Abstract:

The renaturation with simultaneous purification of recombinant human interferon-γ (rhIFN-γ) expressed as inclusion bodies in Escherichia coli (E.coli) was accomplished by the stationary phase of hydrophobic interaction chromatography (STHIC) with the end group of poly(ethylene glycol) （PEG）(PEG200) packed in a chromatographic column and a chromatographic pie by nonlinear gradient, separately. In order to provide more selections for the chromatographic separation of rhIFN-γ from different sources, the chromatographic behavior of rhIFN-γ in reversed-phase liquid chromatography, ion-exchange chromatography and immobilized-nickel affinity chromatography were also studied. The fraction of the renatured and purified rhIFN-γ from HIC was desalted by the size exclusion chromatography, subsequently freeze-dried to powder. With matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), monomeric and dimeric rhIFN-γ were found in the powder due to the freeze-dried process and their relative molecular masses were 17184.0 and 34204.4, respectively. With the bioactivity assay by cytopathic effect inhibition (CPEI), the specific bioactivity of rhIFN-γ was 9.5×108 IU/mg, which was higher than that of the required criteria in the pharmacopoeia of China, because the presence of dimeric rhIFN-γ which has much higher specific bioactivity than its monomer in the powder. The obtained mass recovery, purity, specific bioactivity of the purified monomeric rhIFN-γ were 93.7%, ＞95%, and 4.3×107 IU/mg, respectively. The results showed that the renaturation with simultaneous purification of rhIFN-γ by PEG200-STHIC is a kind of efficient method.

Key words: high performance hydrophobic interaction chromatography (HPHIC), protein folding liquid chromatography (PFLC), purification , renaturation, recombinant human interferon-γ (rhIFN-γ)