High-yield expression of human vascular endothelial growth factor VEGF(165) in Escherichia coli and purification for therapeutic applications

作者：Pizarro Shelly A^*; Gunson Jane; Field Matthew J; Dinges Rachel; Khoo Stefanie; Dalal Milind; Lee Michael; Kaleas Kimberly A; Moiseff Kathryn; Garnick Susan; Reilly Dorothea E; Laird Michael W; Schmelzer Charles H

来源：Protein Expression and Purification, 2010, 72(2): 184-193.

DOI：10.1016/j.pep.2010.03.007

• Bacterial expression system
• HIC
• Mixed-mode resin
• Protein refolding

Vascular endothelial growth factor (VEGF(165)) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9 g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30 +/- 6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells.

• 出版日期2010-8