Song S.,Peking Union Medical College |
Jiang W.-H.,Fanzhou Biopharmagen Corporation |
Jiang Y.-P.,Peking Union Medical College |
Jiang Y.-P.,Fanzhou Biopharmagen Corporation
Chinese Journal of Biomedical Engineering | Year: 2012
The aim of this work is to produce, refold, and purify rhG-CSFa in large scale and determine its bioactivity. Using site-direct and additional mutagenesis, a structurally modified derivative of human G-CSF termed G-CSFa was constructed and fermented in a 40 liter bio-reactor. The rhG-CSFa inclusion bodies were extracted and refolded by a serial dialysis. The refolded G-CSFa was then purified by an ion-exchange column and size-exclusive chromatography, and the purity of rhG-CSFa was then analyzed by HPLC. Purified rhG-CSFa was tested for its biological activity in vitro using a G-CSF-dependent cell (M-NFS - 60) assays. The 31.2% expressed G-CSFa protein yield was achieved and 11.36% refolding yield was obtained after optimizing the protein refolding conditions. The purity of rhG-CSFa was 99.11% after purification. In vitro studies demonstrated that rhG-CSFa induced a much higher proliferation rate of M-NFS - 60 cell lines than wild-type G-CSF at the same concentrations. The large-scale of recombinant G-CSFa in M15 strain was expressed with high yield, purity, and its biological activity and stability was confirmed. The success of large-scale production of G-CSFa facilitates the clinical application of this potential drug.
Jiang Y.,Fanzhou Biopharmagen Corporation |
Jiang W.,Fanzhou Biopharmagen Corporation |
Qiu Y.,Fanzhou Biopharmagen Corporation |
Dai W.,New York University
Journal of Hematology and Oncology | Year: 2011
Background: Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, and differentiation of neutrophilic granulocyte precursors, Recombinant G-CSF has been used for the treatment of congenital and therapy-induced neutropenia and stem cell mobilization. Due to its intrinsic instability, recombinant G-CSF needs to be excessively and/or frequently administered to patients in order to maintain a plasma concentration high enough to achieve therapeutic effects. Therefore, there is a need for the development of G-CSF derivatives that are more stable and active in vivo. Methods. Using site-direct mutagenesis and recombinant DNA technology, a structurally modified derivative of human G-CSF termed G-CSFa was obtained. G-CSFa contains alanine 17 (instead of cysteine 17 as in wild-type G-CSF) as well as four additional amino acids including methionine, arginine, glycine, and serine at the amino-terminus. Purified recombinant G-CSFa was tested for its in vitro activity using cell-based assays and in vivo activity using both murine and primate animal models. Results: In vitro studies demonstrated that G-CSFa, expressed in and purified from E. coli, induced a much higher proliferation rate than that of wild-type G-CSF at the same concentrations. In vivo studies showed that G-CSFa significantly increased the number of peripheral blood leukocytes in cesium-137 irradiated mice or monkeys with neutropenia after administration of clyclophosphamide. In addition, G-CSFa increased neutrophil counts to a higher level in monkeys with a concomitant slower declining rate than that of G-CSF, indicating a longer half-life of G-CSFa. Bone marrow smear analysis also confirmed that G-CSFa was more potent than G-CSF in the induction of granulopoiesis in bone marrows of myelo-suppressed monkeys. Conclusion: G-CSFa, a structurally modified form of G-CSF, is more potent in stimulating proliferation and differentiation of myeloid cells of the granulocytic lineage than the wild-type counterpart both in vitro and in vivo. G-CSFa can be explored for the development of a new generation of recombinant therapeutic drug for leukopenia. © 2011 Jiang et al; licensee BioMed Central Ltd.