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Biotechnol Bioeng. 2018 Oct;115(10):2565-2575. doi: 10.1002/bit.26798. Epub 2018 Aug 13.

Improving the production of recombinant human bone morphogenetic protein-4 in Chinese hamster ovary cell cultures by inhibition of undesirable endocytosis.

Author information

1
Department of Biological Sciences, KAIST, Daejeon, Republic of Korea.
2
Institute of Biomaterial and Medical Engineering, Cellumed, Seoul, Republic of Korea.

Abstract

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.

KEYWORDS:

Chinese hamster ovary (CHO) cells; bone morphogenetic protein (BMP)-4; dextran sulfate (DS); heparan sulfate proteoglycan (HSPG); internalization

PMID:
30011067
DOI:
10.1002/bit.26798

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