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Abstract

The stability of recombinant human interferon alpha-2b (rhIFNα-2b) remains a great challenge for Pharmaceutical science. In previous research we constructed open reading frame encoding rhIFNα-2b and produced the protein in Pichia pastoris (P. pastoris). This research was aimed to study the stability of rhIFNα-2b in three parameters: temperature, pH and shelf life. The rhIFNα-2b was overproduced by using buffered methanol complex medium (BMMY) at 30 °C for 48 h with 2% of methanol as inducer. Filtration of protein was used by minimateTM tangential flow filtration system with molecular weight cut off (MWCO) 5 kDa. Purification of rhIFNα-2b was performed by immobilized affinity chromatography column using AKTA purifier system. Colorimetric bicinchoninic acid assay informed that the yield of purified rhIFNα-2b was 10.92 mg/L (OD600 = 2.3). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot analyses confirmed that the protein was rhIFNα-2b with 24 kDa in size. Matrix assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) mass spectrometry identified the protein as hIFNα-2b with 22% of amino acid coverage. Non reducing SDS-PAGE and Image J software analyses showed that temperature increment, acidic and basic pH as well as shelf life length caused protein aggregation and degradation. 3-[4.5-dimethylthiazol-2il]-2.5-diphenyltetrazolium bromide (MTT) assay informed that the aggregation and degradation reduced the anti-proliferative activity of rhIFNα-2b on human breast cancer MCF-7 cell line. To conclude, all parameters give an impact on rhIFNα-2b stability with the most influencing parameter was temperature at 25 °C. These data can be used to develop rhIFNα-2b formulations as therapeutic protein.

Keywords

rhIFNα-2b Pichia pastoris stability aggregation degradation

Article Details

How to Cite
Syubbanul Wathon, Sri Budiarti, Popi Hadi Wisnuwardhani, Neng Herawati, Adi Santoso, & Ratih Asmana Ningrum. (2015). Stability of Recombinant Human Interferon Alpha-2b Produced In Methylotropic Yeast Pichia pastoris . International Journal of Research in Pharmaceutical Sciences, 6(4), 312-320. Retrieved from https://pharmascope.org/ijrps/article/view/1252