Immobilized metal affinity chromatography (IMAC) has been the most effective single-step preparation tool for highly pure recombinant proteins --- naturally rich in histidine/cysteine residues and those with a histidine residue cluster engineered to their N- or C-terminus via recombinant DNA technology. As with other affinity chromatography techniques, IMAC is robust requiring minimal optimization of purification condition. It is typically performed at physiological pH with good tolerance of a wide variety of chemicals, which is crucial to maintaining the structural integrity and biological activity of target protein molecules. Proteins prepared with IMAC are often pure enough for structural and functional characterization studies. In recent years, the application of immobilized metal affinity chromatography has been extended to the manufacture of protein therapeutics, vaccines, and Adeno-associated virus (AAV). Nuvia IMAC is a rigid macroporous high-capacity resin designed for His-tagged protein manufacture at high flow rate. We have developed platform for the purification of a vaccine-grade recombinant protein from an E. coli lysate. The target molecules are captured by Nuvia IMAC, and subjected to polish purification by two mixed-mode chromatography steps with Nuvia cPrime and Ceramic Hydroxyapatite (CHT), respectively. This workflow facilitates the effective removal of hazardous contaminants, endotoxins and leached Ni2+, as well as the significant reduction of host cell proteins and DNAs. Nuvia IMAC also works robustly in the presence of denaturing reagents, such as high concentration of urea or guanidine hydrochloride. In this presentation, we will demonstrate the successful capture of a green fluorescent protein (GFP) in the presence of 8 M urea, its on-column refolding by gradual elimination of urea, and the eventual elution of molecules with fluorescence comparable to native GFP.

Nuvia IMAC: A metal affinity chromatography media for large-scale manufacturing of recombinant proteins under native and denaturing conditions

2019 Spring National Meeting

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