title: An approach for increasing the mass recovery of proteins derived from inclusion bodies in biotechnology.
abstract: A method for increasing the mass recovery of therapeutic proteins produced by E. coli using liquid chromatography was investigated. Recombinant human interferon-gamma (rhIFN-gamma) produced by E. coli was selected as a model therapeutic protein, and hydrophobic interaction chromatography (HIC) was performed as a model for liquid chromatography. Using seven types of stationary phase hydrophobic interaction chromatography (STHIC) with different end groups, the effect of the stationary phase on the mass recovery during protein folding by liquid chromatography (LC) and the causes of mass loss of rhIFN-gamma during its folding with simultaneous purification were investigated. Also strategies for increasing mass recovery are proposed. The results demonstrate that the mass recovery of rhIFN-gamma increases with the decreasing hydrophobicity for six STHIC with end groups of PEG-200, PEG-400, PEG-600, PEG-1000, furfural, and phenyl, except for PEG-1000. However, for oxethyl and PEG-600, even though the same diol end group is bonded to PEG-600, so long as the PEG-600 is modified by acetyl chloride, it can effectively enhance the mass and bioactivity recovery of rhIFN-gamma compared to the PEG-600 column. The effect of sample size including both mass and volume on the mass recovery of the rhIFN-gamma was also investigated. Last, redissolving the target protein that has irreversibly adsorbed to the stationary phase and re-injecting it onto the column is an approach for increasing mass recovery.
authors: Institute of Modern Separation Science, Key Laboratory of Modern Separation Science in Shaanxi Province, Northwest University, Xi'an 710069, China.
authors: Dan
authors: D
authors: Wu
authors: Chaozhan
authors: C
authors: Wang
authors: Xindu
authors: X
authors: Geng
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D007371:Interferon-gamma / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D007371:Interferon-gamma