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G-CSF 的定点 PEG 化修饰

G-CSF 的定点 PEG 化修饰. 报告人：南京大学 谭小军 二〇〇八年十一月. G-CSF ( 粒细胞集落刺激因子 ). G-CSF 生理功能 促进造血干细胞向中性粒细胞增殖、分化 动员成熟中性粒细胞从骨髓进入外周 G-CSF 的应用 抗肿瘤化疗、放疗以及骨髓移植手术后重要的辅助药物 G-CSF 的应用现存问题 体内半衰期短 易被酶水解和肾脏清除 多次注射引起不良反应. PEG( 聚乙二醇 ). 具有良好的生物相容性 无毒性 无抗原性 修饰后的蛋白和多肽类药物的药代动力学和药效学性质能够得到改善

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G-CSF 的定点 PEG 化修饰

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  1. G-CSF的定点PEG化修饰 报告人：南京大学 谭小军 二〇〇八年十一月

  2. G-CSF (粒细胞集落刺激因子) • G-CSF生理功能 促进造血干细胞向中性粒细胞增殖、分化 动员成熟中性粒细胞从骨髓进入外周 • G-CSF的应用 抗肿瘤化疗、放疗以及骨髓移植手术后重要的辅助药物 • G-CSF的应用现存问题 体内半衰期短 易被酶水解和肾脏清除 多次注射引起不良反应

  3. PEG(聚乙二醇) • 具有良好的生物相容性 • 无毒性 • 无抗原性 • 修饰后的蛋白和多肽类药物的药代动力学和药效学性质能够得到改善 • 在国内外已经得到比较多的应用

  4. 项目概要 • 概括和总结现有蛋白质和多肽类药物的化学修饰方法； • 选用合适的PEG对重组人粒细胞集落刺激因子（rhG-CSF）进行选择性的定点修饰 • 改善rhG-CSF的各项性能，例如延长其在人体内的半衰期，降低其免疫原性而尽量不影响其生物活性，提高其临床应用价值

  5. mPEG-PAL（单甲氧PEG丙醛） 最佳修饰反应条件的摸索！ mPEG-PAL一端的甲氧基相对惰性 G-CSF的Ｎ端氨基与侧链氨基的等电点差 =〉mPEG-PAL另一端醛基在偏酸性条件下特异性地与蛋白质N端氨基发生反应

  6. 正交实验确定最佳反应条件 The Optimal Conditions: • Time：48 hours • pH：5.0 • Temperature：20 ℃ • Molar ratio PEG/rhG-CSF：10:1 Figure 1. SDS-PAGE analysis of the orthogonal samples stained with Coomassie Blue. Lane 1-9: nine samples in the orthogonal tests corresponding to the test number.

  7. 最佳条件反应结果 A B Figure 2. Much mono-PEGylated rhG-CSF was obtained under the optimal conditions (48 h, pH5.0, molar ratio of mPEG to rhG-CSF 10: 1; 20 ℃), more than under the conditions of Test 5 or 7 in the orthogonal tests.

  8. 放大反应分离纯化 Figure 3. The profile of cation exchange fast protein liquid chromatogram (ÄCTA FPLC) of the PEGylated rhG-CSF. The average molecular weights of mPEG-PAL used are (A) 20 kDa and (B) 5 kDa, respectively.

  9. PEG(5 kD)-G-CSF纯品 Figure 4. The FPLC profile of PEG(5 kD)-G-CSF after purification.

  10. PEG(20 kD)-G-CSF纯品 Figure 5. The FPLC profile of PEG(20 kD)-G-CSF after purification.

  11. Mono-PEG-G-CSF纯品 Figure 6. SDS-PAGE analysis of the purified mono-PEG-G-CSF stained with Coomassie brilliant Blue. LANE A: Marker LANE B: Mono-PEG(5 kDa)-G-CSF LANE C: Mono-PEG(20 kDa)-G-CSF LANE D: G-CSF

  12. Mono-PEG-G-CSF体外活性 Figure 7. PEGylation Retained the Activity of rhG-CSF in vitro. (Although The relative activities of PEG(5kD)-G-CSF and PEG(20kD)-G-CSF was down-regulated to about 67% and 40%, respectively, of that of unmodified rhG-CSF.)

  13. Mono-PEG-G-CSF热稳定性 Figure 8. PEGylation increased the thermal stability of rhG-CSF.Left: pH 7.0 in 37 ℃;Right: pH 7.0, at 60 ℃

  14. Mono-PEG-G-CSF pH稳定性 Figure 9. PEGylated rhG-CSF had distinctly higher acid-base stability than unmodified rhG-CSF. Left: pH 2.2; Right: pH 11.0.

  15. Mono-PEG-G-CSF体外稳定性 Figure 10. PEGylation significantly enhanced proteolytic stability of rhG-CSF.Left:human serum; Right: trypsin.

  16. Mono-PEG-G-CSF的体内活性 Figure 11. The activity of mono-PEG-G-CSF to increase leukocyte numbers in the peripheral blood of Kunming mice is significantly higher than that of unmodified rhG-CSF.

  17. 结论 • PEG化修饰表面上降低了rhG-CSF的体外比活 • 但是显著增加了rhG-CSF的体内外稳定性，起到体内缓释作用，大大延长了体内作用时间，显著提高了其体内活性 巨大的临床应用前景！

  18. 致谢 • 感谢导师陈钧辉教授的悉心指导 • 感谢聂永军老师、沈杰师兄的热情帮助 • 感谢苏州中凯生物公司免费提供rh-G-CSF样品 • 感谢南京大学创新计划项目管理办公室的辛勤管理与大力支持 谢谢各位专家与同学！

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