Endocytic regulation serves a critical role in modulating the extracellular level of signaling molecules, such as bone morphogenetic proteins (BMPs). Unfortunately, endocytosis may result in poor yields of recombinant human BMP‐4 (rhBMP‐4) from Chinese hamster ovary (CHO) cell cultures. When rhBMP‐4 was incubated with CHO cells, rhBMP‐4 was actively internalized into cells. Cell surface bound heparan sulfate proteoglycans (HSPGs) served as the major receptors for rhBMP‐4 internalization. Removal of cell surface heparan sulfate (HS) by heparinases or reduction of HSPG synthesis by knockdown of xylosyltransferase2 (xylt2) in CHO cells decreased internalization of rhBMP‐4. In addition, treatment with endocytosis inhibitors (chlorpromazine, genistein, and dynasore) identified a clathrin‐ and dynamin‐dependent endocytic pathway as the major route for rhBMP‐4 internalization. To enhance product yield by minimizing rhBMP‐4 internalization in recombinant CHO (rCHO) cell cultures, we have tested various strategies to reduce HSPG synthesis (knockdown of xylt2 and sodium chlorate treatment) or inhibit the binding of rhBMP‐4 to cell‐surface‐bound HSPGs (supplementation with heparin or dextran sulfate [DS]). Among these approaches, DS, which is a linear anionic sulfated polysaccharide with similarity to HS chains, was the most effective in enhancing rhBMP‐4 production in rCHO cell cultures. Compared with the control cultures, DS addition to the culture medium (1.0 g/L) resulted in 1.4‐fold and 2.3‐fold increases in maximum rhBMP‐4 concentration in batch and fed‐batch cultures, respectively. Taken together, the addition of DS, an effective competitor of HSPGs, improved rhBMP‐4 production in rCHO cell cultures through blockage of rhBMP‐4 internalization.
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