Cell line development and inhibition of undesirable endocytosis for enhancing human bone morphogenetic protein production in recombinant CHO cells

Abstract

With the development of recombinant DNA technology, the development of therapeutic proteins has increased dramatically, and due to its high specificity and low immunogenicity, it is attracting attention as a substitute for chemically synthesized drugs. In general, Chinese hamster ovary (CHO) cells are preferred among various hosts for the production of therapeutic proteins because they ensure post-translational glycosylation and are suitable for large-scale suspension culture.Bone morphogenetic protein (BMP) are known to be essential elements that stimulate bone formation in vivo, and have the potential as therapeutic proteins that help rapid recovery during fracture and implant insertion. According to the global aging trend and the increase in the obese population, the demand for BMPs is gradually increasing, but rhBMP productivity in CHO cells is still reported to be relatively low compared to the amount of antibody production. Therefore, it is necessary to develop a large-scale culture process using CHO cell lines. Previous studies have shown that rhBMP-4 in CHO cells is uptaken intracellularly by heparan sulfate proteoglycans (HSPGs) located in the cell membrane. Therefore, it is needed to find out whether rhBMP-2 and rhBMP-7, which are currently in high demand, are targets for intracellular uptake of CHO cells, and to overcome low productivity by blocking intracellular uptake. When purified rhBMP-2 and rhBMP-7 were cultured with the CHO cell line, it was observed that only rhBMP-2, not rhBMP-7, was actively internalized into the cells. Confocal microscopy analysis revealed that HSPG in the cell membrane acts as a direct receptor for this intracellular uptake of rhBMP-2. It was also found that the cell surface HSPG-mediated endocytosis of rhBMP-2 occurred through both the clathrin- and caveolin-dependent pathways. Heparin and dextran sulfate (DS), which are structurally similar to heparan sulfate (HS) chain of HSPG were used as competitive inhibitors to prevent intracellular uptake. When heparin and DS were treated with CHO-BMP-2 cells, respectively, the production amount increased by up to 19 and 22 times, respectively. In contrast, the production of rhBMP-7, which was not internalized into the rCHO cells, did not dramatically increase upon addition of DS and heparin. Recombinant growth/differentiation factor-5 (rhGDF-5) is one of the BMP family and is recognized as a potential therapeutic agent. For rhGDF-5 production, a production cell line was developed using two CHO host cells, dihydrofolate reductase (DHFR) positive CHO-K1 and DHFR-deficient DG44. In adherent culture, rhGDF-5 molecules were shown to be significantly degraded with the decay phase only in DG44-derived clones. Therefore, CHO-K1-derived GDF-5 producing clones were selected as appropriate rhGDF-5 producing cells. Afterwards, 7 members of proprotein converatases (PCs) was transiently expressed to find appropriate PCs for rhGDF-5 maturation. Among them, cell membrane anchored PC5/6 or PC5/6ΔC which can be secreted from cells, were efficiently induced rhGDF-5 maturation as much as furin. As a result, a clone overexpressing PC5/6ΔC was constructed to increase the yield of mature rhGDF-5 in adherent culture.In previous studies, it was observed that intracellular uptake of BMPs resulted in a decrease in productivity in CHO cells, and heparin or DS effectively enhanced production by interfering with intracellular uptake. Based on this, it was investigated whether rhGDF-5 was uptaken by CHO cells, and the relationship between the chain length of HS and the internalization of rhGDF-5 was studied. Purified rhGDF-5 was incubated with CHO cell to confirm the cellular uptake of rhGDF-5 by FACS and confocal microscopy analysis. This endocytosis is mediated by HSPG, and the length of the HS chain played an important role in introducing rhGDF-5 into the cell. Therefore, short-length of heparin or DS could not interfere with the cellular uptake of rhGDF-5 by HSPG at all, or high doses of them were required to prevent the internalization. The longer the length, the greater the blocking effect of internalization, and the blocking effect occurred even at a low concentration of long-length of heparin or DS. Unfractionated heparin or 40, 200 kDa of DS, found out through screening assay, were treated with CHO-GDF-5 cells and the maximum productivities were enhanced by 10-, 6- and 11-times, respectively.