摘要
为了进行硫代反义寡核苷酸药物FT19的质量控制研究,建立了用阴离子交换高效液相色谱(AX-HPLC)和毛细管凝胶电泳(CGE)分析自行合成的FT19有关物质的方法。设计合成了FT19的硫代不完全序列(P=O)1和短序列(n-x)并将它们作为已知杂质。在AX-HPLC上,使用的分析柱为DNAPacPA-100(4mm×250mm);流动相A为10mmol/LNaOH-0.1mol/LNaCl,流动相B为10mmol/LNaOH-3mol/LNaCl;梯度洗脱条件为流动相B液在8min内从60%升至100%;流速1mL/min;柱温40℃;检测波长为260nm。CGE所用毛细管规格为内径100μm,总长度为31cm,有效长度为20cm;电泳缓冲溶液为三羟甲基氨基甲烷(Tris)-硼酸-7mol/L尿素,pH8.5;采用电动进样,进样电压-10kV;分离胶为250g/L的聚丙烯酰胺;检测波长为254nm。结果表明,FT19与硫代不完全的(P=O)1序列在AX-HPLC上能够达到基线分离,与短序列(n-1)在CGE上能达到基线分离。说明AX-HPLC和CGE联合应用能够很好地分析FT19中的有关物质。
A method using anion exchange high performance liquid chromatography（AX-HPLC）and capillary gel electrophoresis（CGE）was established for the analysis of the related substances in phosphorothioate antisense oligonucleotide FT19 for quality control.The FT19 and its analogs of partial phosphodiester compounds（P=O）1 as well as deletion sequences（n-1）were analyzed.AX-HPLC with the DNA Pac PA-100 column（4 mm×250 mm）was used for the separation of phosphorothioate modifications.The size of the capillary column used in the CGE was 31 cm × 100 μm with an effective length of 20 cm.It was found that full phosphorothioate and partial phosphodiester oligonucleotides of the same length were successfully separated,and the deletion sequences（n-1）and the full-length sequence n were successfully separated in CGE.The results demonstrated that the related substances of phosphorothioate oligonucleotides can be well analyzed by AX-HPLC and CGE.
出处
《色谱》
CAS
CSCD
北大核心
2008年第5期540-543,共4页
Chinese Journal of Chromatography
基金
国家科技攻关项目(2004BA519A35).
关键词
阴离子交换高效液相色谱
毛细管凝胶电泳
硫代反义寡核苷酸
有关物质
：anion exchange high performance liquid chromatography（AX-HPLC）
capillary gel electrophoresis（CGE）
phosphorothioate oligonucleotide
related substances
