HPLC法测定注射用重组人白介素-11中的相关蛋白

Determination of related proteins in recombinant human interleukin-11 for injection by HPLC

目的:建立测定注射用重组人白介素-11中相关蛋白的高效液相色谱法,对主要杂质进行鉴别。方法:采用HPLC法分离分析注射用重组人白介素-11相关蛋白,利用Agilent 1200型高效液相色谱仪,使用Sepax Bio-C4色谱柱(300?,250 mm×4.6 mm,5μm),以0.1%三氟醋酸水溶液为流动相A,70%乙腈溶液(含0.075%三氟醋酸)为流动相B,进行梯度洗脱,流速1.0 mL·min-1,检测波长220 nm,柱温40℃;采用UPLC-QTOF法分析重组人白介素-11相关蛋白,使用Waters BEH-C4色谱柱(300?,2.1 mm×100 mm,1.7μm),以0.1%甲酸水溶液为流动相A,0.1%甲酸乙腈溶液为流动相B,进行梯度洗脱,对主要相关蛋白进行鉴定。结果:UPLC-QTOF鉴定了3个主要相关蛋白,杂质1、2和3,相对分子质量分别为5 488.446、4 576.214和14 489.558。建立的HPLC测定相关蛋白方法具有良好的结果,与《中华人民共和国药典》2015年版三部中重组人白介素-11原液的纯度方法(HPLC法)比较,杂质检出能力更强。结论:建立的HPLC法简便,灵敏度高,专属性好,能有效反应药品质量。

Objective:To establish an HPLC method for determination of related proteins in recombinant human interleukin-11 for injection to indentify major impurities.Methods:Recombinant human interleukin-11 for injection related proteins were separated and analyzed by Agilent 1200 HPLC.A Sepax Bio-C4 column(300?,250 mm×4.6 mm,5μm)was adopted for the study with gradient solution.The mobile phase A was 0.1%trifluoroacetic acid,the mobile phase B was 70%acetonitrile(containing 0.075%trifluoroacetic acid).The flow rate was 1.0 mL·min-1,the detection wavelength was 220 nm,and the column temperature was 40℃.UPLC-QTOF method was used to analyze recombinant human interleukin-11 related prsteins.A Waters BEH-C4 column(300?,2.1 mm×100 mm,1.7μm)was adopted for the study with gradient solution.The mobile phase A was 0.1%formic acid,the mobile phase B was 0.1%formic acid in acetonitrile.The main related proteins were identified.Results:UPLC-QTOF identified three main related proteins,impurities 1,2 and 3,with relative molecular mass of 5 488.446,4 576.214 and 14 489.558,respectively.The established HPLC method for determination of related proteins had good results.Compared with the purity method(HPLC method)of recombinant human interleukin-11 stock solution in 2015 edition of the Chinese pharmacopoeia Volume III,the impurity detection ability was stronger.Conclusion:The established HPLC method is simple,sensitive and specific,which can effectively reflect the quality of the drugs.