Chen Huanhuan, Li Ninghuan, Xie Yueqing, Jiang Hua, Yang Xiaoyi, Cagliero Cedric, Shi Siwei, Zhu Chencen, Luo Han, Chen Junsheng, Zhang Lei, Zhao Menglin, Feng Lei, Lu Huili, Zhu Jianwei
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
Jecho Laboratories, Inc., Frederick, MD, 21704, USA.
Appl Microbiol Biotechnol. 2017 Jul;101(13):5267-5278. doi: 10.1007/s00253-017-8265-x. Epub 2017 Apr 8.
It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.
据文献记载，通过尺寸排阻色谱法（SEC）从大肠杆菌中纯化包涵体可能有利于后续重组蛋白的复性和回收。然而，上样量和柱成本高昂限制了其在生物制药蛋白大规模生产中的应用。我们报道了一种新方法，即在变性条件下使用聚乙二醇（PEG）沉淀来替代SEC，以快速纯化含有重组治疗性蛋白的包涵体。以重组人白细胞介素15（rhIL-15）为例，将rhIL-15的包涵体溶解于7M盐酸胍中，通过添加PEG 6000使rhIL-15沉淀。发现最终浓度为5%（w/v）的PEG 6000最适合沉淀目标蛋白并提高回收率和纯度。与先前报道的S-200尺寸排阻纯化方法相比，PEG沉淀更易于放大规模，且实现了相同的蛋白产量和产品质量。PEG沉淀还将生产时间缩短了约50%，并降低了95%的材料成本。经过复性和进一步纯化后，rhIL-15产品纯度很高，并且与rhIL-15参考标准品具有相当的生物活性。我们的研究表明，在变性条件下对包涵体进行PEG沉淀作为一种从大肠杆菌蛋白表达系统生产生物制药的工艺具有巨大潜力。
