Human cell lines: A promising alternative for recombinant FIX production
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Published on: July 17, 2021
Aline de Sousa Bomfim1, Marcela Cristina Corrêa de Freitas2, Virgínia Picanço-Castro3
1Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil; Center for Cell-based Therapy and Regional Blood Center, University of São Paulo, 2501 Tenente Catão Roxo Street 14051-140, Ribeirão Preto, SP, Brazil.
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.
