Improved process for production of recombinant yeast-derived monomeric human G-CSF

The human granulocyte colony-stimulating factor (hG-CSF) was efficiently se creted at high levels in fed-batch cultures of recombinant Saccharomyces ce revisiae. However, the secreted recombinant hG-CSF (rhG-CSF) was shown to e xist as large multimers in the culture broth due to strong hydrophobic inte raction. 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 t he rhG-CSF was secreted to extracellular broth in the presence of a non-ion ic surfactant (Tween SO) in the culture media, the multimerization of the s ecreted rhG-CSF was efficiently prevented in the fed-batch cultures. Also, the monomer fraction and secretion efficiency of rhG-CSF were significantly increased at the higher culture FH (6.5). Without using any denaturing age nts, the secreted rhG-CSF monomer was easily purified with high recovery yi eld and purity via a simple purification process under acidic conditions, c onsisting of diafiltration, cation exchange, and gel filtration chromatogra phy. A lyophilization process devoid of intermonomer aggregation was also d esigned using effective stabilizing agents.