title: Industrial case study: evaluation of a mixed-mode resin for selective capture of a human growth factor recombinantly expressed in E. coli.
abstract: Mixed-mode chromatography resins are gaining popularity as effective purification tools for challenging feedstocks. This study presents the development of an industrial application to selectively capture recombinant human vascular endothelial growth factor (rhVEGF) on Capto MMC from an alkaline feedstock. Capto MMC resin contains a ligand that has the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. VEGF is a key growth factor involved in angiogenesis and has therapeutic applications for wound healing. In this process, it is expressed in Escherichia coli as inclusion bodies. Solids are harvested from the cell lysate, and the rhVEGF is solubilized and refolded at pH 9.8 in the presence of urea and redox reagents. The unique mixed-mode characteristics of Capto MMC enabled capture of this basic protein with minimal load conditioning and delivered a concentrated pool for downstream processing with >95% yields while reducing host cell protein content to <1.2%. This study explores the impact of loading conditions and residence time on the dynamic binding capacity as well as the development of elution conditions for optimal purification performance. After evaluating various elution buffers, l-arginine HCl was shown to be an effective eluting agent for rhVEGF desorption from the Capto MMC mixed-mode resin since it successfully disrupted the multiple interactions between the resin and rhVEGF. The lab scale effort produced a robust chromatography step that was successfully implemented at commercial manufacturing scale.
authors: Genentech, Inc. Process Research and Development, 1 DNA Way, MS 75, South San Francisco, CA 94080, USA.
authors: Kimberly A
authors: KA
authors: Kaleas
authors: Charles H
authors: CH
authors: Schmelzer
authors: Shelly A
authors: SA
authors: Pizarro
mesh_terms: D001120:Arginine / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007475:Ion Exchange Resins / Q000737:chemistry*; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D042461:Vascular Endothelial Growth Factor A / Q000302:isolation & purification*
chemical_list: D007475:Ion Exchange Resins; D042461:Vascular Endothelial Growth Factor A; D012965:Sodium Chloride; D012685:Sepharose; D001120:Arginine