Human cell lines: A promising alternative for recombinant FIX production

https://doi.org/10.1016/j.pep.2015.11.023Get rights and content

Highlights

  • Evaluation the recombinant human FIX expression in human cell lines.

  • 293T cells produced only 28% of the total secreted protein active.

  • SK-Hep-1-FIX cells produced higher levels of biologically active protein.

Abstract

Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca+2 and Mg+2 ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca+2 and 1 mM Mg+2 resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

Introduction

Hemophilia B is an X-linked recessive bleeding disorder characterized by quantitative or qualitative deficiency of clotting FIX [1]. The prevalence of the disease is 1/30,000 men in the world [2]. The current treatment of hemophilia A and B is based on protein replacement therapy through plasma-derived coagulation factors or recombinant protein produced in murine cells [3]. The commercial recombinant FIX has been available for 16 years, and it is currently produced in Chinese hamster ovary (CHO) cells [4]. At the moment, there are four recombinant products: Benefix, Rixubis, Ixinity and Alprolix. These last two were approved in 2015, and Alprolix is the first to use Fc fusion technology binding the FIX molecule to a recombinant human immune globulin molecule to extend half-life [5]. Although these products have been showing great results and good pharmacokinetic profiles, they exhibit non-human pattern post-translational modifications (PTMs).

Murine cell lines present two important differences in the glycosylation pattern compared to human cells: Gal•1-3Galβ1-(3) 4GlcNAc (α-Gal) epitopes and N-glycolylneuraminic (Neu5Gc) sialic acid, which are not present in humans [6], [7]. Due to non-human pattern post-translational modifications, some studies have suggested that recombinant FIX products may be associated with a higher incidence of inhibitors directed against FIX (alloantibodies). In this case, patients do not respond to the infusion of the deficient factor and have unwieldy bleeding episodes [8], [9], [10], [11].

A new trend in the market of recombinant proteins is the use of human cells as an alternative for the production of human therapeutic proteins. This system is capable of producing recombinant proteins with post-translational modifications identical to its natural counterpart and it might reduce possible immunogenic reactions [7], [8], [9], [10], [11], [12].

In fact, Octapharma has launched in the market a novel recombinant human FVIII (rhFVIII) as the first native, unmodified rhFVIII product manufactured in a human cell line. Studies in previously treated patients indicate that Human-cl rhFVIII is effective and safe in the prevention and treatment of bleeding episodes, and so far, none of the treated patients has developed inhibitors or allergic reactions [13].

The aim of this study was to evaluate the potential of rhFIX expression in two human cell lines. We evaluated the human embryonic kidney 293T cell line, which is used to produce human recombinant factor VIII, and a hepatic cell line (SK-Hep-1) because FIX is mainly synthesized in the liver. Additionally, we described, for the first time, the characterization of SK-Hep-1 cell line producing rhFIX. High levels of rhFIX were produced in 293T and SK-Hep-1 cell lines, being the latter a great producer of this clotting protein.

Section snippets

Cloning of human factor IX into a bicistronic lentiviral vector

Full-length human factor IX (hFIX) cDNA contained in the plasmid pCMV5FIX was acquired from American Type Culture Collection (catalogue number 79871). Primers for the coding region of hFIX were designed to contain the restriction sites for the enzymes SalI and SmaI. Kozak sequence was also included in the forward primer. The oligonucleotides used for hFIX PCR were sense primer 5′-GGA TCC GCC GCC ACC ATG CAG CGC GTG AAC ATG ATC-3′ and antisense primer 5′-GGA TCC CTT TCA TTA AGT GAG CTTTG-3´.

rhFIX expression in 293T and SK-Hep-1 human cell lines

The two human cell lines, 293T and SK-Hep-1, were transduced with pCIGWS-FIX lentiviral vector to express rhFIX (Fig. 1a, b). Both cells showed similar transduction efficiency, 84.5% and 82%, respectively, determined by EGFP expression (Fig. 1c, d). The areas represented by P2 refer to cells selected by sorting that were used for subsequent analysis.

The populations with high levels EGFP expression were expanded and the production of FIX was characterized.

Characterization of secreted rhFIX protein

The biological activity of rhFIX

Discussion

In our study, we characterized two rhFIX-producing human cell lines developed using lentiviral vector. It is important to emphasize that for the first time the human liver cell line SK-Hep-1 has been used for the expression of recombinant FIX protein.

The analysis of rhFIX coagulation activity showed that the transduced human cell lines were able to produce biologically active rhFIX. However, SK-Hep-1-FIX produced higher levels of the biologically active protein when compared with 293T-FIX.

This

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors would like to acknowledge the National Counsel of Technological and Scientific Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico) – CNPq (132609/2011-8 and 383229/2013-8), Financiadora de Fundos e Projetos – FINEP (01.07.0652.00), Sao Paulo Research Foundation (Fundação de Amparo a Pesquisa do Estado de São Paulo) – FAPESP (2012/04629-8), the National Institute of Science and Technology in Stem Cells and Cell Therapy – INCTC (CNPq 573754/2008 and FAPESP

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