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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium

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Author(s):
Eguia, Fara A. P. [1, 2] ; Mascarelli, Daniele E. [1] ; Carvalho, Eneas [3] ; Rodriguez, Gretel R. [4] ; Makiyama, Edson [5] ; Borelli, Primavera [5] ; Lieberman, Celia [1] ; Ho, Paulo Lee [6] ; Barazzone, Giovana C. [1] ; Goncalves, Viviane M. [1]
Total Authors: 10
Affiliation:
[1] Inst Butantan, Lab Desenvolvimento Vacinas, Ave Vital Brasil 1500, BR-05503900 Sao Paulo, SP - Brazil
[2] Univ Sao Paulo, Programa Interunidades Biotecnol, Sao Paulo, SP - Brazil
[3] Inst Butantan, Lab Bacteriol, Sao Paulo, SP - Brazil
[4] Univ Sao Paulo, Inst Ciencias Biomed, Dept Imunol, Sao Paulo, SP - Brazil
[5] Univ Sao Paulo, Lab Hematol, Fac Ciencias Farmaceut, Sao Paulo, SP - Brazil
[6] Inst Butantan, Ctr BioInd, Sao Paulo, SP - Brazil
Total Affiliations: 6
Document type: Journal article
Source: Applied Microbiology and Biotechnology; v. 105, n. 1, p. 169-183, JAN 2021.
Web of Science Citations: 0
Abstract

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 center dot Few papers report the final recovery of the purification process from inclusion bodies.center dot The process developed led to high purity and reasonable recovery compared to literature.center dot Nartograstim biological activity was demonstrated in mice using a neutropenia model. (AU)

FAPESP's process: 18/10384-4 - Synthesis and immunological evaluation of relevant bioconjugates to human health
Grantee:Giovana Cappio Barazzone
Support Opportunities: Regular Research Grants
FAPESP's process: 16/50413-8 - Pulmonary delivery of a targeted mucosal nanocarrier vaccine for pneumonia
Grantee:Viviane Maimoni Gonçalves
Support Opportunities: Regular Research Grants