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Protein Expr Purif ; 51(2): 147-56, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16908190

ABSTRACT

Process development and optimization studies were performed in order to improve the purification process of (rhIFN-gamma). The objective was to generate material with higher purity and quantity. An in-process control screening was developed to obtain the optimal condition for column chromatographic purification by measuring LPS, nucleic acids, rhIFN- gamma, monomer and its covalent dimers. A new resin screening method was applied to select optimal resin for each of the chromatographic columns. The resulting process used Butyl and Q-Sepharose, refolding and SP-Sepharose for purification of IFN-gamma. Effects of different process conditions such as cell lysis, removal of impurity and oxygen concentration were evaluated. Removal of impurities was evaluated by washing of inclusion bodies with 1% Triton X-100 and 3M urea and different chromatography steps. The results reveal that Triton removed about 43% of the LPS but urea had no effect on removal of nucleic acids and LPS. Further analysis show that removal of impurities by column chromatography decreases aggregation and increases the process yield. Oxygen concentration was identified as parameter that could have a significant impact on covalent dimers formation, as an unacceptable pharmaceutical form of rhIFN-gamma. On the basis of small-scale studies, optimum operating conditions were chosen and the purification process was successfully scaled-up to a pilot scale process with step yield and product quality that were better than previous reports.


Subject(s)
Interferon-gamma/isolation & purification , Anilides , Azo Compounds , Bacteriolysis , Chromatography, Gel , Decision Support Techniques , Escherichia coli/metabolism , Humans , Inclusion Bodies/metabolism , Oxygen/pharmacology , Protein Folding , Protein Renaturation , Quality Control , Recombinant Proteins , Solubility
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