离子交换色谱法测定反义寡核苷酸药物CT102的含量

Development of ion-exchange chromatography method for determination of phosphorothioate antisense oligonucleotide drug CT102

目的建立反义寡核苷酸药物CT102的离子交换色谱含量测定方法。方法对不完全硫代序列杂质5'(P=O)、3'(P=O)与CT102全长序列的分离情况进行了考察,采用DNA Pac PA-100阴离子交换柱(4 mm×250 mm);流动相A为1mmol/L乙二胺四乙酸(EDTA)二钠/25 mmol/L三羟基氨基甲烷(Tris),5%乙腈(V/V);流动相B为1 mmol/LEDTA二钠/25mmol/L Tris/1.5 mol/L溴化钠,5%乙腈(V/V);洗脱梯度为0～15 min,40%B～100%B;柱温50℃;流速为1 ml/min;检测波长260 nm。结果合成过程中产生的主要杂质不完全硫代序列(P=O)能够实现与CT102全长序列的分离,CT102在75～1200μg/ml范围内呈良好的线性关系,r2=0.9999,仪器精密度良好,日内精密度RSD为0.64%,日间精密度RSD值为1.19%,检测限为5μg/ml,最低定量限为15μg/ml。结论该方法准确可靠,体系稳定,经济简便,不完全硫代杂质与主峰分离良好,可用于CT102原料药的含量测定,也为规模化制备和终产品的质量控制提供可靠保障。

Objective To develop an ion-exchange high-performance liquid chromatography（IE-HPLC） method for the quantification of the phosphorothioate antisense oligonucleotide drug CT102.Methods CT102 and its analogs of phosphodiester sequences 5’（P = O） and 3’（P = O） were synthesized to study the separation conditions.Separation was performed on a DNA Pac PA-100 column（4mm × 250mm）;mobile phase A was 1 mmol / L EDTA /25 mmol / L Tris and 5% acetonitrile（V / V）;mobile phase B was 1 mmol / L EDTA /25 mmol / L Tris /1.5 mol / L sodium bromide and 5% acetonitrile（V / V）;the gradient was from 40% to 100% of solution B in 15 min;the column temperature was set on 50 ℃;the flow rate was 1.0 ml / min;and the detection wave length was 260 nm.Results Under the optimal conditions,the impurities 5’（P = O） and 3’（P = O） could be well separated from CT102.Good liner relationship was obtained at the range of 75-1200 μg / ml,r2 = 0.9999.The precision result was desirable and the RSD of the intraday and the interday were 0.64% and 1.19%.The limit of detection was 5 μg / ml and the limit of quantification was 15 μg / ml.Conclusion The proposed IE-HPLC method is sensitive,specific,accurate and stable,with good resolution between（P = S /O） and CT102.It has been successfully applied to the quantification of CT102 and it could also be used for the quality control of the purification procedure and the final product of CT102.