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Development of production process and characterization of periplasmic interferon-α2a expressed in Escherichia Coli

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Author(s):
Paulo Victor Sarmento Dias
Total Authors: 1
Document type: Master's Dissertation
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Instituto de Pesquisas Energéticas e Nucleares (IPEN/BT)
Defense date:
Examining board members:
Carlos Roberto Jorge Soares; Sergio Luiz Dalmora; Geraldo Santana Magalhães
Advisor: Carlos Roberto Jorge Soares
Abstract

IFN-&alpha;2 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-&alpha;2a 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-&alpha;2a and interferon-&alpha;2b. 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 &mu;g (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 &mu;g. This rapid methodology allows the application of the RP-HPLC as a powerful tool to monitor the production yield and quality of periplasmic Interferon &alpha;2 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 &mu;g/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 type: Scholarships in Brazil - Master