Effect of culture media and fermentation process on the refolding and purification of rh-GCSF

Abstract

Human granulocyte-colony stimulating factor (h-GCSF) is used to mitigate neutropenia caused by chemotherapy. E. coli is the most common host for h-GCSF production due to its high yield and versatile culture strategies. However, overproduction of h-GCSF in E. coli often results in the formation of inclusion bodies (IBs) in the cytoplasm. The efficiency of refolding and purifying recombinant h-GCSF (rh-GCSF) produced as IBs depends on the expression strategy, induction, and cell growth conditions. This study investigated how different culture media and fermentation processes affect the refolding of IBs and the purification of rh-GCSF. The purified samples were compared to Neupogen(R) and PDgrastim reference standards using Western blotting, size exclusion chromatography, and reverse-phase high-performance liquid chromatography. The analysis confirmed that all proteins were correctly refolded and exhibited a purity exceeding 90 after purification. The highest protein recovery percentage and purity of rh-GCSF with the lowest endotoxin content was achieved using M9 media in fed-batch culture.