Aline Sousa Bomfim1,2,3, Marcela Cristina Corrêa de Freitas4, Virgínia Picanço Castro4, Mario Abreu Soares Neto4, Ricardo Pádua5, Dimas Tadeu Covas4,6, Elisa Maria Sousa Russo7,4. 1. Department 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. 2. Laboratory of Biotechnology, Center for Cell-Based Therapy and Regional Blood Center, University of São Paulo, São Paulo, Brazil. line.sousab@gmail.com. 3. Department of Clinical, Toxicological and Food Science Analysis, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Café Avenue, Block R, Room 7, Ribeirão Preto, SP, ZIP 14040-903, Brazil. line.sousab@gmail.com. 4. Laboratory of Biotechnology, Center for Cell-Based Therapy and Regional Blood Center, University of São Paulo, São Paulo, Brazil. 5. Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, São Paulo, Brazil. 6. Department of Medical Clinic, Faculty of Medicine of Ribeirão Preto, University of São Paulo, São Paulo, Brazil. 7. Department of Clinical, Toxicological and Food Science Analysis, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, São Paulo, Brazil.
Abstract
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.
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 Bmice, 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.
Entities:
Keywords:
Blood coagulation factors; Hemophilia B; Human cell line SK-Hep-1; Recombinant human factor IX
Authors: R Hartmann; M Dockal; W Kammlander; E Panholzer; G A Nicolaes; C Fiedler; J Rosing; F Scheiflinger Journal: J Thromb Haemost Date: 2009-07-28 Impact factor: 5.824
Authors: J Chang; J Jin; P Lollar; W Bode; H Brandstetter; N Hamaguchi; D L Straight; D W Stafford Journal: J Biol Chem Date: 1998-05-15 Impact factor: 5.157