrated that these pr oteins bound specifically and with high affinity to heparan-containing site s on the surface of the CHO-K1 cells. We conclude that the S-Sepharose incl uded in the culture medium captures the BPI protein products as they are se creted and protects them from degradation and/or irreversible binding to ce ll surfaces. This method has been scaled up to a manufacturing process in l arge (2750 liter) fermenters for pharmaceutical production. (C) 2000 Academ ic Press.