Nickel‐ion substituted hydroxyapatite matrices for metal‐affinity chromatographic purification of recombinant proteins

Hydroxyapatite (HAp) is a calcium phosphate ceramic, widely used as a matrix for protein chromatography. The crystal structure of HAp is amenable to a wide range of substitutions, thus allowing for the alteration of its properties. In this study, nickel‐ion substituted HAp (NiSHAp) was synthesized using a wet‐precipitation method, followed by spray drying. This resulted in the structural incorporation of nickel ions within well‐defined microspheres, which were suitable for chromatographic applications. The chromatographic experiments were conducted with NiSHAp and compared with spray‐dried hydroxyapatite (SHAp) matrices. Protein purification experiments were conducted using refolded recombinant L‐asparaginase (L‐Asp), which was produced as inclusion bodies in Escherichia coli. The results showed that NiSHAp effectively adsorbed L‐Asp, which was selectively eluted using a phosphate buffer, surpassing the efficiency of imidazole‐based elution. In contrast, SHAp showed weaker binding and lower selectivity. The significance of this study lies in developing a scalable NiSHAp matrix for protein purification, especially for large‐scale applications. The NiSHAp matrix offers a cost‐effective alternative to commercial immobilized metal affinity chromatography (IMAC) adsorbents, especially for purifying His‐tagged proteins. This innovative approach exhibits the advantages of mixed‐mode chromatography by combining the properties of hydroxyapatite and IMAC in a single matrix, with the potential of improved industrial‐scale protein purification.