Improved process for production of recombinant yeast-derived monomeric human G-CSF.
The human granulocyte colony-stimulating factor (hG-CSF) was efficiently secreted at high levels in fed-batch cultures of recombinant Saccharomyces cerevisiae. However, the secreted recombinant hG-CSF (rhG-CSF) was shown to exist as large multimers in the culture broth due to strong hydrophobic interaction. It was hardly monomerized even by urea at high concentration. This multimer has been reported to diminish specific receptor-binding activity of hG-CSF and causes undesirable problems in the downstream process. When the rhG-CSF was secreted to extracellular broth in the presence of a non-ionic surfactant (Tween 80) in the culture media, the multimerization of the secreted rhG-CSF was efficiently prevented in the fed-batch cultures. Also, the monomer fraction and secreted efficiency of rhG-CSF were significantly increased at the higher culture pH (6.5). Without using any denaturing agents, the secreted rhG-CSF monomer was easily purified with high recovery yield and purity via a simple purification process under acidic conditions, consisting of diafiltration, cation exchange, and gel filtration chromatography. A lyophilization process devoid of intermonomer aggregation was also designed using effective stabilizing agents.
Bae
C S
CS
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Taejon, South Korea.
Yang
D S
DS
Lee
J
J
Park
Y H
YH
D001704:Biopolymers / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D003470:Culture Media; D004355:Drug Stability; D005285:Fermentation; D005612:Freeze Drying; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D001704:Biopolymers; D003470:Culture Media; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor