Improving purification of recombinant human interferon gamma expressed in Escherichia coli; effect of removal of impurity on the process yield.
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.
Mohammadian-Mosaabadi
Jafar
J
Biochemistry Group, Department of Biology, Faculty of Science, Tarbiat Modares University, Tehran, P.O. Box 14115-175, Iran.
Naderi-Manesh
Hossein
H
Maghsoudi
Nader
N
Nassiri-Khalili
Mohammad-Ali
MA
Masoumian
Mohammad Reza
MR
Malek-Sabet
Narges
N
D000813:Anilides; D001391:Azo Compounds; D001433:Bacteriolysis; D002850:Chromatography, Gel; D003661:Decision Support Techniques; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D007371:Interferon-gamma / Q000302:isolation & purification*; D010100:Oxygen / Q000494:pharmacology; D017510:Protein Folding; D020673:Protein Renaturation; D011786:Quality Control; D011994:Recombinant Proteins; D012995:Solubility
D000813:Anilides; D001391:Azo Compounds; D011994:Recombinant Proteins; C506598:1-(2-methoxy-5-nitrophenylazo)-2-hydroxy-3-(3-nitrophenylcarbamoyl)naphthalene; D007371:Interferon-gamma; D010100:Oxygen