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.

固定金属亲和层析（IMAC）是一项主要用于研发实验室的技术，用于纯化含有工程组氨酸标签的蛋白质。尽管通常使用这种色谱法，但由于树脂和金属螯合剂的不同组合可导致色谱性能和产品质量结果高度可变，因此可能会出现问题。为了生成可靠的IMAC纯化过程，通过用多组氨酸标记的双特异性蛋白生成穿透曲线，研究了树脂和金属螯合剂组合的结合差异。统计分析最佳结合组合，以确定色谱参数对操作的影响。另外，建立了平衡吸收等温线，以进一步阐明色谱参数对蛋白质结合的影响。已发现，对于在CHO细胞中表达的蛋白质，Millipore Sigma的Fractogel EMD螯合物（M）含锌²⁺和GE的预充电Ni Sepharose Excel表现出最高的结合能力。当蛋白质在HEK-293中表达时，GE的IMAC Sepharose 6 Fast Flow充满了Co ²⁺或Zn ²⁺结合了最大量的蛋白质。该研究进一步确定了树脂批次的金属结合能力，树脂负载的蛋白质容量以及蛋白质上的多组氨酸标签残基的比例均影响了色谱性能和产品质量。这些发现使开发健壮和可扩展的过程成为可能。将CHO表达的细胞培养产物直接以高容量直接加载到可变金属结合亲和力Fractogel EMD螯合物（M）上。250 mM咪唑洗脱条件确保产物包含单体4和6-组氨酸标记的双特异性蛋白。