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

• Published in: Applied microbiology and biotechnology 2021, Vol.105 (1), p.169-183
• Main Authors: Eguia, Fara A. P., Mascarelli, Daniele E., Carvalho, Eneas, Rodríguez, Gretel R., Makiyama, Edson, Borelli, Primavera, Lieberman, Celia, Ho, Paulo Lee, Barazzone, Giovana C., Gonçalves, Viviane M.
• Format: Article
• Language: English
• Subjects: Animals; Anion exchange; Anion exchanging; Anti-Bacterial Agents; Antibiotics; Biological activity; Biomedical and Life Sciences; Bioreactors; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Biotechnology industry; Cation exchange; Cation exchanging; Cell culture; Centrifugation; Chemical properties; Chromatography; Colonies; Colony-stimulating factor; Contaminants; Control; Culture media (Biology); Cytokines; Dialysis; Dilution; E coli; Escherichia coli; Escherichia coli - genetics; Gel electrophoresis; Granulocyte colony-stimulating factor; Granulocyte Colony-Stimulating Factor - biosynthesis; Granulocytes; Humans; Inclusion bodies; Leukocytes (granulocytic); Life Sciences; Lysis; Mice; Microbial Genetics and Genomics; Microbiology; Mutation; Neutropenia; Production management; Production processes; Purification; Purity; Recombinant Proteins - biosynthesis; Recovery; Sodium lauryl sulfate; Solubilization; Structural integrity
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-020-11014-y

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