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

Human bone morphogenetic protein 2 (hBMP2) is routinely used in medical applications as an inducer of osteoformation. The recombinant production of BMP2 is typically performed using stable Chinese hamster ovary (CHO) cell lines. However, this process is inefficient, resulting in low product titers. In contrast, transient gene expression (TGE), which also enables the production of recombinant proteins, suffers from short production times and hence limited total product amounts. Here, we show that TGE-based BMP2 production is more efficient in HEKsus than in CHOsus cells. Independently of the cell lines, a bicistronic plasmid co-expressing EGFP and BMP2 facilitated the determination of the transfection efficiency but led to inferior BMP2 titers. Finally, we used a high cell density transient transfection (HCD-TGE) protocol to improve and extend the BMP2 expression by performing four rounds of serial transfections on one pool of HEKsus cells. This repeated transient transfection (RTT) process in HEKsus cells was implemented using EGFP as a reporter gene and further adapted for BMP2 production. The proposed method significantly improves BMP2 production (up to 509 ng/10(6) cells) by extending the production phase (96-360 h). RTT can be integrated into the seed train and is shown to be compatible with scale-up to the liter range.