Abstract
Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins.
Highlights
Biosimilars are biologic medical products that are made by living organisms using recombinant DNA technology [1,2]
Recombinant (r) hG-CSF produced by Escherichia coli (E. coli) has similar biological activity to that of the native human protein, but differs in that it contains an Nterminal methionine residue and is not glycosylated [12]
To gain rhG-CSF of high quality, isoforms that are detected in reverse phase (RP)-high-performance liquid chromatography (HPLC) step during rhG-CSF purification are needed to separate through additional methods [18,19], such as ion exchange chromatography [20], hydrophobic interaction chromatography [21], and size exclusion chromatography (SEC) [22]
Summary
Biosimilars are biologic medical products that are made by living organisms using recombinant DNA technology [1,2] One of these biosimilars is granulocyte-colony stimulating factor (GCSF). Recombinant (r) hG-CSF produced by Escherichia coli (E. coli) has similar biological activity to that of the native human protein, but differs in that it contains an Nterminal methionine residue and is not glycosylated [12]. The expression of rhG-CSF in E. coli frequently results in the formation of insoluble aggregates, which are called inclusion bodies (IBs). These aggregates are solubilized in a buffer containing chaotropic agents such as urea [13,14] or guanidine [15]. To gain rhG-CSF of high quality, isoforms that are detected in reverse phase (RP)-high-performance liquid chromatography (HPLC) step during rhG-CSF purification are needed to separate through additional methods [18,19], such as ion exchange chromatography [20], hydrophobic interaction chromatography [21], and size exclusion chromatography (SEC) [22]
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