摘要
目的建立聚乙二醇化重组人粒细胞刺激因子(PEGylated recombinant human granulocyte colony-stimulating factor,PEG-rh G-CSF)平均修饰度的高效液相色谱蒸发光散射(high performance liquid chromatography coupled with evaporative light scattering detection,HPLC-ELSD)检测方法,并进行验证。方法采用Jupiter 5u C4 300A色谱柱(250 mm×4.6 mm,5μm),以含0.1%三氟乙酸(TFA)的水溶液为A流动相,含0.1%TFA的乙腈为B流动相,流速1 mL/min,在35℃柱温条件下经分段线性洗脱的方式分离样品,蒸发光散射检测器漂移管温度90℃,载气流速2.0 L/min,用外标法和双对数校正曲线定量溶液中的游离PEG含量。采用蛋白酶K消化去除PEG-rhG-CSF的蛋白部分后,经相同方法测定溶液中的总PEG含量,并计算结合PEG的含量和PEG的平均修饰度。对方法的专属性、线性、精密性、准确性进行验证,并确定检测限(LOD)和定量限(LOQ)。结果本实验建立的HPLC-ELSD法可将PEG与PEG-rh G-CSF及其他辅料成分有效分离;PEG在50~400μg/mL范围内,上样量对数值与相应峰面积对数值之间呈良好的线性关系,拟合回归方程为:Y=1.969 X+0.652,r^2=0.999;精密性试验RSD为1.9%;LOD和LOQ分别为66和109μg/mL;PEG的平均回收率为98.1%。结论本实验建立的方法具有良好的专属性、精密度及准确性,可用于测定PEG-rh G-CSF的平均修饰度。
Objective To develop and validate a method for determination of the average modification extent of PEGylated recombinant human granulocyte colony-stimulating factor（PEG-rh G-CSF） by high performance liquid chromatography coupled with evaporative light scattering detection（HPLC-ELSD）. Methods The HPLC-ELSD was carried out on a Jupiter 5u C4 300A column（250 mm × 4. 6 mm,5 μm）at a column temperature of 30 ℃ and a flow rate of 1. 0 mL/min. The mobile phase was gradient elution with mixture of trifluoroacetic acid（TFA）-water（0. 1 ∶ 99. 9）（A） and trifluoroacetic acid（TFA）-acetonitrile（0. 1 ∶ 99. 9）（B）. The drift tube temperature of ELSD system was set at 90 ℃ while the flow rate of nebulizing gas was 2. 0 L/min. The free PEG content in the solution was determined by external standard method and double logarithmic calibration curve. After removal of the protein portion of PEG-rh G-CSF by protease K,the total PEG content in the solution was determined by the same method,and the amount of binding PEG and the average extent of PEG modification were calculated. The mothod was validated for specificity,linearity,precision and accuracy,and determined for limit of detection（LOD） and limit of quantitation（LOQ）. Results Free PEG was separated from PEG-rh G-CSF and other excipient ingredients effectively by the developed method. The logarithm of peak area from ELSD was linearly correlated to the logarithm of the injected quantity of PEG at a range of 50 - 400 μg/m L. The regression equation was fitted as follows：Y = 1. 969 X ＋ 0. 652,r^2 = 0. 999. The RSD in precision test was 1. 9%. The limit of detection（LOD）and limit of quantitation（LOQ）of the method were 66 and 109 μg/mL respectively. The average recovery of PEG was 98.1%. Conclusion The developed method showed high specificity,precision and accuracy,which might be used for the determination of average modification extent of PEG-rh G-CSF.
作者
李永红
史新昌
李响
韩春梅
饶春明
LI Yong-hong;SHI Xin-chang;LI Xiang;HAN Chun-mei;RAO Chun-ming(National Institutes for Food and Drug Control,Key Laboratory for Standardization of Method for Quality Control of Biotechnical Products,Beijing 100050,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2018年第7期758-762,共5页
Chinese Journal of Biologicals
基金
国家科技重大专项课题(2015ZX09501008-001)
关键词
聚乙二醇
重组人粒细胞刺激因子
平均修饰度
高效液相色谱法
蒸发光散射检测
PEG
Recombinant human granulocyte colony-stimulating factor （rhG-CSF）
Average modification extent
High performance liquid chromatography （HPLC）
Evaporative light scattering detection （ELSD）
