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目的建立融合蛋白FP3等电点毛细管等电聚焦(Capillary isoelectric focusing,cIEF)电泳分析方法。方法采用中性涂层的毛细管,有效长度为20 cm,总长度为30 cm,内径为50μm;分离温度20℃;检测波长280 nm;20 Psi压力进样99 s,电压20 kV分离聚焦20 min,电压25 kV化学迁移25 min。比较不同两性电解质(PharmalyteTM3-10和AmpholineTM3.5-10)、增溶剂类型及浓度、聚焦电压(15、20、30 kV)对样品分离效果的影响,并验证系统的适应性、方法的重现性及系统的稳定性。结果建立的cIEF方法可使样品的电荷异构体有效分离,样品主峰与次主峰的分离度为1.249(USP)。AmpholineTM3.5-10可使样品的各个电荷异质性组分更有效地分离;6 mol/L尿素能提供良好的增溶环境;20 kV聚焦电压即可获得良好的分离效果。连续5次进样,主峰和次主峰迁移时间相对标准偏差(RSD)分别为1.29%和1.32%,样品主区带等电点范围为6.33～6.90,样品在24 h内检测结果稳定。结论建立了融合蛋白FP3等电点cIEF分析方法,该方法具有分离度高、重现性好、方法稳定和结果准确等优点,为该类制品的质量控制提供了更好的手段。
OBJECTIVE: To establish a Capillary Isoelectric Focus (cIEF) electrophoresis assay for the fusion protein FP3. Methods The capillary of neutral coating was used. The effective length was 20 cm, the total length was 30 cm and the inner diameter was 50 μm. The separation temperature was 20 ℃. The detection wavelength was 280 nm. The sample was injected with 20 Psi pressure for 99 s and voltage was 20 kV. , Voltage 25 kV chemical migration 25 min. The effects of different ampholytes (PharmalyteTM3-10 and AmpholineTM3.5-10), solubilizer type and concentration, focusing voltage (15, 20 and 30 kV) on the separation efficiency of the samples were compared and the adaptability of the system and the method reappearance Sexual and systemic stability. Results The established cIEF method can effectively separate the charge isomers of the sample. The resolution of the major and minor peaks of the sample is 1.249 (USP). AmpholineTM3.5-10 can separate each charge heterogeneity component of the sample more effectively; 6mol / L urea can provide a good solubilization environment; 20kV focus voltage can get a good separation effect. The relative standard deviations (RSDs) of 1.29% and 1.32% for the main peak and the sub-main peak migration time were 5 injections. The isoelectric point range of the main zone was 6.33-6.90. The detection results were stable within 24 h. Conclusion The cIEF analysis method of the isoelectric point of fusion protein FP3 is established. The method has the advantages of high resolution, good reproducibility, stable method and accurate results, which provides a better method for the quality control of these products.