title: Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.
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.
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Huanhuan
authors.initials: H
authors.lastname: Chen
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Ninghuan
authors.initials: N
authors.lastname: Li
authors.affiliation: Jecho Laboratories, Inc., Frederick, MD, 21704, USA.
authors.forename: Yueqing
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Jecho Laboratories, Inc., Frederick, MD, 21704, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
authors.forename: Xiaoyi
authors.initials: X
authors.lastname: Yang
authors.affiliation: Jecho Laboratories, Inc., Frederick, MD, 21704, USA.
authors.forename: Cedric
authors.initials: C
authors.lastname: Cagliero
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Siwei
authors.initials: S
authors.lastname: Shi
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Chencen
authors.initials: C
authors.lastname: Zhu
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Han
authors.initials: H
authors.lastname: Luo
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Junsheng
authors.initials: J
authors.lastname: Chen
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Menglin
authors.initials: M
authors.lastname: Zhao
authors.affiliation: Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Feng
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China. roadeer@sjtu.edu.cn.
authors.forename: Huili
authors.initials: H
authors.lastname: Lu
authors.affiliation: Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China. jianweiz@sjtu.edu.cn.|Jecho Laboratories, Inc., Frederick, MD, 21704, USA. jianweiz@sjtu.edu.cn.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Zhu
mesh_terms: D001702:Biopharmaceutics / Q000379:methods; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D019409:Interleukin-15 / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011092:Polyethylene Glycols / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000627:therapeutic use
chemical_list: D019409:Interleukin-15; D011994:Recombinant Proteins; D011092:Polyethylene Glycols
keywords: Denature; Inclusion bodies; Interleukin-15; PEG precipitation; Therapeutic protein expression and purification