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
Appl Microbiol Biotechnol, v. 105, p. 169-183, nov. 2021
Article
em En
| SES-SP, SESSP-IBPROD, SES-SP
| ID: bud-3345
Biblioteca responsável:
BR78.1
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.
Texto completo:
1
Coleções:
06-national
/
BR
Contexto em Saúde:
2_ODS3
Base de dados:
SES-SP
/
SESSP-IBPROD
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Ano de publicação:
2021
Tipo de documento:
Article