Stable and high-level production of recombinant Factor IX in human hepatic cell line

Author(s)

Femandes, Andrielle de Castilho ^{[1, 2, 3]} ; Fontes, Aparecida ^{[1, 2, 3]} ; Gonsales, Nathalia ^{[1, 2, 3]} ; Swiech, Kamilla ^{[2, 3]} ; Picanco-Castro, Virginia ^{[2, 3]} ; Faca, Sandra ^{[2, 3]} ; Covas, Dimas ^{[1, 2, 3]}

Affiliation

^[1] Univ Sao Paulo, Dept Clin Med, Fac Med Ribeirao Preto, BR-05508 Sao Paulo - Brazil

^[2] Natl Inst Sci & Technol Stem Cell Therapy, Ctr Cell Therapy, Ribeirao Preto - Brazil

^[3] Natl Inst Sci & Technol Stem Cell Therapy, Reg Blood Ctr, Ribeirao Preto - Brazil

• Document type: Journal article
• Source: Biotechnology and Applied Biochemistry; v. 58, n. 4, p. 243-249, JUL-AUG 2011.
• Web of Science Citations: 8

Abstract

Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries. (AU)

• FAPESP's process: 08/04264-4 - Transplant of hepatic stellate cells producing human coagulation factor IX in NOD/SCID mice
• Grantee: Andrielle de Castilho Fernandes
• Support Opportunities: Scholarships in Brazil - Doctorate