Preparation and Characterization of Site-Specific Fatty Chain-Modified Recombinant Human Granulocyte Colony Stimulating Factor

Xu-Dong Wang; Wei-Jia Yu; Jia-Hui Liu; Jie Du; Kang-Nan Chen; Qin-Qin Hu; Wen-Long Sun; Guo-Qing Ying

doi:10.3389/fbioe.2022.923059

Preparation and Characterization of Site-Specific Fatty Chain-Modified Recombinant Human Granulocyte Colony Stimulating Factor

Front Bioeng Biotechnol. 2022 May 23:10:923059. doi: 10.3389/fbioe.2022.923059. eCollection 2022.

Authors

Xu-Dong Wang¹, Wei-Jia Yu¹, Jia-Hui Liu¹, Jie Du¹, Kang-Nan Chen¹, Qin-Qin Hu¹, Wen-Long Sun², Guo-Qing Ying¹

Affiliations

¹ College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, China.
² Institute of Biomedical Research, School of Life Sciences, Shandong University of Technology, Zibo, China.

Abstract

The clinical use of recombinant human granulocyte colony-stimulating factor (rhG-CSF) is limited by its short serum half-life. In this study, a long-acting strategy for site-specific modification of rhG-CSF with 1-pentadecyl-1H-pyrrole-2,5-dione (C15 fatty chain-maleimide, C15-MAL) was studied in mixed DMSO-aqueous solutions. The factors influencing the conjugation reaction were investigated and optimized, and a high yield of the desired product (C15-rhG-CSF) was achieved. Subsequently, C15-rhG-CSF product was efficiently purified using preparative liquid chromatography, and further characterized. Circular dichroism spectroscopy analysis showed that the secondary structure of C15-rhG-CSF had no significant difference from unmodified rhG-CSF. C15-rhG-CSF retained 87.2% of in vitro bioactivity of unmodified rhG-CSF. The pharmacokinetic study showed that the serum half-life of C15-rhG-CSF in mice was 2.08-fold longer than that of unmodified rhG-CSF. Furthermore, C15-rhG-CSF by single-dose subcutaneous administration showed better in vivo efficacy than those of both PEG_10k-rhG-CSF by single-dose administration and rhG-CSF by multiple doses administration. This study demonstrated the potential of C15-rhG-CSF being developed into a novel drug candidate as well as an efficient process for the development of long-acting protein and peptide drugs.

Keywords: fatty chain; fatty chain modification; long-acting rhG-CSF; recombinant human granulocyte colony stimulating factor; rhG-CSF; site-specific modification; thiol modification.