应用RP-HPLC法检测重组猪干扰素α成品蛋白含量

Application of RP-HPLC assay to detect the protein content of finished products of recombinant porcine interferon α

目的：建立测定重组猪干扰素α成品中蛋白含量的反相高效液相色谱法（RP-HPLC）,并进行验证。方法：应用RP-HPLC法测定重组猪干扰素α标准品及供试品的蛋白含量,并对方法的线性、精密度、稳定性、重复性进行验证。采用已建立的RP-HPLC检测方法测定7批重组猪干扰素α样品的蛋白含量,与Lowry法的检测结果进行比较。结果：重组猪干扰素α在6.141 7-25μg范围内,与峰面积呈现良好的线性关系（R2=0.985 9）。用所建立的方法测定同一标准品,进样6次,计算得到RSD（相对标准偏差）为1.7%;同一批样品进样12次,检测供试品的干扰素α蛋白含量,计算得到RSD为3.9%;同一批样品,取6份分别进样,检测供试品的干扰素α蛋白含量,计算得到RSD为0.9%。结论：建立了用RP-HPLC法测定重组猪干扰素α蛋白含量的方法。该方法简便、精密度、稳定性、重复性好,将蛋白分离成单一组分峰,紫外定量测定各峰吸光度,集定量与定性、纯度检验合一,定量范围可小于10μg,测定体积只需几微升,可用于重组猪干扰素α成品蛋白含量检测。

Objective： To establish a reversed-phase high performance liquid chromatography（ RP-HPLC）method to determine the protein content of finished products of recombinant porcine interferon α and verify the method. Methods： The linearity,precision,stability,and repeatability of the RP-HPLC method were verified. Then the RP-HPLC method was used to determine the protein contents of the standard substance of recombinant porcine interferon α and 7 batches of samples. The determination result was compared with that by Lowry assay. Results：The content of recombinant porcine interferon had good linear relationship with the peak area within the range of6. 141 7 ～ 25 μg（ R2= 0. 985 9）. The RSD of 6 injections of the same standard solution was 1. 7%,that of 12 injections of the same test sample was 3. 9%. and that of 6 samples of the same batch was 0. 9%. Conclusion： A method for determining the protein content of recombinant porcine interferon α by RP-HPLC was established. The method is simple and has good sensitivity（ LOD lower than 10 μg）,precision,stability,and repeatability,with small sample volume. It can be used for the protein content detection of finished products of recombinant porcine interference α.