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| Author(s): |
Rafael Tagé Biaggio
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2018-10-29 |
| Examining board members: |
Kamilla Swiech Antonietto;
Elisa Maria de Sousa Russo;
Aldo Tonso
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| Advisor: | Kamilla Swiech Antonietto |
| Abstract | |
Human cell lines have attracted great interest as a plarform for recombinant therapeutic proteins production, due their ability to perform complex posttranslational modification in a similar manner to human proteins. These proteins do not carry immunogenic epitopes as occurs with proteins produced in mammalian cells. These therapeutic proteins should be produced in a animal-free process avoiding virus and prion contamination, as recommended by regulatory agencies for quality control. Thus, this work aims the production of recombinant blood coagulation factor VII (rFVII) used in the treatment of hemophiliacs with inhibitors in human cell lines adapted to serum-free suspension cultures. Human cell lines SK-Hep-1, HKB-11 and Huh-7 were adapted to suspension and serum-free media. These adapted cells were transiently transfected with p1054-GFP lentiviral vector and the polyethyleneimine reagent. However, low transfection efficiency in SK-Hep-1 and Huh-7 cells showed that these cells are difficult to transfect by this method, and even transfection of HKB-11 cell was only possible after varying some parameters, resulting in a 49,5% HKB-11 GFP-positive cells. Stable transfection was assessed and adapted cells were transduced with lentivirus particles containing p1054-FVII vector in one cycle (MOI=1). Percentages of GFP-positive cells above 35% were observed in three modified human cell lines. Transduced cells were sorted by FACS and more than 90% of GFP-positive cells were obtained. The expression of rFVII were evaluated by ELISA test after vitamin K supplementation, however, it was not possible to detect FVII levels in the 48 hour culture supernatant. Cells were transduced again with a second lentivirus cycle (MOI = 2). ELISA quantification of the 48 hour culture supernatant detected 240,96 ng/mL, 217,42 ng/mL and 78,46 ng/mL total FVII, respectively, in the cultures of HKB-11-F7-2C, SK-Hep-1-F7-2C and Huh-7-F7-2C cells. Relative expression of mRNA by RT-PCR was also observed in the three cultures assessed. In parallel, a proteomic analysis of adapted and non-adapted cells was performed in triplicate. Proteins related to cellular metabolism, cytoskeletal structure, apoptosis, cell cycle and cell growth, against oxidative and osmotic stress, antioxidant action were found. (AU) | |
| FAPESP's process: | 14/01404-0 - EVALUATION OF GROWTH AND RECOMBINANT PROTEIN PRODUCTION OF HUMAN CELL LINES ADAPTED TO SERUM-FREE SUSPENSION CULTURES |
| Grantee: | Rafael Tagé Biaggio |
| Support Opportunities: | Scholarships in Brazil - Doctorate |