A novel peptide to enhance recombinant BMP-2 production in mammalian cell cultures

Abstract

Materials and methods Two stable cell lines expressing the hBMP2 gene, CHOBMP2 and HEK-BMP2, were cultured in the presence of IND-1 in short-term (24 h, multi-well) and long-term (two-month, perfusion flasks) cultures. The rhBMP-2 produced was characterized by Western blot and its activity assessed using the C2C12 cell-based assay. The amount of proBMP-2 and mature BMP-2 produced was quantified by ELISA. The mRNA level of BMP-2 and furin in cells treated with or without IND-1 was compared by real-time RT-PCR. Cellular uptake of IND-1 was estimated by measuring the fluorescence of cell lysates following incubation with FITC labeled IND-1. Cellular PC activity post IND-1 incubation was measured using the Boc-RVRR-AMC substrate. Furin-specific siRNA was used to knock down the furin expression in CHO-BMP2 cells and its effect on the rhBMP-2 production was determined. Results Stably transfected CHO-BMP2 cells secreted 36 kDa rhBMP-2 dimers that were biologically active. In 24 h cell cultures, IND-1 treated cells produced significantly greater amounts of proBMP-2 (≥ 10-fold, P <0.001 )a nd mature BMP-2 (≥ 3-fold, P <0.001) in their conditioned medium (Figure 1). In long-term CHO-BMP2 culture, IND-1 continued to increase the yields of BMP-2 (≥ 50%) and proBMP-2 (≥ 2-fold) without affecting cell growth or viability. IND-1 treatment had no effect on the mRNA level of BMP-2 and furin, indicating IND-1 affects rhBMP-2 yield post-transcriptionally. While IND-1 was taken up by the cells and inhibited PC activity when added directly to the cell lysates, cells cultured with IND-1 showed no changes in their PC activity at doses 50 times higher than required to affect BMP-2 yields. Furthermore, knockdown of furin at both the mRNA (≥ 80%, P <0.001) and the protein level (≥ 70%, P <0.001), did not affect rhBMP-2 yields. These results suggest that furin inhibition is most l ikely not the mechanism by which IND-1 enhances rhBMP-2 yields. Conclusions The addition of a novel peptide IND-1 to the cell culture medium significantly enhanced the yields of both pro- and mature BMP-2 in stably transfected CHO and HEK cell lines. These increases were sustainable over an extended time period with regular IND-1 treatments. However, the enhanced rhBMP-2 yield is unlikely due to the well-established role of polyarginines as furin inhibitors.