荧光胺衍生化法测定3种聚乙二醇化重组人生长激素的平均修饰率  

A fluorometric assay to determine the average modification rate of three kinds of PEG-rhGHs

作　　者：李晶[1] 何辉[2] 程速远[1] 梁成罡[1] 

机构地区：[1]中国食品药品检定研究院,北京100050 [2]武汉大学,武汉430072

出　　处：《药物分析杂志》2014年第8期1368-1373,共6页Chinese Journal of Pharmaceutical Analysis

基　　金：重大新药创制"科技部重大专项课题"生物技术药物质量标准和质量控制技术平台(课题编号:2012ZX09304010)资助;中检院学科带头人基金"PEG修饰药物质控关键技术与方法的标准化研究"(课题编号:2011X1)资助

摘　　要：目的:通过对游离氨基进行荧光胺衍生化,测定3种不同结构聚乙二醇化重组人生长激素(PEG-rhGH)的平均修饰率。方法:采用基质辅助激光解吸附飞行时间质谱(MALDI-TOF-MS)测定PEG-rhGH的相对分子质量,求得理论平均修饰率。通过激发波长与发射波长的选取、正交试验、游离PEG干扰试验、反应稳定性考察等方法学考察,建立荧光胺衍生法测定PEG-rhGH平均修饰率。结果:从质谱法可知PEG-rhGH的理论修饰度为10%。方法学考察结果显示,激发波长与发射波长分别为390 nm与480 nm,溶液pH对试验结果的影响最大,低浓度的PEG对试验结果无干扰,在反应开始8 min之内荧光强度稳定。荧光胺法测得3种不同结构PEG-rhGH的修饰度分别为8.8%、9.8%及9.5%,与理论值接近。结论:荧光胺法可作为测定PEG-rhGH平均修饰率的方法,3种PEG化重组人生长激素的平均修饰率为10%。Objective： To determine the average rate of 3 different PEG modifications of rhGH by deriving free amines with fluorescamine. Methods： MALDI- TOF mass spectrometry was used to detect relative molecular mass of PEG,rhGH and the conjugations to calculate the theoretical rate of PEG modification. A fluorometric method was established by optimization and validation through selection of the excitation and emission wavelength,an orthogonal experiment,an interference test of free PEG and the stability study of the reaction. Results： The theoretical value of modification rate was 10%,which was validated by MALDI- TOF mass. Validation results were as below： The excitation and emission wavelengths were 390 nm and 480 nm,respectively. The pH value of solution had the greatest effect on the result. PEG with low content had no effect on the result. The relative fluorescence unit was stable within the first 8 minutes. The actual modification rates of three different PEG- rhGHs were 8. 8%,9. 8% and 9. 5%,respectively,which were close to the theoretical value. Conclusion： The fluorometric derivatization method is suitable for determination of the average rate of modification for PEG- rhGH. The average modification rate of 3 kinds of PEG- rhGHs is 10%.

关 键 词：重组人生长激素(rhGH) 聚乙二醇化 平均修饰率 荧光胺衍生化法 基质辅助激光解吸附飞行时间质谱(MALDI-TOF-MS) 游离聚乙二醇 

分 类 号：R917[医药卫生—药物分析学]