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Updated: Dec 2, 2025
High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension
Published on: December 28, 2015
Generation of hyperfunctional recombinant human factor IX variants expressed in human cell line SK-Hep-1
Aline Sousa Bomfim1,2,3, Marcela Cristina Corrêa de Freitas4, Virgínia Picanço Castro4
1Department of Clinical, Toxicological and Food Science Analysis, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, São Paulo, Brazil. line.sousab@gmail.com.
Objective:
To develop recombinant factor IX (FIX) variants with augmented clotting activity.
Results:
We generated three new variants, FIX-YKALW, FIX-ALL and FIX-LLW, expressed in SK-Hep-1 cells and characterized in vitro and in vivo. FIX-YKALW showed the highest antigen expression level among the variants (2.17 µg-mL), followed by FIX-LLW (1.5 µg-mL) and FIX-ALL (0.9 µg-mL). The expression level of FIX variants was two-five fold lower than FIX-wild-type (FIX-WT) (4.37 µg-mL). However, the biological activities of FIX variants were 15-31 times greater than FIX-WT in the chromogenic assay. Moreover, the new variants FIX-YKALW, FIX-LLW and FIX-ALL also presented higher specific activity than FIX-WT (17, 20 and 29-fold higher, respectively). FIX variants demonstrated a better clotting time than FIX-WT. In hemophilia B mice, we observed that FIX-YKALW promoted hemostatic protection.
Conclusion:
We have developed three improved FIX proteins with potential for use in protein replacement therapy for hemophilia B.
