New Expression Method and Characterization of Recombinant Human Granulocyte Colony Stimulating Factor in a Stable Protein Formulation

Abstract

Human recombinant granulocyte colony stimulating factor (rhG-CSF) is widely used in hematology and oncology for the treatment of neutropenia, for the restoration of neutrophil production after bone marrow transplantation, for myelodysplastic syndromes, and aplastic anemia. The E. coli expression system is commonly used for fast recombinant production of rhG-CSF at a large scale. We have applied a novel autoinduction method for the batch expression of rhG-CSF to study whether this new system would increase cell mass and target-protein yield compared to conventional E. coli cell culture and induction with isopropyl β-D-thiogalactopyranoside (IPTG). We could demonstrate 3-fold higher culture densities and a 5-fold higher protein yield compared to IPTG induction without the need to monitor cell growth in a shortened 24 h expression procedure. rhG-CSF expressed in autoinduction media was successfully extracted from E. coli inclusion bodies and refolded by dialysis. After size exclusion chromatography (SEC) purification, rhG-CSF showed similar conformation, biological activity and aggregation profile compared to the commercially available biosimilar TEVAgrastim(®) (TEVA Pharma AG). Expression by autoinduction is suggested as a cost- and time-effective method for rhG-CSF production.

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

“…This method without inducers such as IPTG is established on a buffered medium that contains glucose as carbon sources. In this study, the expression of rhG-CSF by temperature shifts allowed over 10-fold higher cell densities when compared to that under IPTG induction [22,32]. However, this was slightly lower than that of Khalilzadeh et al obtained by 64g/L [34].…”

“…pET expression system has been extensively used for the production of rhG-CSF in E. coli [26,31,32]. Meanwhile, because we have already accumulated technology for the efficient expression system of a target gene, using fed-batch culture [23,24], we chose pPT expression system containing the growth rate-dependent rrnB P2 promoter instead of pET system, [13,23,33].…”

Optimizing Granulocyte Colony-Stimulating Factor Transcript for Enhanced Expression in Escherichia coli

“…Several studies have earlier utilized different methodologies for improving the yield of G-CSF protein. Most of the studies have focused on modification of media composition, IPTG use (Vanz et al, 2008;Boubeva et al, 2012), ethanol utilization (Mishra et al, 2020), optimization of fermentation (Kim et al, 2014), or subsequent purification strategy/methodology (Wang et al, 2008;Li et al, 2012;Vemula et al, 2015) for improving the yields of G-CSF protein. Codon optimization of G-CSF for improved expression has been extensively used for expression in yeast (Maity et al, 2016) and E. coli (Gomes et al, 2012).…”

Development of Autonomously Replicating Viral Rna to Express the Recombinant Human Granulocyte Colony-Stimulating Factor

“…Industrially produced G-CSF is widely used for treatment of immunosuppressive conditions and neutropenia (low levels of neutrophils) and for restoring hematopoiesis after chemotherapy or radiotherapy. For hospitalized patients recovering from surgery or chemotherapy, the use of G-CSF is extremely important to prevent bacterial infections (Triozzi et al, 2012;Boubeva et al, 2012). In the US, sales of G-CSF (all brands) in 2010 amounted to 605 million dollars.…”

“…In the US, sales of G-CSF (all brands) in 2010 amounted to 605 million dollars. At present, most brands of G-CSF are produced in bacteria (Triozzi et al, 2012;Boubeva et al, 2012;Li et al, 2012). Production of G-CSF in cultured mammalian cells provides the product with better pharmacological properties.…”