Hepatoma Cell Line HuH-7 : a Promising New Cellular System for Recombinant FIX Production.
Authors: Enjolras Nathalie , Dargaud Yesim , Plantier Jean-Luc , Simpson Heli , Guillaume Florine , Negrier Claude 
Published: October 2019

Abstract
Abstract 4202
Hemophilia B is characterized by a deficiency of coagulation factor IX (FIX) which is synthesized by the liver. CHO cells commonly used for the production of recombinant coagulation factors have a limited capacity for post-translational modifications leading to incomplete γ-carboxylation, decreased phosphorylation and sulfation of the recombinant coagulation proteins. In addition, studies in animal models and data from clinical studies in hemophilic patients, have shown that recombinant FIX has a lower recovery than plasma-derived preparations. In the present study, the human hepatoma cell line HuH-7 was assessed for the production of a recombinant FIX molecule with post-translational modifications similar to those of the plasma-derived FIX.

Methods
Human hepatoma cell line HuH-7 was transfected with native FIX cDNA directed by CMV promoter, and cellular clones stably expressing FIX were obtained. Benefix®, Mononine® and a protein extract from human liver were used as controls. Cellular expression was analyzed by northern blot, and FIX production was quantified by ELISA. Procoagulant activities were measured by one-stage clotting and thrombin generation (TGT) assays. Secreted and intracellular FIX were visualized by western blot using an antibody directed against FIX. Intracellular trafficking of FIX was further analyzed using inhibitors of proteasome, lysosome and Golgi pathways. Glycosylation profile was determined after successive digestions of lysates by neuraminidase and N-Glycosydase-F.

Results
Non transfected HuH-7 cells did not express FIX and northern blot did not show any signal corresponding to FIX mRNA. Transiently transfected HuH-7 cells studies revealed the ability to secrete 0.1μg/ml/24h of FIX. The specific activity, measured by a one-stage clotting assay, reached 200% in comparison with Benefix® or rFIX produced by CHO cells. TGT confirmed the high procoagulant activity showing a 2.5 fold increased thrombin generating capacity in comparison with FIX produced by CHO cells, in the same experimental conditions. The secreted molecule appeared as a single band on SDS-PAGE, and presented the same mobility than Benefix® and Mononine®. Following enzymatic digestion, the migration profile suggested that glycosylation and sialylation were similar to Benefix®.
Western blot showed that FIX from cell lysate was retrieved as three bands. The upper band, presenting a lower signal than the two others, had the same molecular weight than the secreted form and the native human FIX extracted from a liver biopsy. The two other bands at different intensities migrated slightly faster than the secreted protein. Brefeldin A significantly blocked intracellular traffic of FIX from ER to the Golgi complex where the protein corresponding to the western blot upper band was dramatically accumulated. No intracellular accumulation of the non-secreted forms was observed, and lysosomal pathway was slightly involved in the degradation of both secreted and non-secreted forms of FIX.

Conclusion
These data demonstrate that HuH-7 cell line is able to produce in serum free medium a biologically active recombinant FIX with a higher specific activity. The protein is correctly synthesized, follows the classical pathway before secretion and therefore is not retained in the cells. N-linked oligosaccharide sialylation is also correctly performed. This preliminary data suggest that HuH7 cells may represent an effective cellular system for rFIX production. Further biochemical analysis of the post-translational modifications and pharmacokinetic properties are required to better characterize the rFIX molecule secreted by HuH7 cells.

Disclosures:
No relevant conflicts of interest to declare.