运用优化的毛细管等电聚焦电泳方法评价_叶酸受体α单抗电荷不均一性

目的: 用优化的cIEF方法分析叶酸受体α单抗(McAb)及其参照品的异构体分布,根据等电点差异,将样品带电荷的异构体分离开来,更好地评价叶酸受体α单抗电荷不均一性。 方法: 毛细管等电聚焦电泳条件:中性涂层的毛细管50 μm×30 cm(有效长度为20 cm);阳极电泳缓冲液200 mmol·L^-1磷酸,阴极电泳缓冲液300 mmol·L^-1氢氧化钠;聚焦电压25 kV,迁移电压30 kV,温度20 ℃ 检测波长280 nm。 结果: 用优化后的方法使样品的电荷异构体能够有效分离,电泳图谱与参照品电泳图谱一致。样品主峰峰面积百分比为60.59%,等电点为7.78,次主峰面积百分比为24.38%,等电点为7.59,主峰与次主峰分离度为1.185(USP)。连续5针进样,主峰面积百分比的RSD为7.05%,等电点的RSD为0.31%;次主峰面积的百分比RSD为5.99%,等电点的RSD为0.69%;待测样品在12 h内稳定。 结论: 采用优化的cIEF方法分析叶酸受体α单抗的电荷不均一性,具有更高的分离度,更好的重现性,为质量控制提供了更准确的手段。

Objective: To optimize the condition of capillary isoelectric focusing(cIEF) method,identified the distribution of charge-based isoforms of folate receptor alpha McAb,which was separated with the difference of isoelectric point by optimized cIEF for quality control. Methods: Neutral-coated capillary column 50 μm×30 cm (effective length 20 cm).Optimum separation was achieved with 200 mmol·L^-1 phosphoric acid (anolyte) and 300 mmol·L^-1 sodium hydroxide (catholyte).The focusing voltage was 25 kV,chemical mobilizing voltage was 30 kV and the capillary temperature was kept constant at 20 ℃ and detected wavelength was 280 nm. Results: The charge-based isoforms of sample were separated effectively and the electropherograms of sample correspond to the reference.The main peak area was 60.59%,pI 7.78,the second peak area was 24.38%,pI 7.59,the resolution (USP) between the main peak and the second peak was 1.185.Sequentialy inject sampe five times,The main peak RSD of area and pI were 7.05% and 0.31%,respectively the second peak RSD of area and pI were 5.99% and 0.69%. Conclusion: The method of optimized cIEF for folate receptor McAb charge heterogeneity has better seperation and repeatbility and it is reliable and feasible for the quality control.