重组人促卵泡激素氧化亚基分析方法研究

Study on analysis method of recombinant human follicle stimulating hormone oxidized subunits

目的:研究建立重组人促卵泡激素(rhFSH)氧化亚基分析方法,制备稳定的氧化亚基分析用系统适用性溶液。方法:通过加入过氧化氢氧化破坏重组人促卵泡激素,后加入甲硫氨酸终止该氧化作用,制备系统适用性溶液。采用RP-HPLC法分离分析重组人促卵泡激素氧化亚基。使用C_(4)色谱柱(250 mm×4.6 mm,5μm);流动相A为0.2 mol·L^(-1)磷酸二氢钾缓冲液(pH 2.5),流动相B为80%乙腈;流速1 mL·min^(-1);柱温30℃;检测波长210 nm;梯度洗脱(0~10 min,82%A→74%A;10~25 min,74%A→60%A;25~26 min,60%A→20%A;26~56 min,20%A;56~56.1 min,20%A→82%A;56.1~75 min,82%A)。结果:加入甲硫氨酸可有效终止氧化作用,制备得到稳定的系统适用性溶液。优化建立的氧化亚基分析方法专属性良好,重复性(RSD=0.22%)、色谱柱耐用性(RSD=3.21%)、36 h内溶液稳定性(RSD=0.65%)结果良好。9批样品中氧化亚基含量范围为0.687%~1.001%。结论:优化建立的氧化亚基分析方法可对重组人促卵泡激素中的氧化亚基进行准确定量,能够用作重组人促卵泡激素的质量研究和放行检验;研究制备的系统适用性溶液可准确地对氧化亚基进行峰定位。

Objective:To study and establish a method for analyzing the oxidized subunits of recombinant human follicle stimulating hormone(rhFSH),and prepare a stable system suitability solution for the analysis of oxidized subunits.Methods:Hydrogen peroxide was added to destroy rhFSH,and then methionine was added to stop oxidating for preparing the system suitability solution.The RP-HPLC method was used to separate and analyze the oxidized subunits of rhFSH.The C_(4)chromatographic column(250 mm×4.6 mm,5μm)was used,mobile phase Awas 0.2 mol·L^(-1)potassium dihydrogen phosphate buffer(pH 2.5),mobile phase B was 80%acetonitrile.Flow rate was 1 mL·min^(-1),column temperature was 30℃,detection wavelength was 210 nm,gradient elution(0-10 min,82%A→74%A;10-25 min,74%A→60%A;25-26 min,60%A→20%A;26-56 min,20%A;56-56.1 min,20%A→82%A;56.1-75 min,82%A).Results:The addition of methionine could effectively terminate the oxidation and a stable system suitability solution was prepared.The optimized analysis method of oxidized subunits had good specificity,repeatability(RSD=0.22%),column robustness(RSD=3.21%),and solution stability within 36 h(RSD=0.65%).The contents of oxidized subunits in 9 batches of samples ranged from 0.687%to 1.001%.Conclusion:The optimized method of oxidized subunits analysis could accurately quantify oxidized subunits in rhFSH,and could be used for quality research and release inspection of rhFSH.The system suitability solution could accurately locate the peaks of oxidized subunits.