Eguia Fara A P, Mascarelli Daniele E, Carvalho Eneas, Rodríguez Gretel R, Makiyama Edson, Borelli Primavera, Lieberman Celia, Ho Paulo Lee, Barazzone Giovana C, Gonçalves Viviane M
Laboratório de Desenvolvimento de Vacinas, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, SP, 05503-900, Brazil.
Programa Interunidades em Biotecnologia, Universidade de São Paulo, São Paulo, SP, Brazil.
Appl Microbiol Biotechnol. 2021 Jan;105(1):169-183. doi: 10.1007/s00253-020-11014-y. Epub 2020 Nov 17.
The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity. KEY POINTS: • Few papers report the final recovery of the purification process from inclusion bodies. • The process developed led to high purity and reasonable recovery compared to literature. • Nartograstim biological activity was demonstrated in mice using a neutropenia model.
粒细胞集落刺激因子（G-CSF）是一种造血细胞因子，在治疗中性粒细胞减少症方面具有重要的临床应用。那托司亭是重组人G-CSF变体。那托司亭在大肠杆菌中作为包涵体（IB）产生，由于其突变，与野生型人G-CSF相比具有更高的稳定性和生物活性。我们在10-L生物反应器中使用自诱导或化学限定培养基开发了那托司亭的生产工艺。细胞裂解、离心、包涵体洗涤和包涵体溶解后，评估了以下三种复性方法：透析过滤、透析以及在两种复性缓冲液中直接稀释。蛋白质免疫印迹法和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳证实两种培养物中18.8-kDa条带均为那托司亭。自诱导培养基产量为1.17 g/L，化学限定培养基产量为0.95 g/L。稀释法复性率最高（99%）。复性后，在将pH值调至5.2的过程中许多杂质蛋白沉淀，纯度从50%提高到78%。将上清液应用于阳离子交换色谱（CEC）后，那托司亭回收率较低，纯度为87%。然而，当将复性溶液应用于阴离子交换色谱然后再进行CEC时，获得了91%-98%的纯度和2.2%的回收率。本研究中描述的纯化工艺可用于获得具有高纯度、结构完整性和预期生物活性的那托司亭。关键点：• 很少有论文报道从包涵体中纯化工艺的最终回收率。• 与文献相比，所开发的工艺具有高纯度和合理的回收率。• 使用中性粒细胞减少症模型在小鼠中证明了那托司亭的生物活性。
