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.
Highlights
Hemophilia B (Christmas disease) is the second most common type of hemophilia and is caused by deficiency of factor IX (FIX), a vitamin K-dependent (VKD) serine protease zymogen (Mr 57,000)
Recombinant human FIX is currently produced from transgenic Chinese hamster ovary (CHO) cells and is commercially available as BeneFIX [3]
Double-transgenic pigs carrying the human FIX [15] and porcine lactoferrin [16] genes driven by the bovine α-lactalbumin promoter were obtained from the National Taiwan University, Taipei, Taiwan [17]
Summary
Hemophilia B (Christmas disease) is the second most common type of hemophilia and is caused by deficiency of factor IX (FIX), a vitamin K-dependent (VKD) serine protease zymogen (Mr 57,000). FIX has a clearly defined four-domain structure that encompasses a γ-carboxyglutamic acid (Gla) rich-domain, two epidermal growth factor-like (EGF-like) domains, and a C-terminal serine protease domain [1]. The protein undergoes extensive posttranslational modification (PTM), including formation of seven disulfide bridges, addition of two N-glycans and six O-glycans, one phosphorylation, one sulfation, one hydroxylation, and up to twelve γ-carboxyglutamic acids [1]. Recombinant human FIX is currently produced from transgenic Chinese hamster ovary (CHO) cells and is commercially available as BeneFIX [3]. This is structurally and functionally similar to plasma-derived FIX (pdFIX), but with minor differences in posttranslational modification, such as sulfation, phosphorylation, and glycosylation [4, 5]
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