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Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli

  • Biotechnological products and process engineering
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Abstract

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.

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Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (No. 81273576 to Lu H., 81473127 to Zhu J.), the Science and Technology Commission of Shanghai Municipality (No. 15431907000 and 15DZ0503700 to Zhu J.), and the Medical and Engineering Cross Research Foundation of Shanghai Jiao Tong University (No. YM2013MS54 and YG2016QN27 to Lu H.). We would also like to thank the Biological Resources Branch (BRB) Preclinical Repository at the National Cancer Institute (Frederick, MD, USA), for supplying rhIL-15 reference.

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Authors and Affiliations

  1. Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China

    Huanhuan Chen, Ninghuan Li, Siwei Shi, Chencen Zhu, Han Luo, Junsheng Chen, Lei Zhang, Menglin Zhao, Huili Lu & Jianwei Zhu

  2. Jecho Laboratories, Inc., Frederick, MD, 21704, USA

    Yueqing Xie, Hua Jiang, Cedric Cagliero & Jianwei Zhu

  3. Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA

    Xiaoyi Yang

  4. Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai, 200240, China

    Lei Feng

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  1. Huanhuan Chen
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  14. Huili Lu
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Contributions

Huanhuan Chen, Ninghuan Li, Siwei Shi, Chencen Zhu, Han Luo, Lei Zhang, Junsheng Chen, Menglin Zhao, Lei Feng, and Huili Lu designed and performed the experiments. Yueqing Xie, Hua Jiang, Xiaoyi Yang, Cedric Cagliero, Huili Lu, and Jianwei Zhu analyzed the data and wrote the manuscript.

Corresponding authors

Correspondence to Huili Lu or Jianwei Zhu.

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This article does not contain any studies with human participants. All animal studies were evaluated and approved by the Animal Care and Use Committee of Shanghai Jiao Tong University, prior to the commencement of any experiments.

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Chen, H., Li, N., Xie, Y. et al. Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli . Appl Microbiol Biotechnol 101, 5267–5278 (2017). https://doi.org/10.1007/s00253-017-8265-x

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