Purification Process Sciences, AstraZeneca, One MedImmune Way, Gaithersburg, MD 20878, USA.
Purification Process Sciences, AstraZeneca, One MedImmune Way, Gaithersburg, MD 20878, USA.
J Chromatogr A. 2020 Oct 11;1629:461505. doi: 10.1016/j.chroma.2020.461505. Epub 2020 Aug 21.
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 Zn 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 Co or Zn 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）是一种主要用于研究和开发实验室的技术，用于纯化含有工程组氨酸标签的蛋白质。尽管这种类型的层析技术很常见，但它可能会出现问题，因为不同的树脂和金属螯合剂组合会导致高度可变的层析性能和产品质量结果。为了生成稳健的 IMAC 纯化工艺，通过生成带有多组氨酸标记的双特异性蛋白的突破曲线来研究树脂和金属螯合剂组合的结合差异。通过统计分析最佳结合组合来确定层析参数对操作的影响。此外，还创建了平衡摄取等温线，以进一步阐明层析参数对蛋白质结合的影响。结果发现，对于在 CHO 细胞中表达的蛋白质，Millipore Sigma 的 Fractogel EMD Chelate（M）带 Zn 电荷和 GE 的预充 Ni Sepharose Excel 显示出最高的结合容量。当蛋白质在 HEK-293 中表达时，GE 的 IMAC Sepharose 6 Fast Flow 带 Co 或 Zn 电荷结合的蛋白质最多。该研究进一步确定了树脂批次的金属结合能力、树脂装载的蛋白质能力以及蛋白质上的多组氨酸标签残基的比例都对层析性能和产品质量有影响。这些发现使开发稳健且可扩展的工艺成为可能。CHO 表达的细胞培养产物直接以高容量加载到具有可变金属结合亲和力的 Fractogel EMD Chelate（M）上。250mM 咪唑洗脱条件确保产品中含有单体 4 和 6-组氨酸标记的双特异性蛋白质。本研究中确定的优化 IMAC 工艺条件可应用于研究和开发实验室中的各种多组氨酸标记蛋白，因为在 HEK-293 或 CHO 细胞中表达的不同分子量和格式的各种多组氨酸标记蛋白都已成功纯化。
