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

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 9g 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.

Evaluation of a multimodal resin for selective capture of CHO-derived monoclonal antibodies directly from harvested cell culture fluid.

This proof-of-concept study examines the applicability of using multimodal chromatography to selectively capture recombinantly produced monoclonal antibodies (mAb) directly from harvested mammalian cell culture fluid (HCCF) with minimal optimization. Capto MMC is a multimodal resin that contains a ligand with the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. Twelve mAb HCCF feedstocks were examined for dynamic binding capacity (DBC) and then two representative feedstocks were selected to develop a systematic approach for elution buffer development. A range of dynamic binding capacities was observed for 10 feedstocks (24-53g/L) and two feedstocks had poor binding properties (<10g/L) despite load conditioning towards a more favorable pH. Analysis of the DBC versus molecular properties showed that the mAb-ligand binding interaction was predominantly charge based. Four separate elution strategies were identified to selectively recover the mAb and could be applied with minimal optimization to other mAb feedstocks. Downstream processing of the Capto MMC pools showed that it is feasible to produce material with comparable purity to a process with affinity capture after two chromatography steps.

Industrial case study: evaluation of a mixed-mode resin for selective capture of a human growth factor recombinantly expressed in E. coli.

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.