【作者】 潘红春；

【导师】 王伯初；

【作者基本信息】 重庆大学 ， 生物医学工程， 2006， 博士

【摘要】 蛋白药物的PEG修饰是第二代生物制药的重要内容,PEG修饰蛋白药物克服了天然蛋白药物的诸多不足,而被赋予了低免疫原性、高酶稳定性、长半衰期等新特性,已逐渐成为临床上广泛应用的新一代高效、低毒的治疗药物。本项目完成了PEG修饰hCH-2项目临床前的大部分基础研究工作,选择了合适的PEG修饰剂,优化并放大验证了修饰工艺和纯化工艺,获得了单链PEG-hCH-2目标产物,建立了单链PEG-hCH-2原液生产的质控方法和质量标准,探索了单链PEG-hCH-2的初步特性,为开发hCH-2的长效PEG修饰药物奠定了基础。具体内容主要包括以下几个方面:（1）利用高效液相色谱技术,建立了一种快速、方便、准确和稳定的定量检测PEG修饰反应液中微量PEG-hCH-2和hCH-2的反相高效液相色谱方法。该方法的分离基础为PEG化hCH-2疏水性的改变,色谱柱为C8反相柱（250mm×4.6mm,300?,5μm）,流动相A为水-TFA（999∶1）,流动相B为100%乙氰,流速为1.0ml/min,线性梯度洗脱和等度洗脱相结合,柱温为30℃,检测波长为280nm。在0.049-0.456mg/ml和0.112-1.796 mg/ml浓度范围内,PEG-hCH-2和hCH-2的浓度与色谱峰峰面积均呈现良好线性关系,回归方程分别为y=2.11×10⁶x-3.30×10⁴和y=1.08×10⁶x-8.45×10³,线性相关系数r分别为0.9997和1.0000,最低检测浓度分别为0.044 mg/ml和0.056 mg/ml,日内和日间RSD（n=6）分别为0.50-2.18%和0.40-2.21%。平均回收率分别为98.01% （n=3,RSD为0.72-2.62%）和97.80% （n=3,RSD为0.56-2.32%）。本方法克服了常用的SDS-PAGE、SPF、TNBS等方法的重现性、精度差以及检测时间长等不足,完全可用于工业化大生产质量控制过程中PEG-hCH-2的测定。（2）根据hCH-2本身的结构特性,选择分子量为20kD的链式琥珀酸琥珀酰亚胺酯类mPEG对hCH-2分子中赖氨酸的ε氨基和/或N末端氨基进行非定点PEG修饰。通过PEG修饰hCH-2的单因素影响研究,获得较适宜的起始pH值为8.0-8.5、摩尔比为1:5、缓冲液浓度为25-50mmol/l、hCH-2浓度为0.5mg/ml和反应时间为4h;利用L₉（3⁴）正交试验对起始pH值、hCH-2与PEG的摩尔比、hCH-2浓度和缓冲液浓度进行了优化,获得优化的起始pH值为8.5、hCH-2与PEG的摩尔比为1:7、hCH-2浓度为0.75mg/ml和缓冲液浓度为50mmol/l;保持温度25℃、反应时间4h、反应总体积0.5ml和转速200r/min,进行优化修饰条件的验证试验研究和放大试验研究,结果表明:优化的PEG修饰工艺条件具有良好的稳定性和可操作性,可使反应液中单链PEG-hCH-2含量可达0.25mg/ml,单链PEG-hCH-2修饰更多还原

【Abstract】 The conjugation of polyethylene glycol (PEG), PEGylation, is an important part of second-generation biopharmaceuticals. It has received increasing attention as a well-established technique that has the capacity to overcome several problems of native proteins for therapeutic uses, and demonstrated reduced immunogenicity, extended circulating half-life, and improved proteolysis stability for these therapeutic agents. PEGylated protein-drugs are gradually becoming new high efficiency and low toxicity therapeutic drugs and extensively applied on clinical trials. In this study, we have accomplished mostly preclinical basic researches of PEGylated hCH-2 such as the selection of eligible PEGylation reagents, the optimization and verification of technologies of PEGylation and purification by amplification experiment, the establishment of methods and requirements for quality control of mono-PEG-hCH-2 and the investigation of preliminary characteristics of mono-PEG-hCH-2, and gained the target product, mono-PEG-hCH-2. These studies have laid the solid foundation for developing the second generation prolonged-release PEGylated drugs of hCH-2. The detailed contents and conclusions are shown as follows:(1) A rapid and reliable reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination of PEG-hCH-2 and hCH-2 in the reaction mixture. PEG-hCH-2 and hCH-2 were separated by HPLC on a C4 column on the basis of the hydrophobicity difference and quantified by ultraviolet detection at 280nm. The chromatography analysis was performed on C8 (250mm×4.6mm i.d., 300?, with a particle size of 5μm) column. The elution strategy was the combination of the linear gradient and isocratic elution with the changes of the mobile phase A and B. The compositions of the mobile phase A and B were 999:1(v/v) water/TFA and 100% acetonitrile at a flow rate of 1.0ml/min. Detection was done by spectrophotometry at 280nm and the column temperature was 30±1°C.For PEG-hCH-2 and hCH-2, the standard curves were linear in the range of 0.049-0.456mg/ml and 0.112-1.796mg/ml, the regression equation were y=2.11×106x -3.30×10~4 (r~2=0.9994, n=5) and y=1.08×10~6x-8.45×10 (r~2=1.0000, n=5), the lower limit of quantitative analysis were established at 0.044mg/ml and 0.056mg/ml, the relative standard deviation (RSD) values of intra-day precision were <1.34% and <2.18% (n=6), the RSD values of inter-day precision were <1.49% and <2.21% (n=6), the RSD values of recovery experiment were <2.62% and <2.32% (n=3), the average recovery rates更多还原