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.

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

“…We were not only able to analyze the abundance and complexity of the proteome as it shifted during the bioprocess and after purification, but we were also able to monitor the abundance of diverse PTMs with site specificity, including the critically important g- Discussion rFIX is commercialized as a replacement therapy for Haemophilia B [8,10]. Considerable effort has been devoted to optimize rFIX bioprocess and expression systems [7,[10][11][12][109][110][111][112][113]. FIX is highly post-translationally modified with a variety of heterogeneous PTMs, and GLA domain gcarboxylation is the most functionally relevant PTM [14,[19][20][21].…”

“…For example, rFIX N-linked glycans (native and engineered) influence rFIX in vivo half-life[94,[116][117][118][119] and may modulate rFIX activation[31], while rFIX EGF-like 1 Oglycans stabilize the domain and likely participate in protein-protein interactions[92,99]. Changes in the expression system and incubation conditions alter the number and variety of PTMs in recombinant proteins[7,10,11,21,70,109,114], and new, unexpected PTMs may appear. Indeed, through a detailed MS proteomic analysis we identified new PTMs on purified rFIX, including a sulfation/phosphorylation on Y45 , several Asp oxidations spread throughout the molecule, and new…”

Industrial production of clotting factors: Challenges of expression, and choice of host cells

“…In mammals, the γ‐glutamylcarboxylation system has a very wide tissue distribution as it has been detected in nearly all cells and tissues isolated [25]. In line with this, academic and industrial laboratories have demonstrated successful expression of VKD protein in active form in several different mammalian cell lines such as AV12‐664, BHK, CHO, COS, HEK293, HepG2, huH‐7, SK‐Hep1 and rat hepatoma [10, 12, 13, 26–29]. Evolutionarily, the γ‐carboxylation system is of ancient origin, predating the mollusks [30].…”

Coagulation Factor IX for Hemophilia B Therapy

“…30 mg/l [37]. It may be increased by 30-50% upon addition of 1 nM of methyl testosterone to the culture medium [38] or doubled after the addition of phorbol 12-myristate, 13-acetate, and calcium ionophore [39]. The appearance of undesired activated FIX in the culture medium can be controlled by decreasing the Ca 2+ ions concentration to 0.5 mM from 1.12 mM [40]; in another study of the same group, it was found that an increase of Ca 2+ ions to 1.3 mM leads to a 30% increase in the production of FIX, without a significant rise in the FIXa level [41].…”

“…Natural FIX is produced by liver cells, and it has been established that the human hepatoma cell line HepG2 produces 1.5 times more recombinant FIX than the human kidney cell line 293 after transfection by the same retroviral vector [39]. Nonvertebrate cultured cells were also evaluated for the expression of FIX, and in a drosophila-derived SF2 cell line a 12-fold increase in functionally active FIX secretion, compared with CHO cells, was detected [49].…”