Recombinant Human Factor IX Produced from Transgenic Porcine Milk

Abstract

Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

In vivo efficacy of human recombinant factor IX produced by the human hepatoma cell line HuH‐7

“…FAM20C‐kinase is present in plasma and it is synthesized by liver implicated in biomineralization, including the small integrin‐binding ligand and liver‐secreted N‐linked glycoproteins like FIX or factor V . Recently, a new rFIX produced by porcine mammalian cells and secreted in porcine milk has been described with improved post‐translational modifications such as activation peptide phosphorylation at 40% . In accordance with this data, we may speculate that the casein‐kinase present in the milk might contribute to the phosphorylation of the molecule.…”

Application of Genetically Engineered Pigs in Biomedical Research

“…The collected milk contained 1 g/L of this protein [48]. Other recombinant human proteins involved in the coagulation process, such as factor VIII [49], factor IX [50,51], von Willebrand factor [52], were also successfully obtained in the porcine mammary gland. Furthermore, the line of transgenic pigs producing functional recombinant human erythropoietin in their milk was demonstrated.…”

Characterization of Enlarged Tongues in Cloned Piglets

“…The WAP protein is specifically expressed in the mammary gland and is controlled by hormones and developmental signals during pregnancy [37]. Many studies have confirmed that the mammary glands of transgenic animals can serve as bioreactors to produce valuable therapeutic proteins [38][39][40].…”