Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia
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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
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 Fara A. P. Eguia 1,2 & Daniele E. Mascarelli 1 & Eneas Carvalho 3 & Gretel R. Rodríguez 4 & Edson Makiyama 5 & Primavera Borelli 5 & Celia Lieberman 1 & Paulo Lee Ho 6 & Giovana C. Barazzone 1 & Viviane M. Gonçalves 1 Received: 3 August 2020 / Revised: 3 November 2020 / Accepted: 9 November 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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 • 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. Keywords G-CSF . Bioreactor cultivation . Inclusion bodies . Refolding . Purification . Recovery
* Viviane M. Gonçalves [email protected] 1
Laboratório de Desenvolvimento de Vacinas, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, SP 05503-900, Brazil
2
Programa Interunidades em Biotecnologia, Universidade de São Paulo, São Paulo, SP, Brazil
3
Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brazil
4
Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil
5
Laboratório de Hematologia, Faculdade de Ciências Farmacêuticas, Universidade de
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