Human hepatoma cell line HuH-7 is an effective cellular system to produce recombinant factor IX with improved post-translational modifications.

Abstract

Recombinant factor IX (rFIX) is increasingly used to treat patients with hemophilia B. CHO (Chinese Hamster Ovary) cells are commonly used for the production of rFIX but they have a limited capacity for introducing post-translational modifications (PTM) leading to incomplete γ-carboxylation, low phosphorylation and sulfation profiles as compared with plasma-derived preparations. Imperfect PTM might have an impact on the activity of Factor IX molecule. Several studies in animal models as well as clinical trials have previously reported a lower recovery of rFIX compared to plasma-derived FIX concentrates. In the present study, we aimed to produce a rFIX having a profile of PTM similar to plasma-derived FIX, using human hepatoma cell line HuH-7. We showed that rFIX produced by HuH-7 cells followed the classical intracellular pathway before secretion. In addition, improved PTM were associated with fully active molecule compared to plasma-derived and recombinant control FIX molecules. Secreted rFIX presented as a single band at the correct molecular weight. HuH-7 cellular clones were obtained and they secreted a biologically active human FIX. FIX was then purified for a detailed evaluation of PTM. Glycosylation and sialylation profiles were similar to plasma-derived and rFIX and mass spectrometry analysis demonstrated the presence of phosphorylated and sulfated forms of rFIX. These data strongly support that HuH-7 cells may represent an effective cellular system for production of rFIX exhibiting PTM similar to plasma-derived FIX.

• Authors: Nathalie Enjolras, Yesim Dargaud, Eloïse Pérot, Florine Guillaume, Michel Becchi, Claude Négrier
• Journal: Thrombosis research (Thromb Res) Vol. 130 Issue 5 Pg. e266-73 (Nov 2012) ISSN: 1879-2472 [Electronic] United States
• PMID: 23021500 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier Ltd. All rights reserved.

Chemical References

• Recombinant Proteins
• Factor IX

Topics

• Animals
• CHO Cells
• Cell Line, Tumor
• Cricetinae
• Factor IX (biosynthesis, genetics, metabolism)
• Glycosylation
• Humans
• Liver Neoplasms (genetics, metabolism)
• Protein Processing, Post-Translational
• Recombinant Proteins (biosynthesis, genetics, metabolism)
• Transfection