Neurturin is a potent neurotrophic factor that has been investigated as a potential therapeutic agent for the treatment of neurodegenerative diseases, including Parkinson's disease, and, more recently, for the treatment of type II diabetes. However, purification of neurturin for clinical applications has been hampered by its low solubility in aqueous solutions. Here we describe the development of a scalable manufacturing process for recombinant neurturin from E. coli. inclusion bodies. Neurturin was refolded from solubilized inclusion bodies by fed-batch dilution refolding with a titer of 90 mg per liter refold and a refold yield of 89%. A two-step purification process using cation exchange and hydrophobic interaction chromatography, followed by formulation using tangential flow filtration resulted in an overall process yield of about 56 mg purified neurturin per liter refold. Solubility of neurturin during the purification process was maintained by the addition of 15% (w/v) glycerol to all buffers. For clinical applications and parenteral administration glycerol was replaced by 15% (w/v) sulfobutyl ether-beta-cyclodextrin (i.e. Captisol) in the drug substance formulation buffer. The final purified product had low or undetectable levels of product-related impurities and concentrations of process-related contaminants such as host cell proteins, host cell DNA, endotoxins and Triton X-100 were reduced more than 10,000-fold or below the limit of detection. Bioactivity of purified recombinant neurturin was demonstrated in a cell-based assay by activation of the MAPK signaling pathway.

中文翻译：

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从大肠杆菌包涵体中重折叠和纯化cGMP级重组人神经营养素。

神经氨酸是一种有效的神经营养因子，已被研究作为治疗神经退行性疾病（包括帕金森氏病）以及最近用于治疗II型糖尿病的潜在治疗剂。但是，由于其在水溶液中的低溶解度，阻碍了神经营养素在临床上的纯化。在这里，我们描述了从大肠杆菌中重组神经营养蛋白的可扩展制造工艺的发展。包涵体。通过补料分批稀释重折叠从溶解的包涵体中重折叠神经氨酸，重均滴度为90 mg / L，重折叠产率为89％。使用阳离子交换和疏水相互作用色谱的两步纯化过程，然后通过使用切向流过滤进行配制，每升复性的总工艺产量约为56 mg纯化的神经营养素。通过在所有缓冲液中添加15％（w / v）甘油来维持纯化过程中神经营养素的溶解度。对于临床应用和肠胃外给药，在药物制剂缓冲液中将甘油替换为15％（w / v）的磺基丁基醚-β-环糊精（即Captisol）。最终纯化的产品具有低水平或无法检测到的产品相关杂质，并且与过程相关的污染物（例如宿主细胞蛋白，宿主细胞DNA，内毒素和Triton X-100）的浓度降低了10,000倍以上或低于检测极限。