Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor
The clinical application of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) is restricted by its short serum half-life. Herein, site-selective modification of the N-terminus of rhG-CSF with PAL-PEG3-Ph-CHO was used to develop a long-acting rhG-CSF. The optimized conditions for...
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Frontiers Media S.A.
2024-03-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2024.1360506/full |
| _version_ | 1797253772164988928 |
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| author | Xu-Dong Wang Zhi-Hao Su Jie Du Wei-Jia Yu Wen-Long Sun |
| author_facet | Xu-Dong Wang Zhi-Hao Su Jie Du Wei-Jia Yu Wen-Long Sun |
| author_sort | Xu-Dong Wang |
| collection | DOAJ |
| description | The clinical application of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) is restricted by its short serum half-life. Herein, site-selective modification of the N-terminus of rhG-CSF with PAL-PEG3-Ph-CHO was used to develop a long-acting rhG-CSF. The optimized conditions for rhG-CSF modification with PAL-PEG3-Ph-CHO were: reaction solvent system of 3% (w/v) Tween 20 and 30 mM NaCNBH3 in acetate buffer (20 mmol/L, pH 5.0), molar ratio of PAL-PEG3-Ph-CHO to rhG-CSF of 6:1, temperature of 20°C, and reaction time of 12 h, consequently, achieving a PAL-PEG3-Ph-rhG-CSF product yield of 70.8%. The reaction mixture was purified via preparative liquid chromatography, yielding the single-modified product PAL-PEG3-Ph-rhG-CSF with a HPLC purity exceeding 95%. The molecular weight of PAL-PEG3-Ph-rhG-CSF was 19297 Da by MALDI-TOF-MS, which was consistent with the theoretical value. The circular dichroism analysis revealed no significant change in its secondary structure compared to unmodified rhG-CSF. The PAL-PEG3-Ph-rhG-CSF retained 82.0% of the in vitro biological activity of unmodified rhG-CSF. The pharmacokinetic analyses showed that the serum half-life of PAL-PEG3-Ph-rhG-CSF was 7.404 ± 0.777 h in mice, 4.08 times longer than unmodified rhG-CSF. Additionally, a single subcutaneous dose of PAL-PEG3-Ph-rhG-CSF presented comparable in vivo efficacy to multiple doses of rhG-CSF. This study demonstrated an efficacious strategy for developing long-acting rhG-CSF drug candidates. |
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| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-24T21:39:22Z |
| publishDate | 2024-03-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-5d9f6ef166334177ba7a38d80d4868b02024-03-21T10:47:21ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852024-03-011210.3389/fbioe.2024.13605061360506Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factorXu-Dong Wang0Zhi-Hao Su1Jie Du2Wei-Jia Yu3Wen-Long Sun4College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, ChinaCollege of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, ChinaCollege of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, ChinaCollege of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, ChinaInstitute of Biomedical Research, School of Life Sciences, Shandong University of Technology, Zibo, ChinaThe clinical application of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) is restricted by its short serum half-life. Herein, site-selective modification of the N-terminus of rhG-CSF with PAL-PEG3-Ph-CHO was used to develop a long-acting rhG-CSF. The optimized conditions for rhG-CSF modification with PAL-PEG3-Ph-CHO were: reaction solvent system of 3% (w/v) Tween 20 and 30 mM NaCNBH3 in acetate buffer (20 mmol/L, pH 5.0), molar ratio of PAL-PEG3-Ph-CHO to rhG-CSF of 6:1, temperature of 20°C, and reaction time of 12 h, consequently, achieving a PAL-PEG3-Ph-rhG-CSF product yield of 70.8%. The reaction mixture was purified via preparative liquid chromatography, yielding the single-modified product PAL-PEG3-Ph-rhG-CSF with a HPLC purity exceeding 95%. The molecular weight of PAL-PEG3-Ph-rhG-CSF was 19297 Da by MALDI-TOF-MS, which was consistent with the theoretical value. The circular dichroism analysis revealed no significant change in its secondary structure compared to unmodified rhG-CSF. The PAL-PEG3-Ph-rhG-CSF retained 82.0% of the in vitro biological activity of unmodified rhG-CSF. The pharmacokinetic analyses showed that the serum half-life of PAL-PEG3-Ph-rhG-CSF was 7.404 ± 0.777 h in mice, 4.08 times longer than unmodified rhG-CSF. Additionally, a single subcutaneous dose of PAL-PEG3-Ph-rhG-CSF presented comparable in vivo efficacy to multiple doses of rhG-CSF. This study demonstrated an efficacious strategy for developing long-acting rhG-CSF drug candidates.https://www.frontiersin.org/articles/10.3389/fbioe.2024.1360506/fullrecombinant human granulocyte colony-stimulating factorrhG-CSFfatty acid chain conjugationsite-selective modificationlong-acting rhG-CSFserum half-life |
| spellingShingle | Xu-Dong Wang Zhi-Hao Su Jie Du Wei-Jia Yu Wen-Long Sun Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor Frontiers in Bioengineering and Biotechnology recombinant human granulocyte colony-stimulating factor rhG-CSF fatty acid chain conjugation site-selective modification long-acting rhG-CSF serum half-life |
| title | Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor |
| title_full | Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor |
| title_fullStr | Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor |
| title_full_unstemmed | Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor |
| title_short | Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor |
| title_sort | site selective fatty acid chain conjugation of the n terminus of the recombinant human granulocyte colony stimulating factor |
| topic | recombinant human granulocyte colony-stimulating factor rhG-CSF fatty acid chain conjugation site-selective modification long-acting rhG-CSF serum half-life |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2024.1360506/full |
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