Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Mol Biol Rep. 2020 Apr;47(4):2861-2869. doi: 10.1007/s11033-020-05404-8. Epub 2020 Mar 29.
Recombinant form of granulocyte colony stimulating factor (G-CSF) was first approved by FDA in 1998 for chemotherapy induced neutropenia. However, despite production of its biosimilars, less expensive production of G-CSF could reduce the overall therapeutic cost. The aim of this study was to evaluate the possibility of producing biologically active recombinant G-CSF via a single step purification procedure mediated by a self-cleavable intein. G-CSF was expressed by E. coli BL21 (DE3) through IPTG induction, followed by its purification using pH optimization on a chitin column. Western blotting, ELISA, size exclusion chromatography, circular diachorism, peptide mapping, and in vitro assays were performed to compare the structural similarity and biological activity of the purified G-CSF with Neupogen™. Protein purification was confirmed by revealing a band of approximately 18.8 kDa on SDS-PAGE. Bioactivity and physicochemical assays based on the US pharmacopeia showed almost identical or acceptable ranges of similarities between recombinant G-CSF and Neopogen™. this study, biologically active soluble recombinant G-CSF was successfully produced with high purity without using chaotropic solvents through a one-step procedure. This shorter and more efficient purification procedure can reduce the cost and time of G-CSF production which makes its industrial production more cost-effective and might be also applicable for production of other biopharmaceuticals.
粒细胞集落刺激因子（G-CSF）的重组形式于 1998 年首次获得 FDA 批准，用于治疗化疗引起的中性粒细胞减少症。然而，尽管已经生产出其生物类似物，但通过使用自切割内含肽进行单一纯化步骤来降低 G-CSF 的生产成本，可以降低整体治疗成本。本研究旨在评估通过自切割内含肽介导的单一纯化步骤生产具有生物活性的重组 G-CSF 的可能性。通过 IPTG 诱导，在大肠杆菌 BL21（DE3）中表达 G-CSF，然后通过在几丁质柱上进行 pH 优化进行纯化。进行 Western blot、ELISA、分子筛、圆二色性、肽图分析和体外测定，以比较纯化的 G-CSF 与 Neupogen™的结构相似性和生物活性。SDS-PAGE 显示蛋白纯化得到了约 18.8 kDa 的条带。根据美国药典进行的生物活性和理化测定表明，重组 G-CSF 与 Neopogen™之间几乎具有相同或可接受的相似性范围。在本研究中，通过一步法成功生产出具有高纯度且不使用变性剂的高生物活性可溶性重组 G-CSF。这种更短、更有效的纯化步骤可以降低 G-CSF 生产成本，使工业生产更具成本效益，并且可能也适用于其他生物制药的生产。
