title: Stable and high-level production of recombinant Factor IX in human hepatic cell line.
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.
authors.affiliation: Department of Clinical Medicine, University of Sao Paulo, Brazil. andrielle_dcf@yahoo.com
authors.forename: Andrielle
authors.initials: A
authors.lastname: de Castilho Fernandes
authors.forename: Aparecida
authors.initials: A
authors.lastname: Fontes
authors.forename: Nathalia
authors.initials: N
authors.lastname: Gonsales
authors.forename: Kamilla
authors.initials: K
authors.lastname: Swiech
authors.forename: Virginia
authors.initials: V
authors.lastname: Picanco-Castro
authors.forename: Sandra
authors.initials: S
authors.lastname: Faca
authors.forename: Dimas
authors.initials: D
authors.lastname: Covas
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D005164:Factor IX / Q000096:biosynthesis* / Q000302:isolation & purification / Q000493:pharmacokinetics; D015870:Gene Expression; D005822:Genetic Vectors; D057809:HEK293 Cells; D002836:Hemophilia B / Q000188:drug therapy; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D010314:Partial Thromboplastin Time; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000493:pharmacokinetics; D012190:Retroviridae; D014161:Transduction, Genetic
chemical_list: D011994:Recombinant Proteins; D005164:Factor IX