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通过高效液相色谱法(HPLC)建立一种测定CN10冻干品中蛋白含量的方法。以0.1 mol/L TrisHCl、0.1 moL/L NaCl、pH 7.2缓冲液作为流动相,色谱柱为TSK2000SWxl(4.6 mm×250 mm)凝胶过滤色谱柱,体积流量为0.65 mL/min,进样体积50μL,检测波长选择280 nm,依据HPLC外标法原理建立CN10含量测定方法,并根据《生物制品质量控制分析方法验证技术审评一般原则》对方法进行相关验证。结果:在该条件下,CN10和人血白蛋白之间有良好的分离度,CN10浓度在0.1~0.6 mg/mL的范围内与其峰面积呈良好的线形关系(R2=0.998 1),最低检出限和最低定量限分别0.01 mg/mL和0.04 mg/mL,方法的重复性RSD(%)为0.38,平均回收率为(100.40±2.28)%。用该方法检测CN10冻干制剂中的CN10蛋白含量时,不受人血白蛋白的影响,具有较高的准确性和专属性。
A method for the determination of protein content in CN10 lyophilized product was established by high performance liquid chromatography (HPLC). The column was TSK2000SWxl (4.6 mm × 250 mm) gel filtration column with 0.1 mol / L TrisHCl, 0.1 mol / L NaCl and pH 7.2 as the mobile phase. The volume flow rate was 0.65 mL / min and the injection volume was 50 μL , Detection wavelength selection 280 nm, according to HPLC external standard method to establish CN10 content determination method, and in accordance with “biological products quality control analysis method validation technical review of the general principles” of the method to verify. Results: There was a good linear relationship between CN10 and human serum albumin under the conditions. The concentration of CN10 in the range of 0.1-0.6 mg / mL was linear with the peak area (R2 = 0.998 1) The limit of detection (RSD) was 0.38 and the average recovery was (100.40 ± 2.28)% with the limits of quantitation of 0.01 mg / mL and 0.04 mg / mL, respectively. CN10 protein content in CN10 lyophilized preparation detected by this method is not affected by human serum albumin and has higher accuracy and specificity.