Immobilized metal affinity chromatography optimization for poly-histidine tagged proteins

Area of Science:

• Biochemistry
• Chemical Engineering
• Protein Purification

Background:

• Immobilized metal affinity chromatography (IMAC) is widely used for purifying histidine-tagged proteins in research and development.
• Variable performance and product quality arise from differing resin and metal chelator combinations in IMAC.
• Developing a robust IMAC process requires understanding the impact of these combinations on protein binding.

Purpose of the Study:

• To investigate and identify optimal resin and metal chelator combinations for robust IMAC purification.
• To statistically analyze chromatographic parameters influencing IMAC performance and product quality.
• To elucidate the impact of chromatographic parameters on protein binding through equilibrium uptake isotherms.

Main Methods:

• Generation of breakthrough curves using a poly-histidine tagged bispecific protein with various resin and metal chelator combinations.
• Statistical analysis of binding data to determine optimal chromatographic parameters.
• Creation of equilibrium uptake isotherms to further understand protein binding characteristics.

Main Results:

• For CHO cell-expressed protein, Fractogel EMD Chelate (M) with Zn²⁺ and Ni Sepharose Excel showed highest binding capacities.
• For HEK-293 expressed protein, IMAC Sepharose 6 Fast Flow with Co²⁺ or Zn²⁺ exhibited greatest protein binding.
• Resin lot metal binding capacity, protein loading capacity, and poly-histidine tag ratio significantly impacted IMAC performance.

Conclusions:

• The study enabled the development of a robust and scalable IMAC process for histidine-tagged proteins.
• Optimized conditions allow direct loading of CHO-expressed product onto Fractogel EMD Chelate (M) at high capacity.
• The findings are applicable to various histidine-tagged proteins expressed in HEK-293 or CHO cells, ensuring monomeric bispecific proteins with specific elution conditions.