聚乙二醇化重组人干扰素α2b位置异构体比例离子交换高效液相色谱测定法的建立及修饰位点分析

Development of ion exchange high performance liquid chromatography for determination of positional isomer ratio of PEGylated recombinant human interferonα2b and analysis of modification sites

目的建立聚乙二醇化重组人干扰素α2b(PEGylated recombinant human interferonα2b,PEG-rhIFNα2b)位置异构体比例的离子交换高效液相色谱(ion exchange high performance liquid chromatography,IEC-HPLC)测定法,并对各异构体修饰位点进行分析。方法色谱柱采用Proteomix SCX-NP10(10.0 mm×250 mm,10μm);流动相A为:1.8 mmol/L一水合柠檬酸-3.3 mmol/L十二水合磷酸氢二钠(pH 5.3),流动相B为:30 mmol/L柠檬酸三钠-35 mmol/L二水合磷酸二氢钠(pH 6.0);线性梯度洗脱:0~180 min(0%B→12%B),180~220 min(12%B→15%B);流速为:0.4 mL/min;柱温为:25℃;进样量:不低于100μg。同时对IEC-HPLC法进行专属性及重复性验证。采用建立的方法测定3批PEG-rhIFNα2b的位置异构体比例,并通过SPSS 19.0软件进行分析,拟定5个位置异构体比例质量标准。采用建立的方法富集5个位置异构体的洗脱峰,进行修饰位点定位。结果空白对照和rhIFNα2b原液无色谱峰出现,对PEG-rhIFNα2b位置异构体测定无干扰,PEG-rhIFNα2b可见5个异构体峰,且分离度良好;同批PEG-rhIFNα2b连续测定3 d,峰1、2、3、4和5面积的总RSD分别为1.96%、2.53%、2.54%、1.05%和1.86%,均<3.0%。PEG-rhIFNα2b的5个位置异构体比例质量标准可拟定为:峰1为5%~9%、峰2为14%~21%、峰3为7%~10%、峰4为30%~34%、峰5为31%~39%。PEG-rhIFNα2b位置异构体峰1修饰位点主要为K31,峰2主要为K134,峰3主要为K131和K164,峰4主要为K83,峰5主要为K121,未检测到N-末端PEG修饰。结论建立了PEG-rhIFNα2b位置异构体比例的IEC-HPLC测定法,该方法具有良好的专属性及重复性,可用于PEG-rhIFNα2b位置异构体比例的测定。

Objective To develop a method for determination of positional isomer ratio of PEGylated recombinant human interferon(PEG-rhIFN)α2 b by ion exchange high performance liquid chromatography(IEC-HPLC),and analyze the modification sites of various positional isomers.Methods HPLC analysis was performed on a Proteomix SCX-NP10(10.0 mm×250 mm,10μm)column.The mobile phase A was 1.8 mmol/L citric acid monohydrate-3.3 mmol/L disodium phosphate dodecahydrate(pH 5.3),while the mobile phase B was 30 mmol/L trisodium citrate dihydrate-35 mmol/L sodium dihydrogen phosphate dehydrate(pH 6.0).Linear gradient elution(0~180 min:0%B→12%B;180~220 min:12%B→15%B)was adopted at a flow rate of 0.4 mL/min,a column temperature of 25℃and a sample load of not less than 100μg.The IEC-HPLC method was verified for specificity and reproducibility.The positional isomer ratio in three batches of PEG-rhIFNα2 b was determined by the developed method,and the result was analyzed by using SPSS 19.0 software to propose the quality standard for ratios of five positional isomers.The elution peaks of five positional isomers were enriched by the developed method to fix the positions of modification sites.Results No chromatographic peak of blank control or rhIFNα2 b appeared,and there was no interference to the determination of positional isomer in PEGrhIFNα2 b.Five peaks of the isomers with good separation degrees were observed.The RSDs of areas of peaks 1,2,3,4 and 5 in the same batch of PEG-rhIFNα2 b determined for 3 consecutive day were 1.96%,2.53%,2.54%,1.05%and 1.86%respectively,all of which were less than 3%.The quality standards for the ratios of five positional isomers in PEG-rhIFNα2 b were proposed as 5%~9%(peak 1),14%~21%(peak 2),7%~10%(peak 3),30%~34%(peak 4)and 31%~39%(peak 5)respectively.The modification sites of positional isomers were mainly K31 for peak 1,K134 for peak 2,K131 and K164 for peak 3,K83 for peak 4,and K121 for peak 5.However,no PEG-modified N-terminus was detected.Conclusion An IEC-HPLC method for determniation of positional isomer ratio of PEG-rhIFNα2 b was developed,which showed high specificity and reproducibility.