Immobilized metal affinity chromatography optimization for poly-histidine tagged proteins

Abstract

Immobilized metal affinity chromatography (IMAC) is a technique primarily used in research and development laboratories to purify proteins containing engineered histidine tags. Although this type of chromatography is commonly used, it can be problematic as differing combinations of resins and metal chelators can result in highly variable chromatographic performance and product quality results. To generate a robust IMAC purification process, the binding differences of resin and metal chelator combinations were studied by generating breakthrough curves with a poly-histidine tagged bispecific protein. The optimal binding combination was statistically analyzed to determine the impact of chromatographic parameters on the operation. Additionally, equilibrium uptake isotherms were created to further elucidate the impact of chromatographic parameters on the binding of protein. It was found that for protein expressed in CHO cells, Millipore Sigma's Fractogel EMD Chelate (M) charged with Zn²⁺ and GE's pre-charged Ni Sepharose Excel displayed the highest binding capacities. When the protein was expressed in HEK-293, GE's IMAC Sepharose 6 Fast Flow charged with either Co²⁺ or Zn²⁺ bound the greatest amount of protein. The study further identified the metal binding capacity of the resin lot, the protein capacity to which the resin is loaded, and the ratio of poly-histidine tag residues on the protein all impacted the chromatographic performance and product quality. These findings enabled the development of a robust and scalable process. The CHO expressed cell culture product was directly loaded at a high capacity onto variable metal binding affinity Fractogel EMD Chelate (M). A 250 mM imidazole elution condition ensured the product contained monomeric 4 and 6-histidine tagged bispecific proteins. The optimized IMAC process conditions determined in this study can be applied to a wide variety of poly-histidine tagged proteins in research and development laboratories as various poly-histidine tagged proteins of differing molecular weights and formats expressed in either HEK-293 or CHO cells were successfully purified.

Keywords: IMAC; Poly-histidine tag; Process optimization; Protein purification; Resin/metal screening.