RP-HPLC法检测CN_(10)蛋白的PEG化修饰率及含量

Determination of modification rate and content of PEGylated CN_(10) by RP-HPLC

目的建立一种简单有效的测定CN10蛋白PEG化修饰率的方法 ,用于PEG化修饰工艺中的质量控制。方法采用反相高效液相色谱(RP-HPLC)法,以TSKgel Octadecyl-4PW作为色谱分离柱,以含0.12%三氟乙酸(TFA)、5%乙腈的水溶液作为A相溶液,以含0.1%TFA的乙腈作为B相溶液,在50℃柱温条件采用分段线性洗脱的方式分离蛋白,并考察CN10蛋白以及PEG化修饰后CN10蛋白的量效关系,根据外标法检测CN10和CN10-PEG的蛋白含量,根据PEG化修饰前后CN10蛋白量效关系的变化推断CN10蛋白的PEG化修饰率。结果 PEG化修饰前后的CN10蛋白经TSKgel Octadecyl-4PW色谱柱层析均能达基线分离,当用214 nm波长检测时,CN10蛋白浓度以及PEG修饰后的CN10蛋白均与其对应峰面积呈现良好的线性关系(r2=0.999 58;r2=0.999 67)。结论建立了一种简单快速的检测CN10蛋白PEG化修饰率的方法 ,此方法具有较高的准确性及专属性,可用于CN10的PEG化修饰工艺的监测。

Objective To establish a simple and effective method to determine the modification rate of PEGylated CN10 protein, which could be used for quality control of EGylation technique. Methods The reversed-phase high performance liquid chromatography （RP-HPLC） was carried out in this study, the separation column is TSKgel Octadecyl-4PW chromatogram column, by using the acetonitrile content of 0.1% TFA as B-phase, when the column temperature is 50 ℃ , by using pieeewise linear elution to separate proteins, meanwhile, studying the dose-effect relationship between CN10 and PEGylated CN10, then adpoted the external standard method to determmine the protein content of CN10 and PEGylated CN10, according to the relationship for change of dose-effect with CN10 before and after PEG modification to deduce the modification rate of PEGylated CN10. Results In performance of column process, before and after PEG modification, CN10 protein achieved baseline separation and was separated well. When we detected the wavelength of 214 nm, both protein concentrations of CN10 and PEGylated CN10 showed a good linear relationship with their corresponding peak area （ r2 =0. 999 8 ;r2 = 0. 998 2 ）. Conclusion A simple and effective method has been established to determine the modification rate of PEGylated CN10 protein, it has a high accuracy and specificity, and could he used in the technology of PEGylated CN10.