Polyarginine peptide IND-1 enhances recombinant human bone morphogenetic protein-2 yield in mammalian cells

Abstract

To improve recombinant human bone morphogenetic protein-2 (rhBMP-2) yield, cell lines stably expressing hBMP2 were cultured in the presence of polyarginine peptide IND-1 and showed up to 6-fold increase in the yield of mature BMP-2. Repeated addition of IND-1 to cell cultures consistently improved BMP-2 yields over 53 days without affecting cell growth and viability. Investigation of its mechanism of action showed that IND-1 inhibited pro-protein convertase (PC) activity when incubated with cell lysates. However, when intact cells were cultured with IND-1, no change in cellular PC activity was observed. Furthermore, knockdown of furin (a prototypical member of the PCs) in cells did not affect their BMP-2 yields, suggesting furin/PC inhibition is unlikely the mechanism by which IND-1 enhances BMP-2 yields. IND-1 as a medium additive thus enhances BMP-2 production in mammalian cell expression systems.

Repeated Transient Transfection: An Alternative for the Recombinant Production of Difficult-to-Express Proteins Like BMP2

“…Using a monocistronic construct, Zhou et al reported BMP2 titers of up to 20 ng/mL for an Flp-In CHO-based recombinant cell line [12]. Recently, Lee's group reported an even higher titer (i.e., 500 ng/mL) using CHO DG44 host cells and methotrexate-mediated gene amplification during the stable cell line creation [11].…”

“…Clinically approved rhBMP2 is known to be produced in stably transfected Chinese hamster ovary (CHO) cells (e.g., 'InductOs ® ', Pfizer, Berlin, Germany) [9]. Product titers of the industrial process are proprietary, but according to the pertinent literature achieving high titers of BMP2 in CHO cell cultivations is challenging [10][11][12], and BMP2 is generally considered a "difficult-to-express" protein in mammalian hosts. Moreover, whereas CHO-derived rhBMP2 seems to be somewhat unstable under cell culture conditions, rhBMP2 produced in the human embryonic kidney cell line HEK293 showed unexpected stability in standard cell culture medium (i.e., DMEM-10% FCS) [13].…”