Inclusion of S-Sepharose Beads in the Culture Medium Significantly Improves Recovery of Secreted rBPI₂₁ from Transfected CHO-K1 Cells

Author： Horwitz A.H. Carroll S.F. Williams R.E. Liu P-s.

Publisher： Academic Press

ISSN： 1046-5928

Source： Protein Expression and Purification, Vol.18, Iss.1, 2000-02, pp. : 77-85

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Abstract

rBPI₂₃, a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills gram-negative bacteria and binds endotoxin. rBPI₂₁, a variant, in which cysteine 132 is changed to alanine, retains the activities of rBPI₂₃. Initial attempts using conventional ion-exchange chromatography to purify rBPI₂₃ from culture supernatants of transfected CHO-K1 cells resulted in lower than expected yields. Also, ELISA of supernatants from CHO-K1 transfectants expressing rBPI₂₃ or rBPI₂₁ yielded variable signals. Results from pulse-chase experiments using [³⁵S]methionine had indicated that rBPI₂₃ could not be detected in the culture medium by 7 h of chase, suggesting that these proteins were degraded and/or bound to cells, media components, or vessel surfaces. To address these issues, we developed a novel process whereby sterile S-Sepharose beads were added directly to the cell culture medium. For attached cells, the beads were added to confluent cultures with serum-free medium for the expression phase, while for suspension-adapted cells, beads were added at the beginning of culture growth. The S-Sepharose was then separated from cells and media and washed, and BPI was eluted with high-salt buffer. This approach yielded up to a 50-fold improvement in recovery of rBPI₂₃ and rBPI₂₁ from roller bottles, shake flasks, and 2-liter fermenters. It also resulted in improved detection and quantitation of secreted rBPI₂₃ and rBPI₂₁ by ELISA. Results of competition binding studies with iodinated rBPI₂₁ in conjunction with unlabeled rBPI₂₁ and rBPI₂₃ or with heparin demonstrated that these proteins bound specifically and with high affinity to heparan-containing sites on the surface of the CHO-K1 cells. We conclude that the S-Sepharose included in the culture medium captures the BPI protein products as they are secreted and protects them from degradation and/or irreversible binding to cell surfaces. This method has been scaled up to a manufacturing process in large (2750 liter) fermenters for pharmaceutical production.