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Key Process Technologies for Industrial Production of Three Therapeutic Proteins with Recombinant Escherichia Coli

Author: GuoWangMing
Tutor: ZuoPeiLin; XuZhiNan
School: Zhejiang University
Course: Biochemical Engineering
Keywords: Erythropoietin mimetic peptide1(EMP1) reeombinant human bone morphogeneticprotein-2(rhBMP-2) recombinant human granulocyte colony stimulating factor(rhG-CSF) soluble expression in vitro refolding hydrophobic interaction chromatography(HIC) prodtictstability
CLC: TQ464.7
Type: PhD thesis
Year: 2012
Downloads: 52
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Abstract


In the present work, several key technologies were developed to produce therapeutic proteins in recombinant Escherichia coli, such as high-efficient expression technology, protein refolding of inclusion body, separation and preparation of protein at pilot scale, and protein stability improvement, etc. Three kinds of recombinant proteins (EMP1, rhBMP-2and rhG-CSF) in different development stages were targeted in order to solve their specific and urgent problems at lab, at pilot plant and in mature markets respectively.Erythropoietin mimetic peptide1(EMP1) is a20-amino acid cyclic peptide which are not found in the primary sequence of EPO, but acts as full agonists and can also stimulate erythropoiesis in mice. In this work, two repeats of EMP1linked by flexible peptide with different length were expressed in E. coli as fusion proteins with thioredoxin or DsbA. Under optimal temperature and IPTG concentration, the highest soluble expression level of fusion proteins was0.83g/L. The soluble part of fusion proteins could be purified by metal chelating affinity chromatography and hydrophobic interaction chromatography with enhanced purity, and the overall process yield was about6.3-7.8%. The two repeat peptide of EMP1was released by enterokinase cleavage of fusion proteins and obtained by followed reversed phase chromatography. The biological activities of the resulted products, EMP1-EMP1(S) and EMP1-EMP1(L) were10-fold greater than monomeric EMP1, the length of flexible linker has negligible effect on the biological activity.The mature bone morphogenetic protein-2(BMP-2) consists of114amino acids and acts as a bioactive disulfide-linked homodimer, or a heterodimer linked with BMP-7. Due to its high hydrophobity, in vitro refolding of insoluble rhBMP-2was low-efficient, and subsequent purification of soluble rhBMP-2was still difficult and expensive due to the employment of heparin sepharose affinity chromatography. In order to solve the above problems, the post-expression processing procedure including inclusion bodies solubilization and in vitro refolding in different solutions were systematically investigated. The results indicated that Gdn-HCl solution (buffer A) was relatively more effective in solubilizing inclusion body, resulting in higher protein recovery and the optimal refolding solution was formulated. Under the optimized conditions, an effective and economic refolding process by simple dilution was established and successfully scaled up with yield higher than70%. The refolding mixture could be subjected to subsequent purification directly without any adjustment or pre-treatment. Then a novel purification process to produce active rhBMP-2homodimer from refolding mixture by hydrophobic interaction chromatography (HIC) was proposed. N, N-dimethylformamide (DMF) was adopted as additive and its concentration was optimized. It was found that5%(V/V) DMF can enhance the resolution of rhBMP-2homodimer most effectively. The rhBMP-2homodimer was purified to homogeneity through two HIC separations at different salt content, the purified rhBMP-2homodimer with high purity exhibited equivalent alkaline phosphatase (ALP) activity to commercial rhBMP-2produced from Chinese hamster ovary cell (CHO) and higher ectopic bone formation capacity. The average process yield was80mg rhBMP-2homodimer per g of wet inclusion bodies. DMF could be totally removed from the purified protein without any detectable trace after dialysis and lyophilization of final product. This efficient refolding and purification procedure was successfully scaled up in the pilot pharmaceutical plant. In the mean time, reversed phase chromatography and size exclusion chromatography were first used to measure the purity of rhBMP-2from E.coli which was validated with capillary electrophoresis and electrophoresis.The recombinant human granulocyte colony stimulating factor (rhG-CSF) is an important therapeutic protein which has been produced at large scale in biotechnology industry. How to guarantee and improve the stability and quality of final product is a practical and important issue for the manufacturers. In this work, a reversed phase chromatography using C4column was applied to determine the purity of rhG-CSF samples with better resolution. Three stages of manufactering process were evaluated to decide their effects on the stability of product. The effects of different sources of ampoules, vials and prefilled syringes on the rhG-CSF stability were compared. After the optimization of preparation condition and the adoption of novel formulation, rhG-CSF products filled in selected ampoule, vial and prefilled syringe exhibited much higher quality and stability than the traditional products.In conclusion, by targeting at three different recombinant proteins with E. coli, the present work not only developed several key technologies for their efficient production, but also pushed the transfer of these therapeutic proteins from research to large-scale production with enhanced productivity and improved stability.

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CLC: > Industrial Technology > Chemical Industry > Pharmaceutical chemical industry > Drug production of biological products > Amino acids,peptides,proteins
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