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.

中文翻译：

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重组人粒细胞集落刺激因子 N 端脂肪酸链的位点选择性缀合

重组人粒细胞集落刺激因子(rhG-CSF)因其血清半衰期短而限制其临床应用。在此，用 PAL-PEG 对 rhG-CSF N 末端进行位点选择性修饰3-Ph-CHO 用于开发长效 rhG-CSF。 PAL-PEG 修饰 rhG-CSF 的优化条件3-Ph-CHO 为：3% (w/v) Tween 20 和 30 mM NaCNBH 的反应溶剂系统3乙酸盐缓冲液（20 mmol/L，pH 5.0）中，PAL-PEG 摩尔比3-Ph-CHO与rhG-CSF的比例为6:1，温度为20°C，反应时间为12小时，从而获得PAL-PEG3-Ph-rhG-CSF产物收率为70.8%。反应混合物通过制备型液相色谱纯化，得到单一修饰产物PAL-PEG3-Ph-rhG-CSF，HPLC纯度超过95%。 PAL-PEG的分子量3MALDI-TOF-MS检测-Ph-rhG-CSF为19297 Da，与理论值一致。圆二色性分析表明，与未修饰的rhG-CSF相比，其二级结构没有显着变化。 PAL-聚乙二醇3-Ph-rhG-CSF 保留了 82.0%体外未修饰的 rhG-CSF 的生物活性。药代动力学分析表明 PAL-PEG 的血清半衰期3-Ph-rhG-CSF在小鼠中为7.404±0.777小时，比未修饰的rhG-CSF长4.08倍。此外，单次皮下注射 PAL-PEG3-Ph-rhG-CSF 具有可比性体内多次剂量的 rhG-CSF 的功效。这项研究证明了开发长效 rhG-CSF 候选药物的有效策略。