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| Author(s): |
Paulo Victor Sarmento Dias
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Pesquisas Energéticas e Nucleares (IPEN/BT) |
| Defense date: | 2017-01-30 |
| Examining board members: |
Carlos Roberto Jorge Soares;
Sergio Luiz Dalmora;
Geraldo Santana Magalhães
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| Advisor: | Carlos Roberto Jorge Soares |
| Abstract | |
IFN-α2 is currently utilized in hepatitis B and C, leukemia, multiple myeloma, hairy cell leukemia, melanoma, Kaposi\'s sarcoma, follicular lymphoma, and renal cell carcinoma therapy, with or without other drugs. In this work, a process for E. coli periplasmic interferon-α2a production and purification utilizing a lambda PL promoter based on constitutive expression was proposed. As a tool for production monitoring, reversedphase high-performance liquid chromatography (RP-HPLC) analysis directly from periplasmic extract was validated. Thus, initially it was described a RP-HPLC methodology for qualitative and quantitative analysis of recombinant human interferon-α2a and interferon-α2b. The method has been set up and validated for accuracy, precision, linearity, sensitivity and specificity. A recovery test indicated a bias of less than 1% and intra-day and inter-day quantitative determinations presented relative standard deviations always < 4 %, while experimental sensitivity was 0.3 μg (RSD = 5 %). Regarding to linearity, the coefficient of determination was 0.998 (p<0.0001), for a range of analyzed interferon mass from 0,62 to 10 μg. This rapid methodology allows the application of the RP-HPLC as a powerful tool to monitor the production yield and quality of periplasmic Interferon α2 right after, or even during the fermentation. The optimum expression temperature was evaluated in flask cultures from 30 to 42 °C. It was observed that the volumetric and specific production were higher for culture temperatures equal or above 35 °C. Thus, considering the potential recombinant protein degradation induced by temperature, 35 °C was well-marked as the optimum temperature for interferon expression. The higher values for specific and volumetric production in culture flasks were 1.04 μg/mL/A600 and 3.45 mg/L respectively. As purification method, it was utilized ionic chromatography followed by size-exclusion chromatography. The final purity and mass recovery was, respectively, 95.3 % and 66 %. The final product was also characterized utilizing SDS-PAGE, western blotting, reversed-phase HPLC, size-exclusion HPLC and mass spectrometry analysis. (AU) | |
| FAPESP's process: | 15/10928-6 - Development of fermentation in a bioreactor for the production of interferon ±-2a secreted into the periplasmic space of Escherichia coli |
| Grantee: | Paulo Victor Sarmento Dias |
| Support Opportunities: | Scholarships in Brazil - Master |