Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1

Abstract

Replacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their expression and activity by enhancement of γ‐carboxylation might be of interest. The expression and functional activity of vitamin K‐dependent (VKD) coagulation proteins rely, in part, on the VKD process of γ‐carboxylation that is mediated by the enzymes γ‐carboxylase and vitamin K epoxide reductase (VKOR). Since the recombinant production of VKD proteins is hampered by the inefficiency of this enzymatic process, we specifically have examined the stable expression of functional blood coagulation factor IX (FIX) in HEK293 cells following transient overexpression of VKORC1 as an important part of VKOR component. Recombinant hFIX‐producing human embryonic kidney (HEK) cells were transfected to overexpress VKORC1. Following reverse transcription polymerase chain reaction (RT‐PCR) analysis, expression efficiency of the active hFIX was analyzed by performing enzyme‐linked immunosorbent assay and coagulation test. In addition, to quantify γ‐carboxylated recombinant FIX, the barium citrate method was used. Overexpression of VKORC1 in FIX‐producing HEK cells, resulting in a 3.2‐fold higher expression of functional FIX, which displayed a 1.4‐fold enhanced specific activity. Moreover, a 3.9‐fold enhanced recovery of fully γ‐carboxylated FIX following barium citrate adsorption was achieved. Collectively, these findings indicate that the overexpression of VKORC1 results in the production of higher levels of functional hFIX in HEK293 cells. The increase of the VKORC1 as a supplier of γ‐carboxylase seems to play a significant role in increasing the amount and efficiency of recombinant FIX production, thereby reducing the production costs.

Effect of prepropeptide replacement on γ-carboxylation and activity of recombinant coagulation factor IX

“…From this we speculate that the increase in γ-carboxylation leads to higher secretion efficiency and a decrease in trapped FIX in the intracellular space, indicating that the secretory machinery is affected by γ-carboxylation. It is in light of other studies that show poor secretion of FIX following γ-carboxylase overexpression (Rehemtulla et al 1993;Hallgren et al 2002;Wajih et al 2005aWajih et al , 2005b or increased secretion of FIX in overexpressed VKOR cells (Wajih et al 2005a(Wajih et al , 2005bHallgren et al 2006, Pakdaman et al 2019, implying that γ-carboxylase acts as an essential factor during protein secretion (McClure et al 1992). Moreover, it was shown that γ-carboxylation is part of the protein secretion pathway (Rishavy and Berkner 2012) and was required for both efficient secretion and anticoagulant activity (McClure et al 1992).…”

Effect of prepropeptide replacement on γ-carboxylation and activity of recombinant coagulation factor IX

“…One day before transfection, 3×10 6 cells were seeded in a volume of 3 ml in 6-well plates, through which the cells were allowed to loosely adhere. The pMT-prepro-PC/FIX construct and the pCoHygro plasmid were transfected into Drosophila S2 cells employing the calcium phosphate coprecipitation method with minor modi cations [19]. After 48 h, the cells were cultured in 300 µg of hygromycin B/ml.…”